[Construction of an integrative recombinant Escherichia coli strain for efficient biosynthesis of chondroitin]

Chondroitin and its derivatives are essential glycosaminoglycans in articular cartilage and connective tissue, with broad applications in the nutraceutical and pharmaceutical industries. Conventional chemical and enzymatic synthesis approaches are often limited by process complexity, low efficiency, and high substrate costs. In contrast, microbial fermentation offers a promising alternative for the sustainable production of chondroitin due to its shorter production cycle, lower cost, and scalability. To obtain a genetically stable chondroitin-producing strain, in this study, an efficient genome-integrated Escherichia coli strain for chondroitin production was developed based on the strain GZ17 previously constructed in our laboratory. First, a chondroitin synthase from Moraxella canis (McCS) was identified through phylogenetic analysis and stably expressed under a strong promoter via genomic integration. Subsequently, key amino acid residues were identified through structural modeling and molecular dynamics simulations, followed by iterative mutagenesis to obtain a highly active mutant, McCS^{S602A/N632S/K637L}. Finally, two copies of the mutant McCS^{S602A/N632S/K637L} gene were integrated into the genome of GZ17, which significantly enhanced chondroitin biosynthesis. The resulting strain, GZ-2ASLM, achieved a chondroitin titer of 7.41 g/L, a glycerol yield of 0.03 g/g, and a productivity of 0.10 g/(L·h) by fed-batch fermentation in a 5-L fermenter. Notably, this engineered strain utilizes glycerol as the sole carbon source for chondroitin production without depending on plasmids. This study provides a robust strategy for developing efficient microbial cell factories for the production of glycosaminoglycans.