代谢工程改造大肠杆菌利用葡萄糖和甲酸共底物合成 l- 苏氨酸

Metabolic engineering of Escherichia coli for l-threonine synthesis with glucose and formate as co-substrates

摘要:

甲酸作为可持续的C1原料，具有来源广泛、水溶性良好等优点，是传统糖基原料的理想替代品。目前，微生物利用甲酸效率低下，导致细胞生长速率缓慢，从而降低了菌株的生产性能。为提升微生物甲酸利用效率，本研究以大肠杆菌BW25113作为底盘菌株，将其改造为甲酸依赖型菌株并引入还原甘氨酸(reductive glycine pathway, rGly)途径，获得甲酸消耗速率为87.2 mg/(g DCW ·h)的菌株WZ-3。在此基础上，对rGly途径中的四氢叶酸循环(tetrahydrofolic acid, THF)与甘氨酸裂解系统(glycine cleavage system, GCS)的表达水平进行组合优化并引入甲酸脱氢酶(formate dehydrogenase, FDH)，获得菌株WZ-15，其甲酸消耗速率提升至158.6 mg/(g DCW ·h)。为了满足甲酸同化所需的NADH与ATP，构建了硫化镉(cadmium sulfide, CdS)人工杂化光合系统，通过捕获光能促进胞内NADH与ATP再生，获得菌株WZ-16，其 OD ₆₀₀与甲酸消耗速率分别提高至1.86、185.5 mg/(g DCW ·h)。在此基础上，通过强化 l-苏氨酸合成路径，获得菌株WZ-21，该菌株在5 L发酵罐中利用30 g/L葡萄糖和5.4 g/L甲酸混合底物可生产6.2 g/L的 l-苏氨酸。本研究成功实现了葡萄糖与甲酸向 l-苏氨酸的转化，为基于C1原料的绿色生物合成提供了新策略。

Abstract:

Formate, as a sustainable C1 substrate, offers advantages including broad availability and favorable aqueous solubility, serving as an ideal alternative to conventional sugar-based carbon sources. Currently, inefficient microbial assimilation of formate leads to slow cellular growth and consequently constrains strain productivity. To address this, we employed Escherichia coli BW25113 as the chassis organism and engineered it into a formate-dependent strain through the introduction of the reductive glycine (rGly) pathway, yielding strain WZ-3 with a formate consumption rate of 87.2 mg/(g DCW ·h). Subsequently, combinatorial optimization of the expression levels of the tetrahydrofolate (THF) cycle and the glycine cleavage system (GCS) within the rGly pathway, coupled with heterologous expression of a formate dehydrogenase, generated strain WZ-15, which achieved an enhanced formate consumption rate of 158.6 mg/(g DCW ·h). To satisfy the NADH and ATP requirements for formate assimilation, we constructed a CdS artificial hybrid photosynthetic system to capture light energy for enhanced regeneration of intracellular NADH and ATP, yielding strain WZ-16 with an OD ₆₀₀ value of 1.86 and the formate consumption rate of 185.5 mg/(g DCW ·h). On this basis, we enhanced the l-threonine biosynthesis pathway to obtain strain WZ-21, which produced 6.2 g/L of l-threonine in a 5 L fermenter with 30 g/L glucose and 5.4 g/L formate as co-substrates. This study successfully achieved the conversion of glucose and formate to l-threonine, thereby offering a novel strategy for the green biosynthesis based on C1 feedstocks.