Current advances in industrial biotechnology are expanding the breadth of what is possible through the use of microbial cell factories. With global resources being exhausted at rapid rates and a heavy reliance on nonrenewable feedstocks, there is pressure to find alternatives using biorenewables. Application of these products is widespread, from reducing petroleum-based demand in the chemical industry, to synthesizing next-generation drug therapeutics at low cost. At the center of this biobased innovation is the need for a workhorse microorganism, capable of efficiently manufacturing these target molecules. In the context of biorenewable compounds, there are many cases where bacteria are not an option due to host toxicity or lack of processing ability, for example, post-translational modification. Baker’s yeast, Saccharomyces cerevisiae, is an ideal candidate as it is well characterized and frequently used industrially. Here we report its use in producing the polyketide triacetic acid lactone (TAL), an important polyketide which can be readily converted to replace fossil-fuel derived counterparts. Due to the complexity inherent in any host cell, metabolic pathway engineering was performed on central carbon metabolism to increase availability of acetyl-CoA and malonyl-CoA metabolites. The computational tool OptKnock in conjunction with other knockout interventions were used to create a strain (BY4741Δprb1Δpyc2Δnte1Δyia6) producing 2.2 g/L TAL, 26% of the theoretical yield on glucose, in fed-batch fermentation. In addition, enzyme engineering of the Gerbera hybrida 2-pyrone synthase (2-PS) catalyzing TAL formation was conducted and then evaluated in our strains. This effort screened 41 2-PS variants and elucidated key residues in the active site and the protein surface, improving catalytic efficiency and/or stability, leading to 2.5-fold increases in TAL levels in S. cerevisiae. The surface-modified variant 2-PS (C35S, C372S) exhibited the highest kcat/Km, over 40 times that of the native synthase. This variant was introduced into an engineered strain (BJ5464Δpyc2Δnte1) and cultivated in glucose or ethanol fed-batch to yield 2.7 g/L and 4.2 g/L TAL, respectively. Fermentation parameters were further improved and feed components modified for high titer (7.6 g/L) and yield (44% of theoretical) of this polyketide. Enhancement of precursor availability was also achieved by using a heterologous pathway from Escherichia coli. A modified E. coli bacterial pyruvate dehydrogenase (PDHm) was introduced in a cofactor-limited (Δzwf1) strain to couple NADPH cofactor balance with the synthesis of the TAL precursor acetyl-CoA. To prevent loss of acetyl-CoA and pyruvate by transport to the mitochondria, recently elucidated pathways were disrupted (Δpor2Δmpc2Δpda1Δyat2). The coupling of all three approaches (Δzwf1Δpor2Δmpc2Δpda1Δyat2+PDHm) elevated titers to 1.6 g/L, the highest by batch cultivation (and 35% of theoretical yield). These strategies are directly applicable to the synthesis of other acetyl-CoA derived compounds, and these studies provide a framework to develop design principles for robust, industrially relevant yeast for the synthesis of polyketides.
