文献类型：期刊文献

英文题名：Rational engineering of Corynebacterium glutamicum for L-Glutamine biosynthesis

作者：Lin, Ying-Tong[1];Chai, Meng[1];Liu, Qing-Hai[1];Ma, Yu-Shu[1];Tao, Xin-Yi[1];Liu, Min[1];Wei, Dong-Zhi[1]

机构：[1]East China Univ Sci & Technol, Newworld Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2026

卷号：227

外文期刊名：BIOCHEMICAL ENGINEERING JOURNAL

收录：;EI(收录号：20254919645511);WOS:【SCI-EXPANDED(收录号:WOS:001633438300003)】；

基金：This study was supported by Shanghai Science and Technology Development Funds (No. 24HC2810400) , the Natural Science Founda-tion of Shanghai (No. 21ZR1417200) , and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering. We thank Prof. Ji-Bin Sun and Prof. Ping Zheng (from Tianjin Insti-tute of Industrial Biotechnology, Chinese Academy of Sciences) for their guidance and support on the work of C. glutamicum system.

语种：英文

外文关键词：L-Glutamine; Metabolic engineering; RBS engineering; SRNA interference; Growth-responsive promoter

摘要：L-Glutamine (L-Gln) was efficiently biosynthesized in Corynebacterium glutamicum using metabolic engineering strategies. Four C. glutamicum strains (ATCC 13032, ATCC 13809, s026, and s9114) were screened, and gamma-glutamyl kinase was deleted using CRISPR/Cas12a system to redirect carbon flux, increasing L-Gln titer from 4.7 g/L to 5.3 g/L. Several glutamine synthetase (GS) were screened from different sources, and Saccharomyces cerevisiaederived GS demonstrated to be with optimal activity. Combined with RBS optimization to enhance translational efficiency, L-Gln titer was increased to 14.62 g/L. Subsequently, a dual strategy of site-directed mutagenesis of GS and small RNA-mediated inhibition of adenylyltransferase were conducted to relieve the adenylylation of GS, increasing L-Gln titer to 19.06 g/L. A "pull-push" strategy was implemented by strengthening glutamate dehydrogenase to enhance L-glutamate precursor supply while blocking L-Gln catabolism via deletion of glutamate synthase and glutaminase, resulting in 28.51 g/L L-Gln. Self-regulatory metabolic control was achieved using a growth-responsive promoter Pcg2705 to downregulate alpha-ketoglutarate dehydrogenase during stationary phase, achieving a shake flask titer of 31.85 g/L. The engineered strain CG17 produced 58.96 g/L L-Gln in a 5 L fedbatch bioreactor, with a yield of 0.31 g/g glucose and productivity of 1.05 g/L/h. The work provides valuable insights for developing high-performance strains for amino acid biosynthesis.