In situ directed evolution of γ-glutamyl kinase in Corynebacterium glutamicum S9114 by innovative EvolvR-mediated mutagenesis and rare codon-based selection to increase L-proline production

Abstract

An efficient evolution and selection system is vital for constructing the high added-value products hyper-producers in fermentation industry. Herein, an innovative EvolvR-mediated mutagenesis was used to diversify the γ-glutamyl kinase (GK)-coding gene proB in C. glutamicum S9114 and an optimized rare codon-based selection was used to isolate the L-proline high-yielding strain with optimal GK variant. Triple-mutant GK^{E74K/G149K/V150M} was identified with high substrate affinity and catalytic efficiency for producing L-proline. The resultant strain C. glutamicum Pro-7 produced 94.2 ± 5.6 g/L of L-proline with productivity of 1.96 g/(L·h) and a yield of 0.316 g/g. Subsequently, the molecular mechanisms involved in high L-proline production in strain Pro-7 was discovered, indicating that ATP and NADPH supply are also the key factors for the L-proline biosynthesis besides GK. Thus, in situ directed evolution of key genes in genome by EvolvR-mediated mutagenesis and rare codon-based selection provides a new direction and reference to construct industrial-strength strains.