Enhanced heparosan biosynthesis in Corynebacterium glutamicum by systematic metabolic engineering and translation-level fine-tuning.

As a widely used anticoagulant, heparin is industrially produced chiefly via animal tissue extraction, which suffers from unstable supply and potential safety hazards. Heparosan shares a similar polysaccharide backbone with heparin and can be converted into heparin under mild enzymatic catalysis. In addition, heparosan exhibits favorable biocompatibility and non-immunogenicity, rendering its efficient, eco-friendly biosynthesis essential. In this study, we systematically engineered Corynebacterium glutamicum, a Generally Recognized as Safe (GRAS) microorganism, to synthesize heparosan via two complementary strategies. First, genome-scale modification was implemented to stably upregulate genes ugd, glmS, and ndk. The heparosan titer of recombinant strain Cg24 increased from 226.37 to 595.39 mg/L. Second, translation-level fine-tuning was implemented to modulate expression of individual genes within the kfiB-kfiC-kfiA cassette by constructing a high-coverage random ribosome binding site (RBS) library, which further lifted heparosan titer to 1161.37 mg/L. In fed-batch fermentation using a 5 L bioreactor with a two-stage growth-production regulation strategy, the recombinant strain Cg24-11 produced 5.36 g/L of heparosan, demonstrating its great potential for efficient heparosan biosynthesis. This combined modification strategy also provides valuable references for constructing high-efficiency cell factories for other complex compounds.