Ethanol production for use as a biofuel is mainly achieved through simultaneous saccharification and fermentation by yeast. Operating at ≥40°C would be beneficial in terms of increasing efficiency of the process and reducing costs, but yeast does not grow efficiently at those temperatures. We used adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype.

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Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73 ± 0.05 mg L(-1) resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85 ± 0.31 mg L(-1) resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39 ± 0.03 mg L(-1). Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57 ± 7.00 mg L(-1). Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41 mg L(-1), respectively.

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