Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 μg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 μg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 μg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.

中文翻译：

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代谢工程和发酵培养基的优化，用于在大肠杆菌中生产维生素B12。

维生素B12是至关重要的精细化学品，广泛用于制药，食品和化学工业，其生产完全取决于微生物发酵。我们之前在大肠杆菌中构建了人工维生素B12的生物合成途径，但工程菌株的收率很低。在这里，我们消除了工程大肠杆菌菌株中维生素B12生物合成途径的代谢瓶颈。在从不同来源筛选cobB基因，优化cobN的表达并定制cobS和cobT的核糖体结合位点后，维生素B12的产量增加到152.29μg/ g干细胞重量（DCW）。下游模块的优化（将椰油酸辅酶II，α，c-二酰胺转化为磷酸腺苷cobinamide）将维生素B12的产量提高至249.04μg/ g DCW。各种等效成分的比较表明，葡萄糖和玉米浆分别是最佳的碳和氮源。最后，采用正交阵列设计确定葡萄糖和氮源（包括玉米浆和酵母提取物）的最佳浓度，从而获得530.29μg/ g DCW的维生素B12产量。这项研究中实现的代谢修饰和发酵条件的优化为进一步提高大肠杆菌中维生素B12的产量提供了基础，并有望加速其工业应用。应用正交阵列设计确定葡萄糖和氮源（包括玉米浆和酵母提取物）的最佳浓度，从而获得530.29μg/ g DCW的维生素B12产量。这项研究中实现的代谢修饰和发酵条件的优化为进一步提高大肠杆菌中维生素B12的产量提供了基础，并有望加速其工业应用。应用正交阵列设计确定葡萄糖和氮源（包括玉米浆和酵母提取物）的最佳浓度，从而获得530.29μg/ g DCW的维生素B12产量。这项研究中实现的代谢修饰和发酵条件的优化为进一步提高大肠杆菌中维生素B12的产量提供了基础，并有望加速其工业应用。