aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes

Li, Lei; Wei, Keke; Liu, Xiaocao; Wu, Yuanjie; Zheng, Guosong; Chen, Shaoxin; Jiang, Weihong; Lü, Yinhua

doi:10.1016/j.ymben.2018.12.001

Cited by 49 publications

(29 citation statements)

References 62 publications

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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%

“…Homologous recombinationbased allelic exchange relies upon endogenous DNA-repair machinery -which is typically inefficient 27 , precluding the construction of even moderately-sized strain libraries. Integrase-mediated recombination technologies [28][29][30][31][32][33][34][35][36][37] have the potential overcome many of the limitations faced by the above genetic tools. Phage integrases (or site-specific recombinases) are enzymes that catalyze recombination between two specific sequences of DNA 34,38 .…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, serine integrases, such as Bxb1 and FC31, have been used across the tree of life and thus are likely to work in the vast majority of organisms. Phage integrase-based tools are often limited to integration of a single DNA fragment 28,29,31,32,35,36,39 or function in a limited range of hosts 30,33,40 . To address these issues and limitations, in this work we developed the Serine-integrase Assisted Genome Engineering (SAGE) system.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Sage Enables Efficient Genome Engineering In Diverse Bacteriamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Elmore

Francis

et al. 2020

Preprint

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“…In addition to the in vivo genome engineering, CRISPR/ Cas9 can be utilized for cloning and refactoring of large SM-BGCs [89,[95][96][97][98][99]. Restriction enzymes or PCR-based cloning strategies are not suitable for manipulation of large-sized DNA fragments due to the limited restriction sites and DNA amplification errors.…”

Section: Editing Streptomyces Genomes and Secondary Metabolite Biosynmentioning

confidence: 99%

“…Although genetic tools based on the CRISPR/Cas system have offered diverse strategies to enhance secondary metabolite production and activate silent SM-BGCs [87,89,90,93,99], further optimization of the CRISPR/Cas system for Streptomyces is still required. An example is that the toxicity of Cas nucleases is disadvantageous for multiplexed applications and CRISPR/Cas bearing systems.…”

Section: Future Perspectivementioning

confidence: 99%

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

Lee¹,

Hwang²,

Lee³

et al. 2019

Journal of Microbiology and Biotechnology

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Recent advances of natural product pesticide milbemycins from Streptomyces

Yang,

Du,

et al. 2024

New Plant Protection

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Milbemycins are a group of 16‐membered macrolides produced by the soil‐dwelling filamentous bacteria Streptomyces. Renowned for their potent acaricidal and insecticidal properties, combined with low toxicity, milbemycins are recognized as eco‐friendly biopesticides, vital for pest control and sustainable agricultural development. Over several decades, milbemycins have been extensively investigated, achieving significant progress, including advancements in their biological activities (such as insecticidal mechanisms and toxicity studies), biosynthetic and regulatory mechanisms, high‐yield strain engineering strategies, and the development of milbemycin‐derived commercial products for agricultural applications. This review discusses recent advances, current limitations, and ongoing and emerging efforts to overcome the limitations of milbemycin research. Finally, future research directions are outlined for the development of superior milbemycin‐producing cell factories to facilitate widespread application in the agricultural field.

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aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes

Cited by 49 publications

References 62 publications

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

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

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

Recent advances of natural product pesticide milbemycins from Streptomyces

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