Coupling optimization of cell growth cycle and key enzyme membrane localization for enhanced synthesis of high molecular weight heparosan by Corynebacterium glutamicum.

High-molecular weight heparosan (HMW-heparosan) is a member of the glycosaminoglycan family. It possesses various chemical and physical properties suitable for a range of high-quality tissue engineering biomaterials, gels, scaffolds, and drug delivery systems. In this study, the HMW-heparosan biosynthesis pathway was engineered in Corynebacterium glutamicum through the introduction of heparosan synthase PmHS2 from Pasteurella multocida combined with overexpression of the key genes ugdA and galU, resulting in the generation of a stable HMW-heparosan-producing strain. Subsequently, to address metabolic flux competition, endogenous glycosyltransferases were systematically deleted to minimize UDP-glucose consumption, leading to a significant increase in HMW-heparosan accumulation. Additionally, cell growth was optimized by overexpressing transcriptional regulators whcD and PnkB, which was found to improve cell growth while creating an improved intracellular environment for biosynthesis. Notably, the critical enzyme heparosan synthase PmHS2 was relocated to the cell membrane by cell membrane display motifs porB, with its stability and catalytic efficiency being significantly enhanced so that the titer of HMW-heparosan reached 1.40 g/L in shake-flasks. Ultimately, the engineered strain was demonstrated to achieve HMW-heparosan production at 7.02 g/L with an average molecular weight (Mw) of 801 kDa in 5 L fed-batch bioreactor. These results demonstrate combinatorial optimization of cell factories, especially cell morphology and membrane localization of key enzymes, is efficacious and likely applicable for the production of other biopolymers.