Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes

Lei, Li; Zheng, Guosong; Chen, Jun; Ge, Mei; Jiang, Weihong; Lü, Yinhua

doi:10.1016/j.ymben.2017.01.004

Cited by 85 publications

(69 citation statements)

References 49 publications

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“…In contrast to the hrdB promoter, the kasO * promoter displayed more effectiveness compared to ermE *, leading to a higher titer of compounds in mutant TG6006 than that of TG6001 (Figure A‐IV). Considering that the duplication of the gene cluster and rate‐limiting genes have been naturally and artificially developed to improve the biosynthetic capacity, we amplified tjhR1 by two copies ( P ermE * ‐ tjhR1‐P kasO * ‐ tjhR1 or P kasO * ‐ tjhR1‐P kasO * ‐ tjhR1 ) and three copies ( P ermE * ‐ tjhR1‐P kasO * ‐ tjhR1‐P kasO * ‐ tjhR1 ) in tandem, yielding mutants TG6008, TG6009, and TG6010, respectively. The corresponding fermentation results revealed that the two‐copy amplification of tjhR1 was more effective than either the one‐copy or three‐copy amplification, and mutant TG6008 ( P ermE * ‐ tjhR1‐P kasO * ‐ tjhR1 ) exhibited a higher yield with more reliable production (Figure A‐V, VI, and VII).…”

Section: Resultsmentioning

confidence: 99%

Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways

Ji¹,

Nie²,

Yin³

et al. 2019

Angewandte Chemie

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One biosynthetic gene cluster (BGC) usually governs the biosynthesis of a series of compounds exhibiting either the same or similar molecular scaffolds. Reported here is a multiplex activation strategy to awaken a cryptic BGC associated with tetracycline polyketides, resulting in the discovery of compounds having different core structures. By constitutively expressing a positive regulator gene in tandem mode, a single BGC directed the biosynthesis of eight aromatic polyketides with two types of frameworks, two pentacyclic isomers and six glycosylated tetracyclines. The proposed biosynthetic pathway, based on systematic gene inactivation and identification of intermediates, employs two sets of tailoring enzymes with a branching point from the same intermediate. These findings not only provide new insights into the role of tailoring enzymes in the diversification of polyketides, but also highlight a reliable strategy for genome mining of natural products.

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Section: Resultsmentioning

confidence: 99%

Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways

Ji¹,

Nie²,

Yin³

et al. 2019

Angewandte Chemie

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“…Serine-integrases are functional in a wide array of eukaryotes, archaea, and bacteria [28][29][30][31][32][33]35,36,40,59 -so SAGE should work in any transformable bacterium where two conditions are met: att sites are integrated into the genome (Fig. 1) and active serine integrases are expressed.…”

Section: Sage Enables Efficient Genome Engineering In Diverse Bacteriamentioning

confidence: 99%

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

Elmore

Francis

et al. 2020

Preprint

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Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural purposes. Synthetic biology can address these limitations, but its application is limited by availability of appropriate tools for sophisticated, high-throughput genome engineering that function in environments where selection for DNA maintenance is impractical. Here we present an orthogonal, Serine-integrase Assisted Genome Engineering (SAGE) system, which enables iterative, site-specific integration of up to 10 different DNA constructs at efficiency on par or better than replicating plasmids. SAGE does not require use of replicating plasmids to deliver recombination machinery, and employs a secondary serineintegrase to excise and recycle selection markers. Furthermore, unlike the widely utilized pBBR1 origin, DNA transformed using SAGE is stable without selection. We highlight SAGE's utility by constructing a 287-member constitutive promoter library with a ~40,000-fold dynamic range in P. fluorescens SBW25. We show that SAGE functions robustly in diverse ⍺and "-proteobacteria, thus providing evidence that it will be broadly useful for engineering industrial or environmental bacteria.

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“…On the other hand, Lei Li et al. integrated five additional copies of the pristinamycin II (PII) BGC in Streptomyces pristinaespiralis , and generated the highest reported PII titers in both flasks and batch fermentations (216% higher than starting strain) .…”

Section: Pathway‐specific Engineering To Exploit Natural Products Biomentioning

confidence: 99%

“…Yunzi Luo et al [70] cloned and refactored a PTM BGC from Streptomyces griseus and discovered several new PTM compounds by introducing six different strong constructive promoters in front of all six genes in the characterized BGC. On the other hand, Lei Li et al integrated five additional copies of the pristinamycin II (PII) BGC in Streptomyces pristinaespiralis, and generated the highest reported PII titers in both flasks and batch fermentations (216% higher than starting strain) [71].…”

Section: Duplication or Higher Order Amplification Of Rate-limiting Ementioning

confidence: 99%

Recent advances in natural products exploitation in Streptomyces via synthetic biology

Zhao

Wang

Luo

2019

Engineering in Life Sciences

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Natural products of microbial origin have proven to be the wellspring of clinically useful compounds for human therapeutics. Streptomyces species are predominant sources of bioactive compounds, most of which serve as potential drug candidates. While the exploitation of natural products has been severely reduced over the past two decades, the growing crisis of evolution and dissemination of drug resistant pathogens have again attracted great interest in this field. The emerging synthetic biology has been heralded as a new bioengineering platform to discover novel bioactive compounds and expand bioactive natural products diversity and production. Herein, we review recent advances in the natural products exploitation of Streptomyces with the applications of synthetic biology from three major aspects, including recently developed synthetic biology tools, natural products biosynthetic pathway engineering strategies as well as chassis host modifications.

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Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes

Cited by 85 publications

References 49 publications

Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways

Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

Recent advances in natural products exploitation in Streptomyces via synthetic biology

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