title: Functional Reconstitution of a Pyruvate Dehydrogenase in the Cytosol of Saccharomyces cerevisiae through Lipoylation Machinery Engineering.
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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, and ‡Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Jiazhang
authors.initials: J
authors.lastname: Lian
authors.affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, and ‡Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000105:Acetyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D002210:Caprylates / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D054889:Fatty Acid Synthase, Type II / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D054878:Lipoylation; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009154:Mutation; D009243:NAD / Q000235:genetics / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D008063:Thioctic Acid / Q000378:metabolism
chemical_list: D002210:Caprylates; D029968:Escherichia coli Proteins; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D000105:Acetyl Coenzyme A; D008063:Thioctic Acid; D054889:Fatty Acid Synthase, Type II; C031492:octanoic acid
keywords: acetyl-CoA; protein lipoylation; pyruvate dehydrogenase; synthetic biology; yeast