Functional Reconstitution of a Pyruvate Dehydrogenase in the Cytosol of Saccharomyces cerevisiae through Lipoylation Machinery Engineering

Jiazhang Lian, Huimin Zhao

Research output: Contribution to journalArticle

Abstract

Acetyl-CoA is a key precursor for the biosynthesis of a wide range of fuels, chemicals, and value-added compounds, whose biosynthesis in Saccharomyces cerevisiae involves acetyl-CoA synthetase (ACS) and is energy intensive. Previous studies have demonstrated that functional expression of a pyruvate dehydrogenase (PDH) could fully replace the endogenous ACS-dependent pathway for cytosolic acetyl-CoA biosynthesis in an ATP-independent manner. However, the requirement for lipoic acid (LA) supplementation hinders its wide industrial applications. In the present study, we focus on the engineering of a de novo synthetic lipoylation machinery for reconstitution of a functional PDH in the cytosol of yeast. First, a LA auxotrophic yeast strain was constructed through the expression of the Escherichia coli PDH structural genes and a lipoate-protein ligase gene in an ACS deficient (acs1Δ acs2Δ) strain, based on which an in vivo acetyl-CoA reporter was developed for following studies. Then the de novo lipoylation pathway was reconstituted in the cytosol of yeast by coexpressing the yeast mitochondrial lipoylation machinery genes and the E. coli type II fatty acid synthase (FAS) genes. Alternatively, an unnatural de novo synthetic lipoylation pathway was constructed by combining the reversed β-oxidation pathway with an acyl-ACP synthetase gene. To the best of our knowledge, reconstitution of natural and unnatural de novo synthetic lipoylation pathways for functional expression of a PDH in the cytosol of yeast has never been reported. Our study has laid a solid foundation for the construction and further optimization of acetyl-CoA overproducing yeast strains.

Original languageEnglish (US)
Pages (from-to)689-697
Number of pages9
JournalACS synthetic biology
Volume5
Issue number7
DOIs
StatePublished - Jul 15 2016

Keywords

  • acetyl-CoA
  • protein lipoylation
  • pyruvate dehydrogenase
  • synthetic biology
  • yeast

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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