State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No.2 Yuanmingyuan West Road, Haidian district, Beijing, 100193, China.
College of Animal Science, Shanxi Agricultural University, Shanxi, 030600, China.
Microb Cell Fact. 2024 Aug 22;23(1):233. doi: 10.1186/s12934-024-02505-y.
Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.
This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.
This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.
甲基丙烯酸甲酯（MMA）是聚甲基丙烯酸甲酯的关键前体，广泛应用于各个行业的透明热塑性塑料。传统的 MMA 生产存在健康和环境风险；因此，柠康酸甲酯作为 MMA 生产的替代细菌化合物前体。以前，通过大肠杆菌 BW25113 获得了最高的柠康酸甲酯产量。然而，通过代谢工程进一步提高柠康酸甲酯产量的研究有限，噬菌体污染是大肠杆菌发酵中一个持续存在的问题。
本研究旨在构建一株能够从葡萄糖生产高柠康酸甲酯产量的抗噬菌体大肠杆菌 BW25113 菌株。首先，筛选了启动子和异源 cimA 基因，并通过过表达来自 Methanococcus jannaschii 的突变 cimA 基因 MjcimA3.7 建立了有效的柠康酸生物合成途径，该基因受大肠杆菌中的 BBa_J23100 启动子调控。随后，通过整合 Ssp 防御系统到基因组中和突变噬菌体感染周期的关键组件，构建了一株抗噬菌体的大肠杆菌菌株。然后，该菌株被工程改造为包括非氧化磷酸戊糖途径，同时去除乙酸合成途径，以增强乙酰辅酶 A 的供应。此外，通过该菌株利用葡萄糖的能力得到了提高，从而增加了柠康酸甲酯的产量。最终，在 80 小时的分批补料发酵后，获得了 110.2 g/L 的柠康酸甲酯。葡萄糖的柠康酸甲酯产率和生产率分别为 0.4 g/g 葡萄糖和 1.4 g/（L·h）。
这是大肠杆菌中报道的最高柠康酸甲酯产量和生产率，无需添加昂贵的酵母提取物和在补料发酵中进行额外诱导，强调了其在生产柠康酸甲酯及其衍生物方面的实际应用潜力。
