title: Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis.
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 1.83gL(-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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Jin
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Linpei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Panhong
authors.initials: P
authors.lastname: Yuan
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: zkang@jiangnan.edu.cn.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Kang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: jchen@jiangnan.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D061249:Batch Cell Culture Techniques; D002807:Chondroitin / Q000096:biosynthesis* / Q000737:chemistry; D004274:DNA, Recombinant / Q000235:genetics; D004187:Disaccharides / Q000096:biosynthesis* / Q000737:chemistry; D005285:Fermentation; D060847:Metabolic Engineering; D008970:Molecular Weight; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004187:Disaccharides; C560363:heparosan; D002807:Chondroitin
keywords: Bacillus subtilis; Chondroitin; Heparosan; Metabolic engineering; Polysaccharides