Enhancing Poly--glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-gamma-glutamic acid (gamma-PGA)-producing strains is an essential strategy for improving gamma-PGA production. Bacillus tequilensis BL01 Delta pgdS Delta ggt Delta sucA Delta gudB:P43-ppc-pyk-gdhA for the efficient synthesis of gamma-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of gamma-PGA by 2.18-fold (3.24 +/- 0.22 g/L) compared to that of B. tequilensis BL01 Delta pgdS Delta ggt Delta sucA Delta gudB (1.02 +/- 0.11 g/L). To further improve the titer of gamma-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the gamma-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 +/- 0.69 g/L gamma-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of gamma-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of gamma-PGA from molasses.