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Biorenewable chemicals such as short and medium chain fatty 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. However, producing these fatty acids in microorganisms can be challenging due to toxicity issues. Octanoic acid (C8) can disrupt the integrity of the cell membrane in yeast, and exogenous supplementation of oleic acid has been shown to help alleviate this. We recently engineered the Saccharomyces cerevisiae enzyme acetyl-CoA carboxylase by replacing serine residue 1157 with alanine to prevent deactivation by phosphorylation. Expression of Acc1 in S. cerevisiae resulted in an increase in total fatty acid production, with the largest increase for oleic acid. In this study, we evaluated the effect of this modified lipid profile on C8 toxicity to the yeast. Expression of Acc1 in S. cerevisiae BY4741 increased the percentage of oleic acid 3.1- and 1.6-fold in the absence and presence of octanoic acid challenge, respectively. Following exposure to 0.9 mM of C8 for 24 h, the engineered yeast had a 10-fold higher cell density relative to the baseline strain. Moreover, overexpressing Acc1 allowed survival at C8 concentrations that were lethal for the baseline strain. This marked reduction of toxicity was shown to be due to higher membrane integrity as an 11-fold decrease in leakage of intracellular magnesium was observed. Due to the increase in oleic acid, this approach has the potential to reduce toxicity of other valuable bioproducts such as shorter chain aliphatic acids and alcohols and other membrane stressors. In an initial screen, increased resistance to n-butanol, 2-propanol, and hexanoic acid was demonstrated with cell densities 3.2-, 1.8-, and 29-fold higher than the baseline strain, respectively. Biotechnol. Bioeng. 2017;114: 1531-1538. © 2017 Wiley Periodicals, Inc.

The native yeast type I fatty acid synthase (FAS) is a complex, rigid enzyme, and challenging to engineer for the production of medium- or short-chain fatty acids. Introduction of a type II FAS is a promising alternative as it allows expression control for each discrete enzyme and the addition of heterologous thioesterases. In this study, the native Saccharomyces cerevisiae FAS was functionally replaced by the Escherichia coli type II FAS (eFAS) system. The E. coli acpS + acpP (together), fabB, fabD, fabG, fabH, fabI, fabZ, and tesA were expressed in individual S. cerevisiae strains, and enzyme activity was confirmed by in vitro activity assays. Eight genes were then integrated into the yeast genome, while tesA or an alternate thioesterase gene, fatB from Ricinus communis or TEII from Rattus novergicus, was expressed from a multi-copy plasmid. Native FAS activity was eliminated by knocking out the yeast FAS2 gene. The strains expressing only the eFAS as de novo fatty acid source grew without fatty acid supplementation demonstrating that this type II FAS is able to functionally replace the native yeast FAS. The engineered strain expressing the R. communis fatB thioesterase increased total fatty acid titer 1.7-fold and shifted the fatty acid profile towards C14 production, increasing it from <1% in the native strain to more than 30% of total fatty acids, and reducing C18 production from 39% to 8%.

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