Dymek Saskia, Jacob Lucas, Pühler Alfred, Kalinowski Jörn
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, Germany.
Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, Germany.
Microorganisms. 2024 Dec 24;13(1):1. doi: 10.3390/microorganisms13010001.
Acarbose, a pseudo-tetrasaccharide produced by sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into sp. These vectors were validated by silencing the known acarbose biosynthesis genes and , as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein-DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production.
阿卡波糖是由sp. SE50/110产生的一种假四糖，是一种α-葡萄糖苷酶抑制剂，用作治疗2型糖尿病的药物。虽然阿卡波糖的生物合成已经阐明，但其调控机制却知之甚少。使用CRISPRi进行基因沉默有助于识别影响阿卡波糖形成的潜在调控因子。为此，建立了两种类型的CRISPRi载体用于sp. SE50/110。pCRISPomyces2i载体可实现可逆沉默，而整合型pSETT4i载体由于其导入sp.的接合时间较短，可为多个靶点提供快速筛选方法。通过沉默已知的阿卡波糖生物合成基因以及它们的调控因子CadC来验证这些载体。产物形成的减少和相应基因相对转录丰度的降低作为成功沉默的证据。这些载体被用于创建一个基于CRISPRi的菌株文库，沉默50个转录调控因子，以研究它们在阿卡波糖生物合成中的潜在影响。这些转录调控基因是从先前涉及蛋白质-DNA相互作用研究的实验中选择的，或者是根据它们的表达谱选择的。鉴定出11个影响阿卡波糖产量的基因。其中7个基因通过CRISPRi介导的敲低显著降低了阿卡波糖的生物合成，而4个基因的敲低则提高了阿卡波糖的产量。
