Systematic metabolic engineering of Corynebacterium glutamicum for enhancing L-isoleucine production

Abstract

L-isoleucine, an essential branched-chain amino acid, enjoys rising demand in the pharmaceutical, food, and feed industries. Corynebacterium glutamicum is a major production host for L-isoleucine production, but its native metabolic regulations limit industrial-scale synthesis. In this study, a L-isoleucine producing strain C. glutamicum YW-8 was engineered to efficiently produce L-isoleucine by finely regulating carbon flux toward its biosynthesis. To do this, promoter replacement was firstly performed to upregulate the expression level of the genes (e.g., ilvBN, ilvA) involved in the L-isoleucine biosynthetic pathway. Then, the genes (e.g., alaT, ldh) involved in the byproduct biosynthesise were knocked out to avoid the by-products accumulation, and the ppc gene was overexpressed to augment pyruvate supply. Subsequently, CRISPRi-mediated repression of the dapA gene was employed to dynamically reduce L-lysine diversion. Finally, the lrp-brnFE operon was overexpressed and the brnQ gene was knocked out to optimize L-isoleucine export. The resulted strain I16 produced (18.5 ± 0.9) g/L L-isoleucine in shake flasks, which was a 3.4-fold higher than that of strain YW-8 (i.e., 5.5 g/L). In addition, strain I-16 showed a significantly reduced L-lysine and L-alanine accumulation and the improved fermentation stability. This study provides feasible technical strategies for the systematic reconstruction and dynamic regulation of complex amino acid metabolic networks, and bears important theoretical and practical significance for promoting the rational design of microbial manufacturing processes.

Keywords

Corynebacterium glutamicum / Metabolic engineering / L-isoleucine production / Dynamic regulation / CRISPR interference (CRISPRi)

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Funding

National Natural Science Foundation of China

RIGHTS & PERMISSIONS

Jiangnan University