Corynebacterium glutamicum for Microbial Production of Chitin Oligosaccharides Using Modular Engineering

Abstract

Microbial fermentation is a potent strategy for eco-friendly and sustainable chitin oligosaccharide (CHOS) production. Nonetheless, hurdles (e.g., imbalanced metabolic flow and the need for uridine diphosphate (UDP)-sugar donor consumption in CHOS synthesis) hinder enhanced and efficient production. In this study, we aimed to use Corynebacterium glutamicum as the foundational organism for de novo CHOS synthesis. Initially, we developed the CHOS synthesis pathway in C. glutamicum, attaining a CHOS titer of 113.34 mg/L. Furthermore, we fortified the uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) synthesis module, vital for CHOS and other functional sugar synthesis, and developed a system for regenerating uridine triphosphate (UTP) precursors. Finally, we performed C. glutamicum-mediated scale-up CHOS production in a 5-L bioreactor yielding a titer of 5.08 g/L. The CHOS chassis strain provides a robust foundation for mass CHOS production via metabolic engineering. Altering the intracellular UDP-sugar donor creation pathway could reportedly significantly enhance CHOS production. We attained the peak concentration of 829.33 mg/L with the heightened expression of glmM, glmU, and the metabolic equilibrium of PCM1 and AmgK. Bacterial growth remained unaffected by the excessive gene expressions or external gene incorporations. In addition, the swift growth and C. glutamicum accumulation in the fermenter led to increased CHOS production, reaching a titer of 5.08 g/L from the recombinant strain CGSL63, being 4.43 times higher than in the case of shake flask fermentation. The engineering strategies used in this study might be helpful for the C. glutamicum-mediated microbial synthesis of functional sugars. The methods applied in this study are broadly applicable for boosting the microbial generation of other valuable functional sugars.