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Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase (crtZ) and β-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for impro...

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Autores principales: Ding, Yong-Wen, Lu, Chuan-Zhen, Zheng, Yan, Ma, Han-Zhang, Jin, Jin, Jia, Bin, Yuan, Ying-Jin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: KeAi Publishing 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9668533/
https://www.ncbi.nlm.nih.gov/pubmed/36408203
http://dx.doi.org/10.1016/j.synbio.2022.10.005
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author Ding, Yong-Wen
Lu, Chuan-Zhen
Zheng, Yan
Ma, Han-Zhang
Jin, Jin
Jia, Bin
Yuan, Ying-Jin
author_facet Ding, Yong-Wen
Lu, Chuan-Zhen
Zheng, Yan
Ma, Han-Zhang
Jin, Jin
Jia, Bin
Yuan, Ying-Jin
author_sort Ding, Yong-Wen
collection PubMed
description The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase (crtZ) and β-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.
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spelling pubmed-96685332022-11-18 Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae Ding, Yong-Wen Lu, Chuan-Zhen Zheng, Yan Ma, Han-Zhang Jin, Jin Jia, Bin Yuan, Ying-Jin Synth Syst Biotechnol Original Research Article The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by β-carotene hydroxylase (crtZ) and β-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway. KeAi Publishing 2022-11-10 /pmc/articles/PMC9668533/ /pubmed/36408203 http://dx.doi.org/10.1016/j.synbio.2022.10.005 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Research Article
Ding, Yong-Wen
Lu, Chuan-Zhen
Zheng, Yan
Ma, Han-Zhang
Jin, Jin
Jia, Bin
Yuan, Ying-Jin
Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title_full Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title_fullStr Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title_full_unstemmed Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title_short Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae
title_sort directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in saccharomyces cerevisiae
topic Original Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9668533/
https://www.ncbi.nlm.nih.gov/pubmed/36408203
http://dx.doi.org/10.1016/j.synbio.2022.10.005
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