Metabolic engineering of Corynebacterium glutamicum WM001 to improve l‐isoleucine production

Abstract

In this study, L-isoleucine production in Corynebacterium glutamicum WM001 was improved by deleting three genes in the genome, replacing the native promoter of ilvA in the genome, and overexpression of five genes in an alr-based auxotrophic complementation expression system. The three genes deleted in the genome are alaT, brnQ, and alr. Deletion of alaT improved L-isoleucine production by increasing the supply of pyruvate, whereas deletion of brnQ improved L-isoleucine production by blocking the uptake of extracellular L-isoleucine. Exchange of the native promoter of ilvA with promoter tac or tacM could contribute to L-isoleucine production by increasing 2-ketobutyric acid; tac is better than tacM for improving L-isoleucine yield. Different combinations of the genes ilvBN, ppnK, lrp, and brnFE were overexpressed in an alr-based auxotrophic complementation expression system to further improve L-isoleucine production, and the best yield after 72-H flask fermentation was obtained from the strain WM005/pYCW-1-ilvBN2-ppnK1. Without addition of any antibiotics, WM005/pYCW-1-ilvBN2-ppnK1 could produce 32.1 g/L L-isoleucine after 72-H fed-batch fermentation, which is 34.3% increase compared with the original strain WM001.

Novel insights into L-serine exporters in Corynebacterium glutamicum from gene mining and functional analysis

“…Fermentation and amino acid export assay results indicate that overexpression of the four exporters could enhance the l -serine efflux. This also confirmed that overexpression of exporters could increase amino acid production in C. glutamicum (13, 20, 35). However, in order to further improve the l -serine titer, cell growth and l -serine accumulation must be balanced, and directed evolution and rational design of amino acid exporters could be employed to enhance l -serine efflux (36).…”

“…The copyright holder for this preprint this version posted July 18, 2022. ; https://doi.org/10.1101/2022.07.13.499997 doi: bioRxiv preprint 20,35). However, in order to further improve the L-serine titer, cell growth and L-serine accumulation must be balanced, and directed evolution and rational design of amino acid exporters could be employed to enhance L-serine efflux (36).…”

“…To date, only four transcription factors involved in amino acid export have been identified in C. glutamicum ; the transcription factor LysG regulating LysE, related to export of l -lysine and l -arginine; the transcription factor CgmR regulating CgmA, related to export of l -arginine; and the transcription factor Lrp regulating BrnFE, related to export of branched-chain amino acids (13, 14, 41). Recent studies have focused on overexpressing transcription factors regulating amino acid exporters, or using them as biosensors for screening strains with high amino acid production (13, 35, 42). Herein, we identified SerE as the major l -serine exporter, but when SerE was overexpressed in SSAAI, the l -serine titer was only increased by 10.5%.…”

Effect of sodium dodecyl sulfate on the production of L-isoleucine by the fermentation of Corynebacterium glutamicum

“…However, pyruvate (PYR) is the direct precursor of Val, and Leu is synthesized by the branching of α-acetylisovaleric acid of the intermediate of the Val synthesis pathway. Therefore, the production of valine and leucine can be reduced by limiting the production of pyruvate[ 1 ].…”

“…L-isoleucine is an essential amino acid for the human body and one of the three branched-chain amino acids[ 1 ]. Dou to its special structure and function, it occupies a particularly important position in human life metabolism.…”