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Construction of Escherichia coli cell factories for crocin biosynthesis

BACKGROUND: Crocin 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. Escherichi...

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Autores principales: Wang, Wen, He, Ping, Zhao, Dongdong, Ye, Lijun, Dai, Longhai, Zhang, Xueli, Sun, Yuanxia, Zheng, Jing, Bi, Changhao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610952/
https://www.ncbi.nlm.nih.gov/pubmed/31277660
http://dx.doi.org/10.1186/s12934-019-1166-1
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author Wang, Wen
He, Ping
Zhao, Dongdong
Ye, Lijun
Dai, Longhai
Zhang, Xueli
Sun, Yuanxia
Zheng, Jing
Bi, Changhao
author_facet Wang, Wen
He, Ping
Zhao, Dongdong
Ye, Lijun
Dai, Longhai
Zhang, Xueli
Sun, Yuanxia
Zheng, Jing
Bi, Changhao
author_sort Wang, Wen
collection PubMed
description BACKGROUND: Crocin 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, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In 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 4.42 mg/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. CONCLUSIONS: A 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-019-1166-1) contains supplementary material, which is available to authorized users.
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spelling pubmed-66109522019-07-16 Construction of Escherichia coli cell factories for crocin biosynthesis Wang, Wen He, Ping Zhao, Dongdong Ye, Lijun Dai, Longhai Zhang, Xueli Sun, Yuanxia Zheng, Jing Bi, Changhao Microb Cell Fact Research BACKGROUND: Crocin 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, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In 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 4.42 mg/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. CONCLUSIONS: A 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-019-1166-1) contains supplementary material, which is available to authorized users. BioMed Central 2019-07-05 /pmc/articles/PMC6610952/ /pubmed/31277660 http://dx.doi.org/10.1186/s12934-019-1166-1 Text en © The Author(s) 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Wang, Wen
He, Ping
Zhao, Dongdong
Ye, Lijun
Dai, Longhai
Zhang, Xueli
Sun, Yuanxia
Zheng, Jing
Bi, Changhao
Construction of Escherichia coli cell factories for crocin biosynthesis
title Construction of Escherichia coli cell factories for crocin biosynthesis
title_full Construction of Escherichia coli cell factories for crocin biosynthesis
title_fullStr Construction of Escherichia coli cell factories for crocin biosynthesis
title_full_unstemmed Construction of Escherichia coli cell factories for crocin biosynthesis
title_short Construction of Escherichia coli cell factories for crocin biosynthesis
title_sort construction of escherichia coli cell factories for crocin biosynthesis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610952/
https://www.ncbi.nlm.nih.gov/pubmed/31277660
http://dx.doi.org/10.1186/s12934-019-1166-1
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