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Metabolic Engineering of Saccharomyces cerevisiae for the Enhanced Production of Biorenewable Fuels and Platform Chemicals

  • Author(s): Leber, Christopher Tyler
  • Advisor(s): Da Silva, Nancy A
  • et al.
Abstract

The production of fuels and chemicals from biorenewable resources is important to alleviate the instability in supply costs, growing environmental concerns and competing policy agendas associated with the use of petroleum feedstock. Many of these petroleum-derived fuels and chemicals can be directly or functionally substituted with bio-produced molecules. Among these compounds, short (SCFAs) and long chain fatty acids (LCFAs) can be utilized as free fatty acids or can fulfill a role as platform molecules using their functional group as a target for chemical catalysis. We have designed Saccharomyces cerevisiae strains to overproduce long and short chain fatty acids by combining novel enzyme and metabolic engineering strategies. LCFAs were increased by targeting three native intracellular processes: b-oxidation, activation (via acyl-CoA synthetases), and neutral lipid recycle. A sextuple mutant (faa1 faa4 fat1 faa2 pxa1 pox1) overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2 g/L extracellular free fatty acids. SCFAs were overproduced by linking a Rattus norvegicus derived TEII to the Homo sapiens derived fatty acid synthase (hFAS). With the use of a Bacillus subtilis derived Sfp phosphopantetheine transferase, extracellular caprylic (C8) and total SCFA acid levels reached 63 mg/L and 68 mg/L, respectively. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L. Further enzyme and pathway engineering strategies were employed to increase SCFA production. By codon optimizing hFAS and targeting the specific degradation pathways for SCFAs in an engineered strain (faa2 ant1 pex11), we were able to increase hexanoic, octanoic, decanoic and total extracellular short chain fatty acids more than 75-, 220-, 120-, and 400-fold, respectively, relative to parent strain BY4741. Our novel combinations of pathway interventions have led to the highest extracellular SCFAs and LCFAs reported for S. cerevisiae. These engineering strategies should also prove useful for the increased production of other fatty acid derived biomolecules in yeast.

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