Journal
ACS SYNTHETIC BIOLOGY
Volume 5, Issue 5, Pages 386-394Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00250
Keywords
metabolic engineering; Saccharomyces cerevisiae; D-xylose; synthetic biology; bioinformatics
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Funding
- Fundacao para a Ciencia e Tecnologia Portugal (FCT) through Project MycoFat [PTDC/AAC-AMB/120940/2010]
- FCT [SFRH/BD/80934/2011]
- national funds through the FCT I.P. [UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569)]
- ERDF through the COMPETE2020 - Programa Operational Competitividade e Internacionalizacao (POCI)
- Fundação para a Ciência e a Tecnologia [SFRH/BD/80934/2011, PTDC/AAC-AMB/120940/2010] Funding Source: FCT
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We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A D-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on D-xylose (0.18 h(-1)) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool.
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