کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1383478 | 1500618 | 2016 | 9 صفحه PDF | دانلود رایگان |
• 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.
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.
Journal: Carbohydrate Polymers - Volume 140, 20 April 2016, Pages 424–432