Enhanced production of poly-γ-glutamic acid via optimizing the expression cassette of Vitreoscilla hemoglobin in Bacillus licheniformis

Poly-γ-glutamic acid (γ-PGA) is a natural polymer with various applications, and its high-viscosity hinders oxygen transmission and improvement of synthesis level. Vitreoscilla hemoglobin (VHB) has been introduced into various hosts as oxygen carrier, however, its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis. Here, we want to optimize the expression cassette of VHB for γ-PGA production. Firstly, our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation (Tat) signal peptides (SPYwbN, SPPhoD and SPTorA) into VHB expression cassette, and the best performance was attained by SPYwbN from Bacillus subtilis, the γ-PGA yield of which was 18.53% higher than that of control strain, and intracellular ATP content and oxygen transfer coefficient (KLa) were increased by 29.71% and 73.12%, respectively, indicating that VHB mediated by SPYwbN benefited oxygen transfer and ATP generation for γ-PGA synthesis. Furthermore, four promoters were screened, and Pvgb was proven as the more suitable promoter for VHB expression and γ-PGA synthesis, and γ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%. Finally, WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation, and 46.39 g/L γ-PGA was attained by glucose feeding, increased by 49.26% compared with the initial yield (31.01 g/L). Taken together, this study has attained an efficient VHB expression cassette for oxygen transfer and γ-PGA synthesis, which could also be applied in the production of other metabolites.