Production of fatty acids using engineered Saccharomyces cerevisiae cells is a challenging task in part due to low efficiency of the native fatty acid biosynthesis pathway. One option for improving production efficiency relies on exploring alternative fatty acid production pathways with either improved kinetics, thermodynamics or yield properties.In this work, we explored the reverse β-oxidation pathway as an alternative pathway for free fatty acid production. Different gene combinations and analysis methods were tested for assessing pathway efficiency when expressed in the yeast Saccharomyces cerevisiae. Even though different alternatives were tested, quantitative analysis showed no improvement or major change in fatty acid production of the tested strains in our conditions. This lack of improvement suggests that the tested pathway designs and constructs are either nonfunctional in the tested conditions or the resulting strains lack a metabolic driving force that is needed for a functional pathway.We conclude that expression of the reverse β-oxidation pathway in S. cerevisiae poses many challenges when compared to expression in bacterial systems. These factors gravely hinder development efforts and success rate for producing fatty acids through this pathway.
Production of triacylglycerols (TAGs) through microbial fermentation is an emerging alternative to plant and animal-derived sources. The yeast Saccharomyces cerevisiae is a preferred organism for industrial use but has natively a very poor capacity of TAG production and storage. Here, we engineered S. cerevisiae for accumulation of high TAG levels through the use of structural and physiological factors that influence assembly and biogenesis of lipid droplets. First, human and fungal perilipin genes were expressed, increasing TAG content by up to 36% when expressing the human perilipin gene PLIN3. Secondly, expression of the FIT2 homologue YFT2 resulted in a 26% increase in TAG content. Lastly, the genes ERD1 and PMR1 were deleted in order to induce an ER stress response and stimulate lipid droplet formation, increasing TAG content by 72% for Δerd1, with an additive effect for both YFT2 and PLIN3 expression. These new approaches were implemented in previously engineered strains that carry high flux of fatty acid biosynthesis and conversion of acyl-CoA into TAG, resulting in improvements of up to 138% over those high-producing strains without any substantial growth effects or abnormal cell morphology. We find that these approaches are not only a major advancement in engineering S. cerevisiae for TAG production, but also highlight the importance of lipid droplet dynamics for high lipid accumulation in yeast.
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