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A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica

[Image: see text] Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipoly...

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Autores principales: Tong, Yingjia, Zhou, Jingwen, Zhang, Liang, Xu, Peng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812646/
https://www.ncbi.nlm.nih.gov/pubmed/33399465
http://dx.doi.org/10.1021/acssynbio.0c00469
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author Tong, Yingjia
Zhou, Jingwen
Zhang, Liang
Xu, Peng
author_facet Tong, Yingjia
Zhou, Jingwen
Zhang, Liang
Xu, Peng
author_sort Tong, Yingjia
collection PubMed
description [Image: see text] Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.
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spelling pubmed-78126462021-01-21 A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica Tong, Yingjia Zhou, Jingwen Zhang, Liang Xu, Peng ACS Synth Biol [Image: see text] Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis. American Chemical Society 2021-01-05 2021-01-15 /pmc/articles/PMC7812646/ /pubmed/33399465 http://dx.doi.org/10.1021/acssynbio.0c00469 Text en © 2021 The Authors. Published by American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
spellingShingle Tong, Yingjia
Zhou, Jingwen
Zhang, Liang
Xu, Peng
A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title_full A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title_fullStr A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title_full_unstemmed A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title_short A Golden-Gate Based Cloning Toolkit to Build Violacein Pathway Libraries in Yarrowia lipolytica
title_sort golden-gate based cloning toolkit to build violacein pathway libraries in yarrowia lipolytica
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812646/
https://www.ncbi.nlm.nih.gov/pubmed/33399465
http://dx.doi.org/10.1021/acssynbio.0c00469
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