Violacein, a blue-violet compound with a wide range of beneficial bioactivities, is an attractive product for microbial production. Currently, violacein production has been demonstrated in several sugar heterotrophs through metabolic engineering; however, the cost of production remains an obstacle for business ventures. To address this issue, the development of host strains that can utilize inexpensive alternative substrates to reduce production costs would enable the commercialization of violacein. In this study, we engineered a facultative methylotroph, Methylorubrum extorquens AM1, to develop a methanol-based platform for violacein production. By optimizing expression vectors as well as inducer concentrations, 11.7 mg/L violacein production was first demonstrated using methanol as the sole substrate. Considering that unidentified bottlenecks for violacein biosynthesis in the shikimate pathway of M. extorquens AM1 would be difficult to address using generic metabolic engineering approaches, random mutagenesis and site-directed mutagenesis were implemented, and a 2-fold improvement in violacein production was achieved. Finally, by co-utilization of methanol and acetate, a remarkable enhancement of violacein production to 118 mg/L was achieved. Our results establish a platform strain for violacein production from non-sugar feedstocks, which may contribute to the development of an economically efficient large-scale fermentation system for violacein production.

紫罗兰素是一种蓝紫色化合物，具有广泛的有益生物活性，是一种有吸引力的微生物生产产品。目前，已经通过代谢工程在几种糖异养生物中证明了紫罗兰素的产生；然而，生产成本仍然是商业企业的障碍。为了解决这个问题，可以利用廉价的替代底物来降低生产成本的宿主菌株的开发将使紫罗兰素的商业化成为可能。在这项研究中，我们设计了一种兼性甲基营养菌 Methylorubrum extorquensAM1，开发用于生产紫罗兰素的甲醇平台。通过优化表达载体和诱导剂浓度，首次证明了使用甲醇作为唯一底物可生产 11.7 mg/L 紫罗兰素。考虑到M. extorquens莽草酸途径中紫罗兰素生物合成的未知瓶颈使用通用代谢工程方法难以解决 AM1，实施了随机诱变和定点诱变，并实现了 2 倍的紫罗兰素产量改进。最后，通过共同使用甲醇和乙酸盐，可以将紫罗兰素的产量显着提高到 118 mg/L。我们的研究结果建立了一个从非糖原料生产紫罗兰素的平台菌株，这可能有助于开发一种经济高效的紫罗兰素生产大规模发酵系统。