Biorenewable chemicals such as medium chain carboxylic acids enable functional or direct substitution of petroleum-derived building blocks, allowing reduction of anthropogenic greenhouse gases while meeting market needs of high-demand products like aliphatic alcohols and alpha olefins. Our lab engineered Saccharomyces cerevisiae for production of free octanoic and decanoic acids from glucose by expression of a heterologous fatty acid synthase and thioesterase. However, in order to further increase titers it is necessary to reduce the toxicity of these products. The main inhibition mechanism of octanoic acid (C8) has been liked to disruption of membrane integrity, which can be alleviated by exogenous supplementation of oleic acid. Recently, our laboratory engineered S. cerevisiae to increase levels of malonyl-CoA, an essential building block for production of polyketides and fatty acids. This was achieved by preventing deactivation via phosphorylation of the enzyme acetyl-CoA carboxylase through introduction of a mutation in the serine residue 1157 (Acc1m). The resulting strain showed a 3-fold increase in production of total fatty acids, with oleic acid levels being significantly higher than the baseline strain. In this study, we demonstrate how overexpression of Acc1m in S. cerevisiae increases oleic acid proportion by 3.1-fold, resulting in improved C8 tolerance through modification of the lipid profile. Overexpressing Acc1m resulted in survival to C8 doses that were lethal for the baseline strain. This marked reduction of toxicity is due to higher membrane integrity, based on the 11-fold decrease of intracellular magnesium leakage observed for the engineered cells. Based on other reports and due to the pronounced increase in oleic acid observed, this approach has the potential to reduce toxicity of other valuable bioproducts like shorter chain aliphatic alcohols. We have also explored alternative strategies to alleviate stress caused by decanoic acid, and have demonstrated increased tolerance through overexpression of membrane transporters.

Strategies for increasing Saccharomyces cerevisiae tolerance to medium-chain fatty acids

253rd National Meeting (2017)

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