Journal: Microbial cell factories

Article Title: Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

doi: 10.1186/s12934-016-0448-0

Figure Lengend Snippet: Fig. 1 Promoter region sequences of PxylA, PlacA and PlacSynth. Nucleotide sequence from repressor to start codon of mCherry are shown (region within dotted square). The mCherry start codon is indicated in italics, preceded by an identical ribosome binding site (RBS; italics) an XbaI restriction site (bold) and an identical 9 nt spacer sequence was introduced upstream of mCherry start codon. The −35 and −10 promoter region were identi- fied (SoftBerry, BPROM) and are underlined. Primer binding sites for negative controls (for construction of negative controls without promoter) are underlined in dashed line. a PxylA; promoter of xylA gene from B. megaterium DSMZ 319 and promoter of repressor XylR. Operator sequences for XylR binding are underlined; cre sites (catabolite-responsive element) are highlighted. b PlacA; endogenous promoter of LacA from L. plantarum 3NSH and promoter of repressor LacR. LacR-binding site was identified (RegPrecise) and underlined, and putative cre-sites are highlighted. c PlacSynth; promoter P2083 from L. buchneri CD034 with artificially integrated operator binding sites with recommended distance of 93 nt (O1 and OiD from E. coli) are underlined (dotted line), terminator of lacI is underlined (solid line)

Article Snippet: Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon).

Techniques: Sequencing, Binding Assay

Journal: Microbial cell factories

Article Title: Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

doi: 10.1186/s12934-016-0448-0

Figure Lengend Snippet: Fig. 2 Maps of plasmids used in this study. Annotations and relevant restriction sites are indicated. a pCDLbu1 initial vector backbone (highlighted region are E. coli specific sequences); b pCDblu1_PxylA_mCherry; c pCDLbu1_PlacA_mCherry; d pCDLbu1_PlacSynth_mCherry; e pCDLbu1ΔEc_P11_ mCherry (constitutive P11 promoter; internal reference plasmid described by Tauer and colleagues [47]); The term ‘ΔEc’ indicates removal of E. coli specific sequences which are highlighted in plasmid A. f pCD256_PlacSynth_mCherry; g pCD256_PlacSynth_T7RNAP; h pCDLbu1ΔEc_PT7_mCherry _TT7_Ery. OripCDLbu1 and miniori256: origins of replication (ori) for L. plantarum 3NSH; pMB1ori: ori for replication in E. coli; CAT chloramphenicol acetyltransferase gene; Amp ampicillin resistance gene; Ery ermI gene encoding resistance to erythromycin; P promoter; T terminator of transcrip- tion. Subscripted characters are specifications. Important restriction sites are indicated

Article Snippet: Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon).

Techniques: Plasmid Preparation

Journal: Scientific Reports

Article Title: Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

doi: 10.1038/s41598-021-83106-2

Figure Lengend Snippet: Structure and function of omphalotin biosynthetic gene cluster and homologous clusters in the basidiomycetes O. olearius , D. bispora and L. edodes. ( A ) Schematic representation of the borosin biosynthetic gene clusters in the basidiomycetes O. olearius (VT 653.13, scaffold _ 169), L. edodes (Le(Bin) 0899 ss11, scaffold_10) and D. bispora (CBS 962.96, scaffold_621). All three clusters code for a precursor (methyltransferase) protein, referred to as OphMA, LedMA and DbiMA1 respectively, and a prolyloligopeptidase, referred to as OphP, LedP and DbiP, respectively. The DNA scaffolds and filtered gene models were taken from https://mycocosm.jgi.doe.gov/ . Double slashes (//) indicate that the sequence of the DNA scaffold continues beyond this position. ( B ) Biosynthesis scheme of omphalotin A and closely related peptides, lentinulin A and dendrothelin A as a combined action of the precursor (methyltransferase) protein and the prolyloligopeptidase. ( C ) Alignment of the C-termini of the precursor (methyltransferase) proteins from O. olearius (OphMA), L. edodes (LedMA) and D. bispora (DbiMA1). Residues differing from the omphalotin core peptide are shown in red. The indicated methylation patterns (green fill) were previously determined by heterologous expression of the respective cDNAs in E. coli , , . CRS = C-terminal recognition sequence.

Article Snippet: Omphalotus olearius (strain DSM3398) was ordered from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ; Leibniz Institute, Germany), Dendrothele bispora (strain CBS 962.96) from the Westerdijk Fungal Biodiversity Institute strain collection (The Netherlands), and Lentinula edodes (strain 4312 from Sylvan, China) was a gift from a local mushroom farm (Fine Funghi AG, Gossau ZH, Switzerland).

Techniques: Sequencing, Methylation, Expressing

Journal: Scientific Reports

Article Title: Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

doi: 10.1038/s41598-021-83106-2

Figure Lengend Snippet: LC–MS analysis of backbone N-methylated peptides extracted from mushrooms O. olearius , L. edodes and D. bispora . ( A – C ) Ion chromatograms depicting some of the peptides species produced by 28 days liquid cultures of the respective fungi. In case of O. olearius ( A ) and D. bispora ( B ), both backbone-N-methylated macrocyclic peptides (M + H + ) and additionally oxidized macrocyclic peptides (M + nO + H) + thereof were observed in the culture supernatant. In case of the L. edodes liquid culture ( C ) , oxidized cyclic peptides were detected in the fungal mycelium. (M + H 2 O + H) + represents the linear form of the macrocyclic peptide. ( D ) Chemical structures of omphalotin A, lentinulin A and dendrothelin A. Residues different from omphalotin A are underlined. Backbone N-methylation is indicated by green circles.

Article Snippet: Omphalotus olearius (strain DSM3398) was ordered from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ; Leibniz Institute, Germany), Dendrothele bispora (strain CBS 962.96) from the Westerdijk Fungal Biodiversity Institute strain collection (The Netherlands), and Lentinula edodes (strain 4312 from Sylvan, China) was a gift from a local mushroom farm (Fine Funghi AG, Gossau ZH, Switzerland).

Techniques: Liquid Chromatography with Mass Spectroscopy, Methylation, Produced