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YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction...

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Autores principales: Guo, Yakun, Dong, Junkai, Zhou, Tong, Auxillos, Jamie, Li, Tianyi, Zhang, Weimin, Wang, Lihui, Shen, Yue, Luo, Yisha, Zheng, Yijing, Lin, Jiwei, Chen, Guo-Qiang, Wu, Qingyu, Cai, Yizhi, Dai, Junbiao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513847/
https://www.ncbi.nlm.nih.gov/pubmed/25956650
http://dx.doi.org/10.1093/nar/gkv464
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author Guo, Yakun
Dong, Junkai
Zhou, Tong
Auxillos, Jamie
Li, Tianyi
Zhang, Weimin
Wang, Lihui
Shen, Yue
Luo, Yisha
Zheng, Yijing
Lin, Jiwei
Chen, Guo-Qiang
Wu, Qingyu
Cai, Yizhi
Dai, Junbiao
author_facet Guo, Yakun
Dong, Junkai
Zhou, Tong
Auxillos, Jamie
Li, Tianyi
Zhang, Weimin
Wang, Lihui
Shen, Yue
Luo, Yisha
Zheng, Yijing
Lin, Jiwei
Chen, Guo-Qiang
Wu, Qingyu
Cai, Yizhi
Dai, Junbiao
author_sort Guo, Yakun
collection PubMed
description It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.
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spelling pubmed-45138472015-07-27 YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae Guo, Yakun Dong, Junkai Zhou, Tong Auxillos, Jamie Li, Tianyi Zhang, Weimin Wang, Lihui Shen, Yue Luo, Yisha Zheng, Yijing Lin, Jiwei Chen, Guo-Qiang Wu, Qingyu Cai, Yizhi Dai, Junbiao Nucleic Acids Res Methods Online It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts. Oxford University Press 2015-07-27 2015-05-08 /pmc/articles/PMC4513847/ /pubmed/25956650 http://dx.doi.org/10.1093/nar/gkv464 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Guo, Yakun
Dong, Junkai
Zhou, Tong
Auxillos, Jamie
Li, Tianyi
Zhang, Weimin
Wang, Lihui
Shen, Yue
Luo, Yisha
Zheng, Yijing
Lin, Jiwei
Chen, Guo-Qiang
Wu, Qingyu
Cai, Yizhi
Dai, Junbiao
YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title_full YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title_fullStr YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title_full_unstemmed YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title_short YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
title_sort yeastfab: the design and construction of standard biological parts for metabolic engineering in saccharomyces cerevisiae
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513847/
https://www.ncbi.nlm.nih.gov/pubmed/25956650
http://dx.doi.org/10.1093/nar/gkv464
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