National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2021 Jan 15;10(1):84-93. doi: 10.1021/acssynbio.0c00456. Epub 2021 Jan 5.
is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in and promoted the metabolic engineering modification of to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.
以其不完全的氧化能力而闻名，已广泛应用于工业生产。然而，与模式微生物相比， 中的遗传工具仍然稀缺，限制了其代谢工程。本研究旨在基于典型的 I-E 型内源性 CRISPR/Cas 系统，开发一个基于 WSH-003 的簇状规则间隔短回文重复干扰（CRISPRi）系统。该系统中的核酸酶 Cas3 自然失活，因此不需要敲除。随后，通过抑制荧光蛋白的表达验证了 CRISPRi 效应，显示出有效的多重基因抑制。最后，将内源性 CRISPRi 系统用于研究中央碳代谢途径，包括戊糖磷酸途径（PPP）和 Entner-Doudoroff 途径（EDP）在 WSH-003 中的作用。这证明了代谢工程的应用。发现 PPP 对细胞生长和基质转化率很重要。CRISPRi 系统的开发丰富了 中的基因调控工具，并促进了 代谢工程的修饰，以提高其性能。此外，它可能对其他遗传上难以修饰的菌株的代谢工程修饰具有意义。
