Tang Xiao-Ling, Chen Lu-Jia, Du Xu-Yuan, Zhang Bo, Liu Zhi-Qiang, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, People's Republic of China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, People's Republic of China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, People's Republic of China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, People's Republic of China.
J Biotechnol. 2020 Feb 10;309:53-58. doi: 10.1016/j.jbiotec.2019.12.018. Epub 2019 Dec 28.
l-Methionine biosynthesis in Eschericha coli consists of multiple unit modules with various enzymes involved and the imbalance between different modules always restricted its productivity. In this study, the key enzymes participating in the pathway were investigated for their effect on l-methionine production and the pivotal enzyme homoserine O-succinyltransferase (MetA) was designed to be regulated. The surface amino acid residues of MetA were effectively modified through site-saturation mutagenesis and single mutants L63F, A28V, P298L and double mutant L63F/A28V were obtained with improved l-methionine productivity. The structure analysis revealed that the involved residues were on the surface loop regions, which was proposed to be conducive to the refolding of MetA and thus reduce the inhibition effect caused by l-methionine. After expression of the selected single mutant L63F in engineered E. coli ΔIJA-HFEBC strain with l-methionine efflux pump and mutated 3-phosphoglycerate dehydrogenase, the l-methionine production was significantly improved, with a final yield of 3528 mg/L. The results demonstrated the efficiency of MetA regulation for enhanced production of l-methionine and meanwhile provided important guidance for further engineering of MetA with increased l-methionine productivity.
大肠杆菌中L-甲硫氨酸的生物合成由多个单元模块组成，涉及多种酶，不同模块之间的不平衡一直限制着其产量。在本研究中，对参与该途径的关键酶对L-甲硫氨酸生产的影响进行了研究，并设计对关键酶高丝氨酸O-琥珀酰转移酶（MetA）进行调控。通过位点饱和诱变有效修饰了MetA的表面氨基酸残基，获得了具有提高的L-甲硫氨酸产量的单突变体L63F、A28V、P298L和双突变体L63F/A28V。结构分析表明，相关残基位于表面环区域，这被认为有利于MetA的重折叠，从而降低L-甲硫氨酸引起的抑制作用。在具有L-甲硫氨酸外排泵和突变的3-磷酸甘油酸脱氢酶的工程大肠杆菌ΔIJA-HFEBC菌株中表达所选的单突变体L63F后，L-甲硫氨酸产量显著提高，最终产量为3528 mg/L。结果证明了对MetA进行调控以提高L-甲硫氨酸产量的有效性，同时为进一步改造具有更高L-甲硫氨酸产量的MetA提供了重要指导。
