Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae

Abstract

Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the acetyl-CoA metabolism by co-transformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10) and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB) and poly(3-hydroxybutyrate) polymerase (phaC) from Ralstonia eutropha H16. Introduction of the acetyl-CoA plasmid together with the PHB plasmid, improved the productivity of PHB more than 16 times compared to the reference strain used in this study, as well as it reduced the specific product formation of side products.

Figure 1

Schematic pathway and plasmid maps for polyhydroxybutyrate production inS. cerevisiae. (a) Acetyl-CoA boost plasmid (pIYC08) containing ADH2: alcohol dehydrogenase, ALD6: aldehyde dehydrogenase, ACS: acetyl-CoA synthetase variant and ERG10: acetyl-CoA acetyltransferase. (b) PHB plasmid (pKK01) containing PHB genes from R. eutropha, phaA: acetyl-CoA acetyltransferase, phaB: NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase and phaC: poly(3-hydroxybutyrate) polymerase. P and T in the plasmid map represent promoter and terminator, respectively.

Figure 2

Measurements of biomass and PHB from shake flask cultivations in a modified minimal medium with 20 g L^-1glucose as carbon source. Strain SCKK005 harbors an empty plasmid (pIYC04) and the PHB plasmid (pKK01), strain SCKK006 harbors an acetyl-CoA boost plasmid (pIYC08) and the PHB plasmid (pKK01), SCKK009 and SCKK010 harbor pIYC08 and pKK01 and carry a CIT2 and MLS1 deletion, respectively.

Figure 3

Fermentation profile ofS. cerevisiaeproducing PHB in aerobic batch bioreactor cultivation using a chemically defined medium with 20 g L^-1glucose as carbon source. (a) SCKK006: S. cerevisiae harboring an acetyl-CoA boost plasmid (pIYC08) and the PHB plasmid (pKK01). (b) SCKK005: S. cerevisiae harboring an empty plasmid (pIYC04) and the PHB plasmid (pKK01).

Figure 4

Time profile of glucose consumption and ethanol, glycerol and acetate formation during batch bioreactor cultivation of a) SCKK006 and b) SCKK005 in a chemically define minimal medium with 20 g L^-1glucose as carbon source. SCKK006: S. cerevisiae harboring an acetyl-CoA boost plasmid (pIYC08) and the PHB plasmid (pKK01). and SCKK005: S. cerevisiae harboring an empty plasmid (pIYC04) and the PHB plasmid (pKK01), respectively.

Figure 5

Comparison of specific fluxes in SCKK006 and SCKK005 during growth on glucose in aerobic batch bioreactor cultivation with 20 g L^-1glucose as carbon source. SCKK006 is S. cerevisiae harboring an acetyl-CoA boost plasmid (pIYC08) and the PHB plasmid (pKK01). SCKK005 is S. cerevisiae harboring an empty plasmid (pIYC04) and the PHB plasmid (pKK01). The fluxes towards the different lipids were calculated from measurement of the lipid composition of the biomass and the maximum specific growth rate. The mean value ± SD from at least triplicate fermentations are reported.