Biotin (Vitamin H or B₇) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.

生物素（维生素 H 或 B ₇）是参与原核细胞和真核细胞的中枢代谢的最重要的辅助因子之一。目前，化学合成是商业化生产的唯一途径。该研究报告了在变异假单胞菌中生物素的有效微生物生产通过多级代谢工程策略： 1级，过表达参与生物素合成的限速酶编码基因（即启动子和核糖体结合位点工程）；第 2 级，解除对生物素生物合成的调节（即删除负调节因子和生物素导入基因）；第 3 级，增加用于生物素生物合成的辅助因子（即 S-腺苷-L-甲硫氨酸和 [Fe-S] 簇）的供应；第 4 级，增加前体庚二酸硫酯的可用性（即从枯草芽孢杆菌中引入 BioW-BioI 途径）。这些干预措施的结合导致了生物素过度生产菌株的建立，生物素的分泌增加了 460 多倍。结合生物过程工程的努力，在摇瓶中以 87.17 mg/L 的最终滴度和在以甘油为碳源的补料分批发酵罐中生产的生物素的最终滴度为 271.88 mg/L。这是迄今为止使用合理设计的微生物细胞工厂报告的最高生物素滴度。