Frontiers in Microbiology
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2018
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IF 4.019
Peng Qinying,Gao Guixi,Lü Jin,Long Qingshan,Chen Xuefei,Zhang Fei,Xu Min,Liu Kai,Wang Yemin,Deng Zixin,Li Zhiyong,Tao Meifeng
Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover “cryptic” secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.
研究领域:
微生物学
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合成生物学
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药物开发
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2018
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IF 2.533
Ming Zhao,Shui-Ling Wang,Xin-Yi Tao,Guo-Ling Zhao,Yu-Hong Ren,Feng-Qing Wang,Dong-Zhi Wei
Due to the lack of powerful gene regulation elements, the engineering development of Streptomyces is often limited. Here, we disclosed that the heterologous σ 70 -dependent promoters, which have been reported as inefficient tools for gene expression in Streptomyces , could be efficiently recognized by Streptomyces housekeeping factor σ hrdB . Therefore, an effective strategy was developed to engineer these promoters for robust gene expression in Streptomyces by fusing them with optimized 5′-untranslation regions (5′-UTRs). As a proof of concept, the widely used P tac in E. coli was engineered by fusing its core promoter region with the 5′-UTR R15 from a relatively powerful Streptomyces promoter P kasO * R15 and resulted in P tac* , the activity of which was 8.1-fold that of P tac and 1.7-fold that of P kasO * R15 in S. lividans TK24. Next, the 5′-UTR R15 was optimized by randomizing the ribosome binding site (RBS). Based on the base biases of those RBSs with higher activity, eight artificial RBSs were rationally designed, and the optimal resulting promoter P tac * RBS3 showed about 2.1, 3.6, and 17.6 times the activity of P tac *, P kasO * R15 , and P tac , respectively, demonstrating that the heterologous P tac was converted into a type of robust Streptomyces promoters. This study thus greatly expands promoter diversity for the engineering of Streptomyces .
研究领域:
合成生物学
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分子遗传学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Ming Zhao,Xiu-Shan Zhang,Liang-Bin Xiong,Kun Liu,Xiang-Fei Li,Yan Liu,Feng-Qing Wang
Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from Streptomyces species. However, due to the complex metabolic switches of Streptomyces, most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from the Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σhrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured with cheap industrial glycerol as a supplementary carbon source, and 14.3 and 46.27 g/L indigoidine could be achieved in a flask and a 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine. This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.
研究领域:
微生物学
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生物工程
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合成生物学
✓ 已验证
DOI
PubMed
高影响因子
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