Niu Kun, Fu Qiang, Mei Zi-Long, Ge Li-Rong, Guan An-Qi, Liu Zhi-Qiang, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China.
ACS Synth Biol. 2023 Feb 17;12(2):492-501. doi: 10.1021/acssynbio.2c00481. Epub 2023 Jan 26.
l-Methionine is the only sulfur-containing amino acid among the essential amino acids, and it is mainly produced by the chemical method in industry so far. The fermentation production of l-methionine by genetically engineered strains is an attractive alternative. Due to the complex metabolic mechanism and multilevel regulation of the synthesis pathway in the organism, the fermentation production of l-methionine by genetically engineered strains was still not satisfied. In this study, the biosynthesis pathway of l-methionine was regulated based on the previous studies. As the competitive pathway and an essential amino acid for cell growth, the biosynthesis pathway of l-lysine was first repaired by complementation of the gene on the genome and then replaced the promoter with the dynamically regulated promoter P to construct a nonauxotroph strain. In addition, the central metabolic pathway and l-cysteine catabolism pathway were further modified to promote the cell growth and enhance the l-methionine production. Finally, the l-methionine fermentation yield in a 5 L bioreactor reached 17.74 g/L without adding exogenous amino acids. These strategies can effectively balance the contradiction between cell growth and l-methionine production and alleviate the complexity of fermentation operation and the cost with auxotroph strains, which provide a reference for the industrial production of l-methionine by microbial fermentation.
L-甲硫氨酸是必需氨基酸中唯一含硫的氨基酸，目前在工业上主要通过化学方法生产。利用基因工程菌株发酵生产L-甲硫氨酸是一种有吸引力的替代方法。由于生物体中合成途径的代谢机制复杂且存在多级调控，基因工程菌株发酵生产L-甲硫氨酸的效果仍不尽人意。在本研究中，基于前期研究对L-甲硫氨酸的生物合成途径进行调控。作为竞争途径和细胞生长必需的氨基酸，首先通过在基因组上互补基因修复L-赖氨酸的生物合成途径，然后用动态调控的启动子P替换启动子，构建非营养缺陷型菌株。此外，对中心代谢途径和L-半胱氨酸分解代谢途径进行进一步改造，以促进细胞生长并提高L-甲硫氨酸产量。最终，在5 L生物反应器中，不添加外源氨基酸时L-甲硫氨酸的发酵产量达到17.74 g/L。这些策略可以有效平衡细胞生长与L-甲硫氨酸生产之间的矛盾，减轻使用营养缺陷型菌株时发酵操作的复杂性和成本，为微生物发酵法工业化生产L-甲硫氨酸提供参考。
