Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces

Zhao, Xuejin; Wei, Weijia; Zong, Yeqing; Bai, Chaoxian; Guo, Xian; Zhu, Hua; Lou, Chunbo

doi:10.1016/j.synbio.2022.05.010

Cited by 7 publications

(7 citation statements)

References 53 publications

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“…In order to facilitate optimization gene expression at the desired level, we developed a rapid, simple, and standardized gene expression assembly vectors based on pTHS, including three constitutive expression vectors and one inducible expression vector compatible with the Goden gate cloning method. Of these, pTHS-XGe, pTHS-XGk and pTHS-XG23 carry constitutive promoters with different strengths ( ermE p*, KasO p*and SP23) [ 22 ], and pTHS-XGSN* contains a tight cumate-inducible promoter P cymR* [ 23 ] ( Fig. 4 A).…”

Section: Resultsmentioning

confidence: 99%

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Zhao,

Zong,

Lou

et al. 2024

Synthetic and Systems Biotechnology

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Section: Resultsmentioning

confidence: 99%

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Zhao,

Zong,

Lou

et al. 2024

Synthetic and Systems Biotechnology

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“…These DNA parts were then combined into a single transcriptional unit (TU), flanked by BsaI restriction sites, and introduced into plasmid pAmp (Level 2). A multigene assembly was then constructed by assembling the multiple TUs into site-specific integrating vectors pTHS or pPAP (Level 3) to facilitate conjugation into Streptomyces . Due to the lack of terminators with distinctive sequences and to avoid repetitive usage in multigene constructs, we recharacterized ten synthetic terminators published previously .…”

Section: Results and Discussionmentioning

confidence: 99%

CUBIC: A Versatile Cumate-Based Inducible CRISPRi System in Streptomyces

Bai,

van Wezel

2023

ACS Synth. Biol.

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Streptomyces, a genus of Gram-positive bacteria, is known as nature’s medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of Streptomyces. We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in Streptomyces. Its broad application is shown by the specific and nondisruptive knockdown of genes involved in growth, development and antibiotic production in various Streptomyces species. To facilitate hyper-efficient plasmid construction, we adapted the Golden Gate assembly to achieve 100% cloning efficiency of the protospacers. We expect that the versatile plug-and-play CUBIC system will create new opportunities for research and innovation in the field of Streptomyces.

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“…In order to facilitate optimization gene expression at the desired level, we developed a rapid, simple, and standardized gene expression assembly vectors based on pTHS, including three constitutive expression vectors and one inducible expression vector compatible with the Goden gate cloning method. Of these, pTHS-XGe, pTHS-XGk and pTHS-XG23 carry constitutive promoters with different strengths ( ermE p*, KasO p*and SP23) (Bai et al, 2015), and pTHS-XGSN* contains a tight cumate-inducible promoter P cymR* (Zhao et al, 2022) (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering inStreptomyces

Zhao,

Zong,

Lou

et al. 2023

Preprint

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Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit are compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

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Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces

Cited by 7 publications

References 53 publications

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

CUBIC: A Versatile Cumate-Based Inducible CRISPRi System in Streptomyces

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering inStreptomyces

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