Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains.
Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States; Energy Biosciences Institute, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
Jiazhang
J
Lian
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
Tong
T
Si
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States; Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, United States.
Nikhil U
NU
Nair
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States; Energy Biosciences Institute, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States; Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States. Electronic address: zhao5@illinois.edu.
Huimin
H
Zhao
D020001:1-Butanol / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000096:biosynthesis / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D060847:Metabolic Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D000105:Acetyl Coenzyme A; D020001:1-Butanol
ATP-dependent citrate lyase; Acetyl-CoA; Acetyl-CoA synthetase; Metabolic engineering; Pyruvate dehydrogenase; n-Butanol