Abstract:

Objective This study explored the feasibility of using engineered Corynebacterium glutamicum to utilize corn stover acid hydrolysate, providing a new approach for the low-cost utilization of lignocellulosic waste. Method Single-factor experiments were conducted to identify components that facilitate the utilization of acid hydrolysate. The endogenous genes ino-1 (encoding inositol-3-phosphate synthase) and msrA (encoding methionine sulfoxide reductase) were overexpressed in Corynebacterium glutamicum to enhance the host’s antioxidant capacity. The expression of the antiporter Mrp1 was enhanced by promoter replacement. Adaptive laboratory evolution and overexpression of the ADY2 gene (encoding a paired protein from Saccharomyces cerevisiae) were then employed to improve host acetate utilization. Finally, the feasibility of using the engineered strain to produce heterologous proteins from acid hydrolysate was validated using the model protein mCherry, α-amylase from Bacillus subtilis, and the camelid antibody fragment VHH. Result The wild-type strain could not grow directly on acid hydrolysate. However, supplementation of the hydrolysate with a nitrogen source or with 1 mmol/L MgSO₄ and 0.1 mmol/L CaCl₂ enabled strain growth. Overexpression of the ino-1 and msrA genes increased the maximum cell density of the host grown in acid hydrolysate by 66.98% and 73.18%, respectively. The evolved strain Evo90 exhibited better growth than the wild type in medium supplemented with 0.8 g/L glucose and 70 g/L sodium acetate. Furthermore, when combined with overexpression of mrp1, ino-1, and ADY2, the engineered strain reached stationary phase in acid hydrolysate approximately 40% faster than the unmodified strain. Ultimately, the engineered strain successfully secreted three heterologous proteins using the hydrolysate. The mCherry yield exceeded that achieved with 35 g/L glucose, and the maximum fluorescence intensity per unit cell density reached approximately 10.18-fold to fermentation with 50 g/L glucose. Conclusion Supplementation of the acid hydrolysate with a nitrogen source or with Mg²⁺ and Ca²⁺ supported normal growth of the strain. Together with engineering modifications that enhanced antioxidant capacity and acetate utilization, the engineered C. glutamicum was able to secrete heterologous proteins using the acid hydrolysate.

Key words: acetate, lignocellulose, alternative carbon sources, green production, Corynebacterium glutamicum