Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis. (20th April 2016)

• Record Type: Journal Article
• Authors: Jin, Peng; Zhang, Linpei; Yuan, Panhong; Kang, Zhen; Du, Guocheng; Chen, Jian
• Is Part Of: Carbohydrate polymers. Volume 140(2016)
• Page Start: 424
• Page End: 432
• Publication Date: 2016-04-20
• Subjects: Polysaccharides -- Chondroitin -- Heparosan -- Bacillus subtilis -- Metabolic engineering; Polysaccharides -- Periodicals; Polysaccharides -- Periodicals; Polysaccharides -- Périodiques; Electronic journals; 547.78
• DOI: 10.1016/j.carbpol.2015.12.065
• Languages: English
• ISSNs: 0144-8617

Abstract

Highlights: First report of chondroitin and heparosan production in metabolically engineered Bacillus subtilis. The expression cassettes kfoC – kfoA and kfiC – kfiA regulated by xylose were constructed and integrated onto genome. Up-regulation of tuaD significantly increased the production and molecular weights of chondroitin and heparosan. Highest titers of chondroitin and heparosan (5.22 g L −1 and 5.82 g L −1, respectively) was obtained in fed-batch cultures. Abstract: 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 1.83 g L −1 and 1.71 g L −1, respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54 g L −1 and 2.65 g L −1 . Moreover, production reached the highest peaks of 5.22 g L −1 and 5.82 g L −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 67.70 kDa, 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.