m neoaurum atcc 25795  (ATCC)


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    Name:
    Mycobacterium neoaurum 3503 NCTC 10818
    Description:

    Catalog Number:
    25795
    Price:
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    Applications:
    Produces L-amino amidase
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    Structured Review

    ATCC m neoaurum atcc 25795
    ( a ) Partial gene cluster encoding the catabolism of sterols in M. <t>neoaurum</t> ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum <t>ATCC</t> 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

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    Images

    1) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    2) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    3) Product Images from "Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes"

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0705-x

    Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements
    Figure Legend Snippet: Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Techniques Used: Permeability, Sequencing, Molecular Weight, Marker, Staining, Fluorescence, Spectrophotometry, Cell Culture, Standard Deviation

    Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv
    Figure Legend Snippet: Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Techniques Used: Sequencing

    4) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    5) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    6) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    7) Product Images from "Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes"

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0705-x

    Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements
    Figure Legend Snippet: Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Techniques Used: Permeability, Sequencing, Molecular Weight, Marker, Staining, Fluorescence, Spectrophotometry, Cell Culture, Standard Deviation

    Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv
    Figure Legend Snippet: Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Techniques Used: Sequencing

    8) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    9) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    10) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    11) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    12) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    13) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    14) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    15) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    16) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    17) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    18) Product Images from "Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes"

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0705-x

    Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements
    Figure Legend Snippet: Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Techniques Used: Permeability, Sequencing, Molecular Weight, Marker, Staining, Fluorescence, Spectrophotometry, Cell Culture, Standard Deviation

    Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv
    Figure Legend Snippet: Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Techniques Used: Sequencing

    19) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    20) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    21) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    22) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    23) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    24) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    25) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    26) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    27) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    28) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    29) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    30) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    31) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    32) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    33) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    34) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    35) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    36) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    37) Product Images from "Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism"

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep21928

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.
    Figure Legend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Techniques Used: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.
    Figure Legend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Techniques Used: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.
    Figure Legend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Techniques Used: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    38) Product Images from "Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes"

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-017-0705-x

    Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements
    Figure Legend Snippet: Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Techniques Used: Permeability, Sequencing, Molecular Weight, Marker, Staining, Fluorescence, Spectrophotometry, Cell Culture, Standard Deviation

    Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv
    Figure Legend Snippet: Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Techniques Used: Sequencing

    39) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    40) Product Images from "Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons"

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-018-0916-9

    The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk
    Figure Legend Snippet: The sequence alignment of known KstD enzymes. DSM1381-KstDs from M. neoaurum DSM 1381, ATCC25795-KstDs from M. neoaurum ATCC 25795, DSM43269-KstDs from R. rhodochrous DSM 43269, Cho1-4-KstDs from R. ruber strain Chol-4, SQ1-KstDs from R. erythropolis SQ1. Active site residues and residues involved in co-ordination of a FAD in SQ1-KstD1 are indicated by number sign. A conserved sequence for FAD-binding region is indicated by asterisk

    Techniques Used: Sequencing, Binding Assay

    Related Articles

    Sequencing:

    Article Title: Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as Causes of Mycobacteremia ▿
    Article Snippet: .. Isolates whose complete 16S rRNA gene sequence was a 100% match to the complete 16S rRNA gene sequence of the type strain of M. neoaurum (ATCC 25795) or M. lacticola (ATCC 9626) or that exhibited > 99.5% identity (difference of 6 bp or less) to the complete 16S rRNA gene sequence of the M. neoaurum type strain (designated M. neoaurum -like) were included in the study. ..

    Activity Assay:

    Article Title: Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons
    Article Snippet: .. Compared with the KstD3 from M. neoaurum ATCC 25795, the KstD3 from M. neoaurum DSM 1381 hardly showed detectable activity on both AD and 4HP [ ]. .. Following the analysis of its aa sequence, it is possible that these observations can be attributed to the eight substitutions near Tyr318 in the catalytic domain of KstD3.

    Construct:

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes
    Article Snippet: .. The strain ΔmmpL3 was constructed by deleting mmpL3 in M. neoaurum ATCC 25795. .. The engineered strain WIIIΔmmpL3 was constructed by deleting mmpL3 in the WIII strain based on the allelic recombination.

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    ATCC m neoaurum
    Neighbor-joining trees of the rpoB gene of M. <t>neoaurum</t> , M. lacticola , and M. neoaurum- like clinical isolates and culture collection strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations. M. aurum is included as
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    Neighbor-joining trees of the rpoB gene of M. neoaurum , M. lacticola , and M. neoaurum- like clinical isolates and culture collection strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations. M. aurum is included as

    Journal: Journal of Clinical Microbiology

    Article Title: Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as Causes of Mycobacteremia ▿

    doi: 10.1128/JCM.00853-10

    Figure Lengend Snippet: Neighbor-joining trees of the rpoB gene of M. neoaurum , M. lacticola , and M. neoaurum- like clinical isolates and culture collection strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations. M. aurum is included as

    Article Snippet: Isolates whose complete 16S rRNA gene sequence was a 100% match to the complete 16S rRNA gene sequence of the type strain of M. neoaurum (ATCC 25795) or M. lacticola (ATCC 9626) or that exhibited > 99.5% identity (difference of 6 bp or less) to the complete 16S rRNA gene sequence of the M. neoaurum type strain (designated M. neoaurum -like) were included in the study.

    Techniques:

    Neighbor-joining trees of 16S rRNA (A) and hsp65 (B) genes of M. neoaurum , M. lacticola , and M. neoaurum -like clinical isolates and culture collection strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations. M. cosmeticum

    Journal: Journal of Clinical Microbiology

    Article Title: Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as Causes of Mycobacteremia ▿

    doi: 10.1128/JCM.00853-10

    Figure Lengend Snippet: Neighbor-joining trees of 16S rRNA (A) and hsp65 (B) genes of M. neoaurum , M. lacticola , and M. neoaurum -like clinical isolates and culture collection strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations. M. cosmeticum

    Article Snippet: Isolates whose complete 16S rRNA gene sequence was a 100% match to the complete 16S rRNA gene sequence of the type strain of M. neoaurum (ATCC 25795) or M. lacticola (ATCC 9626) or that exhibited > 99.5% identity (difference of 6 bp or less) to the complete 16S rRNA gene sequence of the M. neoaurum type strain (designated M. neoaurum -like) were included in the study.

    Techniques:

    ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Journal: Scientific Reports

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    doi: 10.1038/srep21928

    Figure Lengend Snippet: ( a ) Partial gene cluster encoding the catabolism of sterols in M. neoaurum ATCC 25795. Genes in the map are color-coded according to assigned or proposed function: orange, mce cluster genes for steroids transportation; green, side-chain degradation genes; blue, nucleus cleavage genes; gray, unassigned function; yellow, gene encoding the transcriptional repressor KstR1. The numbers, e.g. 55 bp and 646 bp, beside the meander lines indicate the spaces between adjacent genes. ( b ) Schematic of the genomic organization of hsd4A homologues in M. neoaurum ATCC 25795 and other mycobacteria. Percentages, such as 71%, 81% and 67%, indicate the amino acid sequence identity of Hsd4A MN in M. neoaurum ATCC 25795 with homologs from other mycobacteria, including M. tuberculosis H37Rv, M. smegmatis str. MC 2 155 and R . jostii RHA1. The location of hsd4A is between the yrbE4B-4A and fadE26 – 27 . The size and direction of genes are indicated by arrows with corresponding length to scale.

    Article Snippet: There are three KstD homologues in M. neoaurum ATCC 25795, and KstD1 was characterized as the major enzyme in our previous work .

    Techniques: Sequencing

    Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Journal: Scientific Reports

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    doi: 10.1038/srep21928

    Figure Lengend Snippet: Phenotypic analyses of the metabolism of sterol by M. neoaurum ATCC25795 and its derivative strains. ( a ) HPLC chromatogram comparison of the products from the transformation of 2 g/l of cholesterol in MYC/02 media at 30 °C by strains M. neoaurum ATCC 25795 (blue), NwIB-XII (red) and XIIp261 hsd4A (green). Cholesterol can be completely degraded by M. neoaurum ATCC 25795 without obvious accumulation of intermediates. The catabolism of cholesterol in NwIB-XII was blocked to accumulate multiple metabolites, including ADD (I), AD (II), TS (III) and BD (IV). The plasmid pMV261- hsd4A was electro-introduced into NwIB-XII, resulting in the XIIp261 hsd4A strain. Strain XIIp261 hsd4A transformed cholesterol to ADD (I) and AD (II). ( b ) Conversion relationship between the metabolites (I to IV). Arrows in purple signify the function of Hsd4A MN deduced from the product phenotypes of strains NwIB-XII and XIIp261 hsd4A . Hsd4A MN can irreversibly catalyze the oxidation of TS to AD and BD to ADD, respectively. AD, androst-4-ene-3,17-dione; ADD, androst-1,4-dien-3,17-dione; BD, boldenone; TS, testosterone.

    Article Snippet: There are three KstD homologues in M. neoaurum ATCC 25795, and KstD1 was characterized as the major enzyme in our previous work .

    Techniques: High Performance Liquid Chromatography, Transformation Assay, Plasmid Preparation

    Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Journal: Scientific Reports

    Article Title: Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

    doi: 10.1038/srep21928

    Figure Lengend Snippet: Schematic diagram of sterol catabolism and the combinatorial construction of HBCs producing strains. ( a ) The construction of strain XIIΔ hsd4A -p261 kstD1 . To construct XIIΔ hsd4A -p261 kstD1 , hsd4A was deleted in a kshAs -null mutant and then kstD1 was overexpressed. The resulting strain XIIΔ hsd4A -p261 kstD1 transformed phytosterols to 1,4-HBC as the major product. ( b ) The construction of strain XIIΔ hsd4A Δ kstD123 . To construct XIIΔ hsd4A Δ kstD123 , hsd4A was deleted in a kshAs -null mutant and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain XIIΔ hsd4A Δ kstD123 transformed phytosterols to 4-HBC as the major product. ( c ) The construction of strain MNΔ hsd4A Δ kstD123 . To construct MNΔ hsd4A Δ kstD123 , hsd4A was deleted in M. neoaurum ATCC 25795 and then kstD1 , kstD2 and kstD3 were knocked out sequentially. The resulting strain MNΔ hsd4A Δ kstD123 transformed phytosterols to 9-OHHBC as the major product. 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one. ( d ) Proposed metabolism of the sterol side chain for the production of C19 and C22 steroids in M. neoaurum ATCC 25795. The conversion from 22HOBNC-CoA to AD was designated as the AD pathway (orange arrows) and has been proposed as the sole pathway of sterol side-chain degradation. Here, a new pathway is proposed: the conversion from 22HOBNC-CoA to 4-HBC (designated as the HBC pathway, blue arrows). Between the two pathways, 22HOBNC-CoA is the branching-node, which leads to AD via the catalysis of Hsd4A MN and leads to 4-HBC via an aldolytic reaction. The β-hydroxybutyryl-CoA moiety of 22HOBNC-CoA and acetoacetyl-CoA moiety of 22OBNC-CoA are labelled in red, which were the active substrates of Hsd4A MN in vitro . 24HOC-CoA, 24-hydroxy-3-oxo-chol-4-en-26-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; OBNC22-CoA, 3-oxo-22,23-bisnorchol-4-ene-22-oyl-CoA; OBNC20FA, 3-oxo-23,24-bisnorchol-4-ene-20-formic acid; OBNC20CA, 3-oxo-23,24-bisnorchol-4-ene-20-carbaldehyde. ( e ) The proposed pathway of cholate side-chain degradation in Pseudomonas sp. strain Chol1 37 . sal , an aldol-lyase; sad , an aldehyde dehydrogenase. Intercepted arrows (red) indicate the blockage of the target reaction by gene deletion. Arrows in purple signify the enhanced KstD1 activity. Compounds in green are the major products accumulated in constructed strains. The compound in brackets is an unstable compound that will lead to the decomposition of steroid nucleus and further degradation. Dashed arrows indicate the proposed way for HBCs formation previously suggested by Szentirmai 32 . Arrows in arrays signify multiple reaction steps.

    Article Snippet: There are three KstD homologues in M. neoaurum ATCC 25795, and KstD1 was characterized as the major enzyme in our previous work .

    Techniques: Construct, Mutagenesis, Transformation Assay, In Vitro, Activity Assay

    Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Journal: Microbial Cell Factories

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    doi: 10.1186/s12934-017-0705-x

    Figure Lengend Snippet: Effects of deleting mmpL3 on cell permeability and the cholesterol utilization. a Evidence for allelic replacement at the mmpL3 locus of M. neoaurum ATCC 25795. The wild-type (WT) 4839-bp sequence was replaced by a 2145-bp fragment ligated with a 1074-bp upstream sequence and a 1071-bp downstream of the mmpL3 ( m ) gene, thus resulting in the mmpL3 -deficient M. neoaurum ( m -mut1 and m -mut2). MWM molecular weight marker. b Effects of MmpL3 disruption on cell permeability. Diluted cell suspensions were stained with fluorescein diacetate (FDA) and then the mixtures were detected by a fluorescence spectrophotometer. c Growth characteristics of the wild-type M. neoaurum ATCC 25795 (WT, squares ), the deficiency strain of mmpL3 in the WT (Δ mmpL3 , open circles ) and the complementation strain of mmpL3 in the Δ mmpL3 (Δ mmpL3 + mmpL3 , triangles ) cultured in MM with 1.0 g/L cholesterol. The control is the medium containing 1.0 g/L cholesterol without inoculum. d Quantitative determination of residual cholesterol from the three strains cultured in MM with 1.0 g/L cholesterol. Data represent mean ± standard deviation of three measurements

    Article Snippet: The strain ΔmmpL3 was constructed by deleting mmpL3 in M. neoaurum ATCC 25795.

    Techniques: Permeability, Sequencing, Molecular Weight, Marker, Staining, Fluorescence, Spectrophotometry, Cell Culture, Standard Deviation

    Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Journal: Microbial Cell Factories

    Article Title: Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes

    doi: 10.1186/s12934-017-0705-x

    Figure Lengend Snippet: Localization of the mmpL3 homologues in the genome of M. neoaurum ATCC 25795 and other mycobacteria. The size and direction of genes from the predicted genome information were displayed as an arrow according to the scale. The percentages, such as 94 and 72%, indicate the sequence identity of mmpL3 from M. neoaurum ATCC 25795 with the homologs in M. neoaurum NRRL B-3805, M. neoaurum VKM Ac-1815D, and M . tuberculosis H37Rv

    Article Snippet: The strain ΔmmpL3 was constructed by deleting mmpL3 in M. neoaurum ATCC 25795.

    Techniques: Sequencing