Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis.
Chondroitin and heparosan, important polysaccharides and key precursors of chondroitin sulfate and heparin/heparan sulfate, have drawn much attention due to their wide applications in many aspects. In this study, we designed two independent synthetic pathways of chondroitin and heparosan in food grade Bacillus subtilis, integrating critical synthases genes derived from Escherichia coli into B. subtilis genome. By RT PCR analysis, we confirmed that synthases genes transcripted an integral mRNA chain, suggesting co expression. In shaken flask, chondroitin and heparosan were produced at a level of 1.83gL( 1) and 1.71gL( 1) respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54gL( 1) and 2.65gL( 1) Moreover, production reached the highest peaks of 5.22gL( 1) and 5.82gL( 1) in 3 L fed batch fermentation, respectively, allowed to double the production that in shaken flask. The weight average molecular weight of chondroitin and heparosan from B. subtilis E168C/pP43 D and E168H/pP43 D were 114.07 and 67.70kDa, respectively. This work provided alternative safer synthetic pathways for metabolic engineering of chondroitin and heparosan in B. subtilis and a useful approach for enhancing production, which can be optimized for further improvement.
Carbohydrate polymers