Engineering Corynebacterium glutamicum for the de novo biosynthesis of tailored poly-γ-glutamic acid

γ-Polyglutamic acid (γ-PGA) is a biodegradable polymer naturally produced by Bacillus spp. that has wide applications. Fermentation of γ-PGA using Bacillus species often requires the supplementation of L-glutamic acid, which greatly increases the overall cost. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing γ-PGA from glucose. The genes encoding γ-PGA synthase complex from B. subtilis (pgsB, C, and A) or B. licheniformis (capB, C, and A) were expressed under inducible promoter P_tac in a L-glutamic acid producer C. glutamicum ATCC 13032, which led to low levels of γ-PGA production. Subsequently, C. glutamicum F343 with a strong L-glutamic acid production capability was tested. C. glutamicum F343 carrying capBCA produced γ-PGA up to 11.4 g/L, showing a higher titer compared with C. glutamicum F343 expressing pgsBCA. By introducing B. subtilis glutamate racemase gene racE under P_tac promoter mutants with different expression strength, the percentage of L-glutamic acid units in γ-PGA could be adjusted from 97.1% to 36.9%, and stayed constant during the fermentation process, while the γ-PGA titer reached 21.3 g/L under optimal initial glucose concentrations. The molecular weight (M_w) of γ-PGA in the engineered strains ranged from 2000 to 4000 kDa. This work provides a foundation for the development of sustainable and cost-effective de novo production of γ-PGA from glucose with customized ratios of L-glutamic acid in C. glutamicum.