Liu Guoqiang, Liu Qi, Song Xiaotong, Jiao Xingzhi, Zhou Wanping, Kang Qianjin, Bai Linquan
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
College of Life Science, Tarim University, Alar 843300, China.
J Agric Food Chem. 2025 May 28;73(21):12845-12855. doi: 10.1021/acs.jafc.5c00613. Epub 2025 May 14.
The α-glucosidase inhibitor acarbose is used in the treatment of type 2 diabetes mellitus. Metabolic engineering is crucial to overcome acarbose production bottlenecks. Herein, a genetic toolkit was developed to enable metabolic engineering in sp.. The attachment/integration (Att/Int) systems of ΦBT1, pSAM2, R4, and TG1 showed conjugation frequencies of 0.98-24.4%. Furthermore, three mutants were constructed by deleting nontarget biosynthetic gene clusters (BGCs) and inserting one to three additional copies of the acarbose biosynthetic gene cluster (). These mutants, with 2, 3, and 4 copies of gene cluster, demonstrated titer increases of 69.4%, 99.3%, and 24.2%, respectively, with a sharply declined titer in the four-copy strain LGQ-17::3. To rescue the acarbose titer in LGQ-17::3, we overexpressed rate-limiting genes , , , , , , or . The overexpression of and resulted in acarbose titer increases of 1.04-fold and 98%, respectively. Furthermore, following fed-batch fermentation optimization in shake flasks, the titer of acarbose in LGQ-17::3::- increased by 1.1-fold to reach 8.12 g/L. This genetic engineering toolkit with multiple Att/Int systems and high conjugation frequencies paves the way for future genetic engineering in sp., and the engineered strain shows excellent potential for industrial application.
α-葡萄糖苷酶抑制剂阿卡波糖用于治疗2型糖尿病。代谢工程对于克服阿卡波糖生产瓶颈至关重要。在此，开发了一种遗传工具包以实现对[具体菌种名称缺失]的代谢工程改造。ΦBT1、pSAM2、R4和TG1的附着/整合（Att/Int）系统显示出0.98 - 24.4%的接合频率。此外，通过删除非目标生物合成基因簇（BGCs）并插入一到三个额外拷贝的阿卡波糖生物合成基因簇构建了三个突变体。这些分别具有2、3和4个基因簇拷贝的突变体，其产量分别提高了69.4%、99.3%和24.2%，而四拷贝基因簇菌株LGQ - 17::3的产量急剧下降。为挽救LGQ - 17::3中的阿卡波糖产量，我们过表达了限速基因[具体基因名称缺失]。[具体基因名称缺失]和[具体基因名称缺失]的过表达分别使阿卡波糖产量提高了1.04倍和98%。此外，在摇瓶中进行补料分批发酵优化后，LGQ - 17::3::-中的阿卡波糖产量提高了1.1倍，达到8.12 g/L。这种具有多个Att/Int系统和高接合频率的基因工程工具包为未来对[具体菌种名称缺失]的基因工程改造铺平了道路，并且工程菌株显示出优异的工业应用潜力。
