Genetically engineering central carbon and nitrogen metabolism in Bacillus paralicheniformis for high γ-PGA production via glutamate-independent pathway.

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with great significance and broad applications. However, its fermentation in Bacillus strains usually requires costly glutamate supplementation. To reduce production costs, we engineered B. paralicheniformis to efficiently produce γ-PGA in a glutamate-independent manner by enhancing de novo glutamate biosynthesis via rocG-mediated pathways and alleviating carbon/nitrogen catabolite repression. In this study, we deleted three key regulatory genes: ccpA (catabolite control protein A), cggR (central glycolytic genes repressor), and tnrA (master regulator of nitrogen assimilation) to relieve repression on the TCA cycle, glycolysis, and γ-PGA biosynthesis pathways, thereby significantly increasing γ-PGA yield and productivity in B. paralicheniformis. In batch fermentation experiments, strains ΔccpA and ΔtnrA achieved γ-PGA yields of 63.86 g/L and 61.38 g/L, with productivities of 19.50 g/(L·h) and 6.93 g/(L·h) during the rapid increase period of γ-PGA production, respectively. To our knowledge, the ΔccpA strain achieved the highest γ-PGA yield and productivity among glutamate-independent producers. Both ΔccpA and ΔtnrA strains demonstrated superior efficiency in converting inexpensive NaNO₃ into high-value γ-PGA compared to the parental strain. Collectively, relieving suppression of carbon metabolism (the TCA cycle and glycolysis) and nitrogen metabolism (nitrate reduction) via genetic engineering holds significant potential for further enhancing the ability to biosynthesize γ-PGA in a glutamate-independent manner. The findings highlight the effectiveness of targeted genetic modifications in improving industrial bioprocesses for cost-efficient γ-PGA production.