文献类型：期刊文献

英文题名：Construction of Escherichia coli cell factories for crocin biosynthesis

作者：Wang, Wen[1,2];He, Ping[1,2];Zhao, Dongdong[1];Ye, Lijun[1];Dai, Longhai[1];Zhang, Xueli[1];Sun, Yuanxia[1];Zheng, Jing[2];Bi, Changhao[1]

机构：[1]Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin 300308, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Key Lab New Drug Design, Sch Pharm, Shanghai 200237, Peoples R China

年份：2019

卷号：18

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000474600900001)】；

基金：This research was financially supported by the Key Research Program of the Chinese Academy of Science (KFZD-SW-215), National Natural Science Foundation of China (31522002, 31861143019) and the Fundamental Research Funds for the Central Universities.

语种：英文

外文关键词：Crocetin; Crocin; Escherichia coli; Saffron stigma; Glycosyltransferase

摘要：BackgroundCrocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, beta-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis.ResultsIn this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS.ConclusionsA heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.