State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Metab Eng. 2018 May;47:314-322. doi: 10.1016/j.ymben.2018.04.006. Epub 2018 Apr 12.
Microbial production of chondroitin and chondroitin-like polysaccharides from renewable feedstock is a promising and sustainable alternative to extraction from animal tissues. In this study, we attempted to improve production of fructosylated chondroitin in Escherichia coli K4 by balancing intracellular levels of the precursors UDP-GalNAc and UDP-GlcA. To this end, we deleted pfkA to favor the production of Fru-6-P. Then, we identified rate-limiting enzymes in the synthesis of UDP-precursors. Third, UDP-GalNAc synthesis, UDP-GlcA synthesis, and chondroitin polymerization were combinatorially optimized by altering the expression of relevant enzymes. The ratio of intracellular UDP-GalNAc to UDP-GlcA increased from 0.17 in the wild-type strain to 1.05 in a 30-L fed-batch culture of the engineered strain. Titer and productivity of fructosylated chondroitin also increased to 8.43 g/L and 227.84 mg/L/h; the latter represented the highest productivity level achieved to date.
从可再生原料中微生物生产硫酸软骨素和类似硫酸软骨素的多糖是一种很有前途和可持续的替代从动物组织中提取的方法。在这项研究中，我们试图通过平衡细胞内 UDP-GalNAc 和 UDP-GlcA 前体的水平来提高大肠杆菌 K4 中糖基化硫酸软骨素的产量。为此，我们删除了 pfkA 以有利于 Fru-6-P 的产生。然后，我们确定了合成 UDP 前体的限速酶。第三，通过改变相关酶的表达，组合优化 UDP-GalNAc 合成、UDP-GlcA 合成和软骨素聚合。与野生型菌株相比，工程菌株在 30-L 分批补料培养物中细胞内 UDP-GalNAc 与 UDP-GlcA 的比例从 0.17 增加到 1.05。糖基化硫酸软骨素的产量和生产强度也分别提高到 8.43 g/L 和 227.84 mg/L/h；后者代表了迄今为止达到的最高生产强度水平。
