Developing a Bacillus licheniformis platform for de novo production of γ-aminobutyric acid and other glutamate-derived chemicals.

Microbial cell factories (MCFs) have emerged as a sustainable tool for the production of value-added biochemicals. However, developing high-performance MCFs remains a major challenge to fulfill the burgeoning demands of global markets. This study aimed to establish the B. licheniformis cell factory for the cost-effective production of glutamate-derived chemicals by modular metabolic engineering. Initially, the glutamate decarboxylase from E. coli was introduced into B. licheniformis DW2 to construct the artificial γ-aminobutyric acid (GABA) pathway. By systematically optimizing the central metabolic pathway, boosting the L-Glu synthesis pathway and improving the cofactor NADPH supply, the strain G35/pHY-P_r5u12-gadB^E89Q/H465A achieved a remarkable yield of 62.9 g/L of GABA in a 5-L bioreactor, representing the highest yield of 0.5 g/g glucose with a significant 49.3-fold increase. Remarkably, bioinformatics analyses and function verification identified the putative glyoxylate to glycolic acid synthesis pathway and KipR, an inhibitor of the glyoxylate cycle, as the rate-limiting steps in GABA production. Additionally, a versatile and robust platform using engineered B. licheniformis for efficient production of diverse glutamate-derived chemicals was established and the titer of 5-aminolevulinic acid, heme and indigoidine was improved by 5.3-, 4.7- and 1.9-fold, respectively. This study not only facilitates extensive application of B. licheniformis for chemical production, but also sheds light on research to improve the performance of other MCFs.