Optimizing streptomyces sp. MD102 for heterologous expression of the cannabinoid biosynthetic pathway

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

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Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression

Science China Life Sciences ◽

10.1007/s11427-013-4504-2 ◽

2013 ◽

Vol 56 (7) ◽

pp. 619-627 ◽

Cited By ~ 17

Author(s):

JinE Li ◽

ZhengYan Guo ◽

Wei Huang ◽

XiangXi Meng ◽

GuoMin Ai ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Streptomyces Sp

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A new monocupin quercetinase of Streptomyces sp. FLA: identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols

Archives of Microbiology ◽

10.1007/s00203-007-0215-z ◽

2007 ◽

Vol 187 (6) ◽

pp. 475-487 ◽

Cited By ~ 20

Author(s):

Hedda Merkens ◽

Sonja Sielker ◽

Karsten Rose ◽

Susanne Fetzner

Keyword(s):

Heterologous Expression ◽

Recombinant Enzyme ◽

Streptomyces Sp

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“Hairy Root” Technology: An Emerging Arena for Heterologous Expression of Biosynthetic Pathway Genes in Medicinal Plants

Reference Series in Phytochemistry - Transgenesis and Secondary Metabolism ◽

10.1007/978-3-319-28669-3_7 ◽

2017 ◽

pp. 295-322 ◽

Cited By ~ 2

Author(s):

Suchitra Banerjee ◽

Sailendra Singh ◽

Pallavi Pandey

Keyword(s):

Medicinal Plants ◽

Heterologous Expression ◽

Hairy Root ◽

Biosynthetic Pathway

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Medelamine C, A New ω-Hydroxy Alkylamine Derivative from Endophytic Streptomyces sp. YIM 66142

Natural Product Communications ◽

10.1177/1934578x1400900129 ◽

2014 ◽

Vol 9 (1) ◽

pp. 1934578X1400900

Author(s):

Ju-Cheng Zhang ◽

Ya-Bin Yang ◽

Hao Zhou ◽

Tian-Feng Peng ◽

Fang-Fang Yang ◽

...

Keyword(s):

Fatty Acid ◽

Biosynthetic Pathway ◽

2D Nmr ◽

Sw480 Cell ◽

Hydroxyl Group ◽

Genus Streptomyces ◽

Streptomyces Sp ◽

Endophytic Streptomyces ◽

Common Fatty Acid ◽

Mcf 7

A new alkylamine derivative and a common fatty acid were isolated from Streptomyces sp. YIM 66142. On the basis of spectral data, including HRMS, NMR and 2D NMR, their structures were determined as medelamine C (1) and isomyristic acid (2). The ω-hydroxyl group in structure 1 is rare in a natural alkylamine. The possible biosynthetic pathway in the genus Streptomyces from isomyristic acid (2) to medelamines is proposed. Compound 1 showed no obvious cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, SW480 cell lines. The ω-hydroxyl and the acetyl at NH in compound 1 decreased its cytotoxicity in comparison with that of medelamine.

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A revised biosynthetic pathway for the cofactor F420in bacteria

10.1101/470336 ◽

2018 ◽

Author(s):

Ghader Bashiri ◽

James Antoney ◽

Ehab N. M. Jirgis ◽

Mihir V. Shah ◽

Blair Ney ◽

...

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Secondary Metabolism ◽

Biosynthetic Pathway ◽

Antibiotic Biosynthesis ◽

Bacterial Persistence ◽

Redox Transformations ◽

Terminal Domain ◽

Primary And Secondary Metabolism ◽

Drug Activation

AbstractCofactor F420plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420has not been fully eluci-dated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we show that the guanylyltransferases FbiD and CofC accept phosphoenolpyruvate, rather than 2-phospho-L-lactate, as their substrate, leading to the formation of the previously uncharacterized intermediate, dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420species when combined with the γ-glutamyl ligase activity of the N-terminal domain. This new insight has allowed the heterologous expression F420from a recombinant F420biosynthetic pathway inEscherichia coli.

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Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods

Molecules ◽

10.3390/molecules26216580 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6580

Author(s):

Charlotte Beck ◽

Tetiana Gren ◽

Francisco Javier Ortiz-López ◽

Tue Sparholt Jørgensen ◽

Daniel Carretero-Molina ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Transcriptional Regulators ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Heterologous Host ◽

Streptomyces Sp ◽

Identification And Characterization

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

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Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1

Microbial Cell Factories ◽

10.1186/s12934-019-1215-9 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Xiaowen Sun ◽

Hui Liu ◽

Peng Wang ◽

Li wang ◽

Wenfeng Ni ◽

...

Keyword(s):

Pichia Pastoris ◽

Heterologous Expression ◽

Biosynthetic Pathway ◽

Initial Conditions ◽

Expression System ◽

Value Added ◽

Theoretical Research ◽

High Yield ◽

Yield Strain ◽

Synthetic Pathway

Abstract Background With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Results Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. Conclusions In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

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