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• The solubility of csABC I was improved with fusion of the MBP tag. • Mutation of I309 V significantly increased the catalytic activity. • OmpA signal peptide achieved efficient secretion of csABC I. • Deletion of a lipoprotein encoding gene lpp increased csABC I secretion. • Chondroitin sulfate oligosaccharides were produced with engineered csABC I. Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp , all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ± 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides. [ABSTRACT FROM AUTHOR]

• An efficient sulfation-modification system for CSA synthesis was constructed. • The soluble expression of C4ST was increased by 30 times. • The stability of AST IV was significantly improved with addition of glycerol. • CSA with high sulfation degree was produced with protein scaffold strategies. • 15 g CSA was produced within 24 h in 1 L system. Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees. [ABSTRACT FROM AUTHOR]