An inducible CRISPRi circuit for tunable dynamic regulation of gene expression in Saccharopolyspora erythraea.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2024-04-01 Epub Date: 2024-01-27 DOI:10.1007/s10529-023-03462-z

Xing Jiang, Xiang Ke, Xiwei Tian, Ju Chu

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Abstract

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

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诱导性 CRISPRi 电路可对红球藻中的基因表达进行可调动态调控。

放线菌是一种革兰氏阳性细菌，以其宝贵的次级代谢产物而闻名。要将放线菌的代谢通量重新导向所需的产品，需要对基因表达进行精确而动态的调控。在本研究中，我们将 CRISPR 干扰（CRISPRi）系统与积雪草诱导型启动子相结合，开发出了一种诱导型基因下调方法，可用于生产红霉素的著名放线菌 Saccharopolyspora erythraea。以 gusA 基因为报告基因验证了积雪草诱导型启动子的功能，并确认了 dCas9 基因的成功诱导表达。然后，利用所开发的可诱导 CRISPRi 策略下调了野生型菌株 NRRL2338 的目标基因 rppA 和高产红霉素菌株 E3 的目标基因 sucC 的表达。通过动态控制 sucC 的表达，菌株 E3 的红霉素产量显著提高。这项研究证明了利用 CRISPRi 和积雪草诱导型启动子进行诱导基因下调的有效性，为优化放线菌天然产物的生产提供了宝贵的见解。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.