Enhancement of Acarbose Production in <i>Actinoplanes</i> sp. QQ-12 via Multiple Engineering Strategies

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 <i>Actinoplanes</i> 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 (<i>acb</i>). These mutants, with 2, 3, and 4 copies of <i>acb</i> gene cluster, demonstrated titer increases of 69.4%, 99.3%, and 24.2%, respectively, with a sharply declined titer in the four-copy <i>acb</i> strain LGQ-17::3<i>acb</i>. To rescue the acarbose titer in LGQ-17::3<i>acb</i>, we overexpressed rate-limiting genes <i>acbC</i>, <i>M</i>, <i>O</i>, <i>N</i>, <i>J</i>, <i>V</i>, <i>I,</i> or <i>S</i>. The overexpression of <i>acbJ</i> and <i>acbS</i> 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<i>acb</i>::<i>stnYp</i>-<i>acbJ</i> 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 <i>Actinoplanes</i> sp., and the engineered strain shows excellent potential for industrial application.