ptyb21  (New England Biolabs)

Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Effect of Amino Acid Substitutions on Biological Activity of Antimicrobial Peptide: Design, Recombinant Production, and Biological Activity

doi: 10.22037/ijpr.2019.112397.13734

Figure Lengend Snippet: The structure of recombinant vector pTYB21 for novel designed AMP (P24). (A) The structure of pTYB21; the important parts are outlined with red box. (B) The sequence of first 5 amino acids at MCS of pTYB21, was added to the N-terminal of P19, indicated in red box

Article Snippet: Cloning and transformation of AMP gene-containing construct The P24 gene was digested with NdeI at 5’ end and EcoRI at the 3´end of the gene for site-directed cloning into the pTYB21.

Techniques: Recombinant, Plasmid Preparation, Sequencing

Journal: Nature Communications

Article Title: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

doi: 10.1038/s41467-022-28883-8

Figure Lengend Snippet: PsPDC1 promotes an alternative branch to 4HPAA for norcoclaurine production. a The PPDC branch pathway (green) can produce 4HPAA via 4HPP, and this missing link is also unreported in P. somniferum . b Structural comparison of classified phenylpyruvate decarboxylase (PPDC) enzymes ARO10 (purple), Azospirillum brasilense PPDC (AbPPDC, PDB ID: 2Q5O [ https://www.rcsb.org/structure/2Q5O ], blue) in comparison to typical Zymomonas mobilis pyruvate decarboxylase (PDC) (ZmPDC, PDB ID: 2WVA [ https://www.rcsb.org/structure/2WVA ], grey) and candidate PPDC sequence PsPDC1 (green). The modeled PsPDC1 active site contains Y332, which is also present in typical PDC enzymes which decarboxylate pyruvate. In this respect, the PsPDC1 active site is distinct from microbial PPDCs, which all contain smaller residues (red) in place of Y332 ( Lactococcus lactis KdcA contains S286 corresponding to Y332). Yet, the presence of Y332 in PsPDC1 does not interfere with the docking of tyrosine into the PsPDC1 active site. c Cross-validation for correct assignment of PPDC model training sequences is performed using SVM models (blue), Random forests models (green), and by comparing sequence homology of each training sequence to a consensus sequence of PPDC training sequences and a consensus sequence of PDC training sequences (grey), as described in the methods section. d SVM-based prediction of putative PPDC sequences visualized in three dimensions by compressing high-dimensional data (Supplementary Table 3 , upper table) into two dimensions (PC1 and PC2) and plotting them together with two-dimensional decision scores. Prediction spaces with two-dimensional decision scores above and below 2 are colored green and grey, respectively. Prediction score trends for truncated PsPDC1 isoform X1 (TrcPsPDC1-IX1, red), PsPDC1 (red), PsPDC2 and Ps2HCLL (2-hydroxyacyl-CoA ligase-like) are similar in high dimensional models (Supplementary Table 3 ). e PsPDC1 mediates in vivo production of 4-hydroxyphenylethanol (tyrosol) through a 4HPAA intermediate (green), in M9 medium supplemented with 1.2 mM 4HPP at 25 °C with 180 rpm shaking. Strain P1-01-AI, which contains PsPDC1 (red), mediates higher tyrosol production than that of strains P2-01-AI and P3-01-AI, which contain PsPDC2 (blue) and Ps2HCLL (magenta), respectively. PsPDC1 (red) mediates downstream production of norcoclaurine (NC) from LB supplemented with 5 mM tyrosine and 3.7 mM dopamine in strain P1-02-AI, at 20–25 °C with 180 rpm shaking. Here, tyrosol is detected after 71 h, and norcoclaurine is detected after 41 h, from filtered and dried culture medium as trimethylsilyl (TMS)-derivatives using GC-MS, as shown in the extracted ion chromatograms (EICs). Detection of PsPDC1 products is replicated in Supplementary Fig. 4 .

Article Snippet: To produce pTYB21-PsTyDC1, pTYB21-PsPDC1, pTYB21-PsPDC2 and pTYB21-Ps2HCLL, PsTyDC1, PsPDC1 , Ps2HCLL , and N-terminal truncated PsPDC2 were PCR amplified and cloned into pTYB21 digested with SapI and BamHI via Gibson assembly (NEB) .

Techniques: Sequencing, In Vivo, Gas Chromatography-Mass Spectrometry

Journal: Nature Communications

Article Title: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

doi: 10.1038/s41467-022-28883-8

Figure Lengend Snippet: Optimization of norcocluarine, reticuline, and N -methylcoclaurine production for analysis of flux through hybrid pathways. a PpDDC-Y79F-F80Y-H181N (PpDDC-T) and PsPDC1 containing strain P1-07-AI (olive green) prefers the norlaudanosoline containing pathway. Combination of PpDDC-T, ARO10 and PsTyDC1 in strain A1-06-AI (dark grey) promotes both norcoclaurine and norlaudanosoline containing pathways. ARO10 expressing strain A1-01-DE3 (light purple) converts tyrosine and dopamine to norcoclaurine and N -methylcoclaurine. N -Methylcoclaurine and reticuline were extracted with ethyl acetate from cultures 40 h after addition of tyrosine together with L-DOPA or dopamine. Tested strains P1-06-DE3 (blue), P1-07-AI (olive green), A1-06-AI (dark grey) and A1-01-DE3 (light purple) each contain Cj6OMT, CjCNMT, Cj4OMT, NCS, plus the indicated genes of the bottom 4 rows. P1-06-DE3 and P1-07-AI contain the same genes, but P1-06-DE3 was induced with only IPTG, without including arabinose for PsPDC1 expression. Cultures containing PpDDC-T and L-DOPA were supplemented with additional sodium ascorbate. The BL21(AI) derived strain P1-07-AI was induced with IPTG and arabinose. For improved N -methylcoclaurine production, A1-01-DE3 was supplemented with the aldehyde reductase/dehydrogenase inhibitor gossypol. Additional culture conditions are described in the methods section. Extracted N -methylcoclaurine and reticuline were TMS-derivatized and analyzed with GC-MS ( t = 40 h, n = 3). After extraction, cultures were stored at 4 °C and stable norcoclaurine titers from culture medium were analyzed with LC-MS ( n = 2). b Isotope profiling of strains P1-02-AI (expressing PsPDC1) and P1-04-AI (expressing PsPDC1 and PsTyDC1), which produce N -methylcoclaurine- d 6 (P1-02-AI - 62 nM, P1-04-AI 160 nM) and reticuline- d 5 from tyrosine- d 4 and L-DOPA- d 3 ( t = 61 h, n = 3). There is a synergistic improvement in BIA production when combining PsPDC1 and PsTyDC1. Here, NCS catalyzes the loss of a deuterium from in vivo generated dopamine- d 3 . c For tracing aromatic isotope flux from tyrosine to norcoclaurine and N -methylcoclaurine, alkaloids were extracted with ethyl acetate from the A1-01-DE3 culture 40 h after addition of tyrosine- d 3 and dopamine, according to the methods section. Extracted alkaloids were TMS-derivatized and analyzed with GC-MS ( n = 3). After extraction, cultures were stored at 4 °C and stable BIA titers from culture medium were analyzed with CE-MS ( n = 3); the fraction of labeled BIA- d 4 and unlabeled BIA from natural tyrosine in the rich TB broth can be quantified. With exception to unlabeled norcoclaurine ( n = 2), all other individual samples were analyzed 3 times ( n = 3) to generate bar graphs in Prism 7, with error bars representing mean values +/− standard deviations. Source data are provided as a Source Data file.

Article Snippet: To produce pTYB21-PsTyDC1, pTYB21-PsPDC1, pTYB21-PsPDC2 and pTYB21-Ps2HCLL, PsTyDC1, PsPDC1 , Ps2HCLL , and N-terminal truncated PsPDC2 were PCR amplified and cloned into pTYB21 digested with SapI and BamHI via Gibson assembly (NEB) .

Techniques: Expressing, Derivative Assay, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, In Vivo, Generated, Labeling

Journal: Nature Communications

Article Title: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

doi: 10.1038/s41467-022-28883-8

Figure Lengend Snippet: PsPDC1 and PsTyDC1 promote the norlaudanosoline pathway from L-DOPA to reticuline. a Pathway expansion of the P. somniferum 4HPAA pathway to a dual norcoclaurine (NC) and norlaudanosoline (NL) pathway. b Strains T1-10-DE3, P1-02-AI and P1-04-AI contain PpDDC, PsONCS3, Cj6OMT, CjCNMT, and Cj4OMT in addition to PsTyDC1 (grey), PsPDC1 (light green) and PsTyDC1 + PsPDC1 (dark green), respectively. Cultures were grown to high density in TB before addition of inducing agent, L-DOPA and ascorbate according to the methods section. 61 h after addition of L-DOPA substrate, PsPDC1-mediated reticuline titers declined, likely due to oxidative degradation. Replicate samples of filtered culture medium were analyzed with CE-MS ( n = 3). Here, 3 samples from individual cultures ( n = 3) were analyzed to generate bar graphs in Prism 7 with error bars representing mean values +/− standard deviations. Source data are provided as a Source Data file.

Article Snippet: To produce pTYB21-PsTyDC1, pTYB21-PsPDC1, pTYB21-PsPDC2 and pTYB21-Ps2HCLL, PsTyDC1, PsPDC1 , Ps2HCLL , and N-terminal truncated PsPDC2 were PCR amplified and cloned into pTYB21 digested with SapI and BamHI via Gibson assembly (NEB) .

Techniques:

Journal: Nature Communications

Article Title: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

doi: 10.1038/s41467-022-28883-8

Figure Lengend Snippet: Replicating norlaudanosoline pathways using homologous enzyme templates. a PsTyDC1 (green) is exchanged with an engineered PpDDC template (blue) via three active site gain-of-function substitutions, Y79F, F80Y, and H181N, to promote DHPAAS activity. In accordance with the resulting increase in AAS activity, these three substitutions result in increased SVM probability scores for AAS prediction, and reduced SVM probability scores for AAAD prediction. Norlaudanosoline (NL) production from L-DOPA was compared using PsTyDC1 in strain T1-10-DE3 ( t = 44 h, n = 3) and PpDDC-Y79F-F80Y-H181N (PpDDC-T) in strain DT-02-DE3 ( t = 40.5 h, n = 2). Culture conditions for each strain are described in the methods section. b PsPDC1 (green) is exchanged with S. cerevisiae ARO10 (purple) for higher PPDC activity in E. coli . Production of norlaudanosoline (NL) from L-DOPA by PsPDC1 in strain P1-02-AI is shown ( t = 44 h, n = 2). Production of norlaudanosoline (NL) from L-DOPA and dopamine by ARO10 in strain A1-01-DE3 is compared ( t = 44 h, n = 3). Culture conditions are described in the methods section. For panels a and b , Samples from individual cultures were analyzed two or three times ( n = 2 or n = 3) to generate bar graphs in Prism 7, with error bars representing mean values +/− standard deviations. c Strain A1-01-DE3 containing ARO10 (purple) metabolizes tryptophan in TB medium to produce an indole 3-acetaldehyde derived indole alkaloid byproduct ( t = 61 h), as indicated by the extracted ion chromatogram (EIC). Strain P1-02-AI containing PsPDC1 (green) did not readily convert indole 3-pyruvate to indole 3-acetaldehyde, as indicated by no detectable indole alkaloid byproduct ( t = 61 h). Source data are provided as a Source Data file.

Article Snippet: To produce pTYB21-PsTyDC1, pTYB21-PsPDC1, pTYB21-PsPDC2 and pTYB21-Ps2HCLL, PsTyDC1, PsPDC1 , Ps2HCLL , and N-terminal truncated PsPDC2 were PCR amplified and cloned into pTYB21 digested with SapI and BamHI via Gibson assembly (NEB) .

Techniques: Activity Assay, Atomic Absorption Spectroscopy, Derivative Assay

Journal: Nature Communications

Article Title: Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

doi: 10.1038/s41467-022-28883-8

Figure Lengend Snippet: Prediction of AAS branch pathway enzymes to produce 4HPAA for norcoclaurine production. a The aromatic acetaldehyde synthase (AAS) branch pathway (green) can produce 4HPAA directly from tyrosine, but this missing link is unreported in P. somniferum . b Structure-based curation of typical aromatic amino acid decarboxylase (AAAD), insect-type AAS and plant-type AAS, as represented by the active site configurations of Pseudomonas putida DDC (PpDDC, blue), Bombyx mori DHPAAS (DHPAAS, grey), and Petroselinum crispum 4HPAAS (Pc4HPAAS, deepteal). AAS candidate PsTyDC1 (green) has a unique active site, while AAS candidate PsTyDC6 (green) has an AAAD-like active site and could not be predicted by a homology or structure-based approach alone. c Cross-validation for correct assignment of AAAD and AAS training sequences is performed using SVM models (blue), Random forests models (green), and by comparing sequence homology of each training sequence to a consensus sequence of AAS training sequences and a consensus sequence of AAAD training sequences (grey), as described in the methods section. d For visual representation, a two-dimensional plot of AAS SVM-based prediction is shown, with positive and negative prediction spaces colored green and white, respectively (left side). Principal component analysis (PCA) is used to compress multi-dimensional data into two dimensions (PC1 and PC2) for a visual representation. Corresponding high-dimensional SVM decision scores from Supplementary Table 1 are shown on the right. Decision scores represent the distance from the SVM prediction boundary. PsTyDC1 and PsTyDC6 score highest for AAS prediction and are colored red. e LC-MS detection of products from Thalictrum flavum norcoclaurine synthase (TfNCS) containing strains T1-01-DE3 (wild-type PsTyDC1 + TfNCS), T1-02-DE3 (PsTyDC1-L205H + TfNCS) and T1-03-DE3 (PsTyDC1-Y98F-F99Y-L205N + TfNCS) (Supplementary Table 2 ), grown in LB supplemented with 1 mM tyrosine and 0.5 mM dopamine, at 28 °C with 180 rpm shaking for 51 h. Selective in vivo production of the downstream AAS product norcoclaurine accompanies the expression of wild-type PsTyDC1 (green), as well as the triple variant of PsTyDC1 with an engineered active site based on that of insect DHPAAS (red). Tyramine is the major product of PsTyDC1-L205H (grey), which contains an engineered active site based on typical AAAD. Similar results are replicated in Supplementary Fig. 2 and Supplementary Fig. 3 .

Article Snippet: To produce pTYB21-PsTyDC1, pTYB21-PsPDC1, pTYB21-PsPDC2 and pTYB21-Ps2HCLL, PsTyDC1, PsPDC1 , Ps2HCLL , and N-terminal truncated PsPDC2 were PCR amplified and cloned into pTYB21 digested with SapI and BamHI via Gibson assembly (NEB) .

Techniques: Atomic Absorption Spectroscopy, Sequencing, Liquid Chromatography with Mass Spectroscopy, In Vivo, Expressing, Variant Assay