Metabolic Engineering Of Corynebacterium Glutamicum For L-isoleucine Production

Branched-chain amino acid L-isoleucine, an essential amino acid with a variety of physiological functions, is widely used in food, pharmaceutical, cosmetics and animal feed. Production of L-isoleucine in industry is mainly performed by microbial fermentation, and Corynebacterium glutamicum is one of the major production strains. L-isoleucine producing C. glutamicum strains are mainly obtained by random mutagenesis; since their genetic background is unknown it is difficult to further improve the yield of L-isoleucine in these strains. Therefore, improving L-isoleucine production in C. glutamicum by metabolic engineering is drawing more attention in recent years. This study investigated the regulation metabolism of elongation factor EF-G and ribosomal recycle factor RRF to L-isoleucine synthesis in C. glutamicum, using L-isoleucine producing strain IWJ001 as starting strain, and increased the yield of L-isoleucine by metabolic engineering. The main conclusions are listed below:(1) Elongation factor EF-G can promote L-isoleucine synthesis in C. glutamicum. Overexpressing fus A gene from the wild-type C. glutamicum ATCC13869 and fus A1 gene from C. glutamicum IWJ001 increased yield of L-isoleucine, and the former increased more than the latter;(2) The impact of ribosome recycle factor RRF, elongation factor EF-Tu and elongation factor EF-Ts on L-isoleucine synthesis in C. glutamicum was studied. RRF could promote L-isoleucine synthesis; EF-Tu inhibited the synthesis of L-isoleucine, but the synthesis of Lthreonine and L-lysine were improved; EF-Ts has no significant effect on the synthesis of amino acids in C. glutamicum;(3) Isoleucine production in C. glutamicum IWJ001 was improved by metabolic engineering. Coexpression of EF-G and RRF increased yields of L-isoleucine and other amino acid in C. glutamicum IWJ001. Coexpression of ilv BNA and ppnk increased L-isoleucine production by 76.5%, with lower levels of byproduct; IWJ001/p DXW-8-fus A-frr-ilv BNA-ppnk were cultured for 72 h in 3L fermentor, L-isoleucine production increased by 39.7%, reaching 28.5 g/L;(4) The mechanism for improving L-isoleucine synthesis in C. glutamicum by overexpression of elongation factor EF-G and ribosomal factor RRF was analyzed. RT-PCR analysis showed that EF-G and RRF could improve the transcription levels of L-isoleucine biosynthesis related genes in C. glutamicum; EF- G and RRF could slightly improve activity of key enzymes in the L-isoleucine biosynthetic pathway. Therefore, improved L-isoleucine synthesis by EF-G and RRF may be due to the increased levels of transcription and translation of key synthases;(5) The stability of antibiotic-free expression system in IWJ001 was determined. Plasmids p JYW-4 and p JYW-4-fus A could stably replicate in IWJ001Δalr in fermentation medium without kanamycin.