Central carbon rewiring of Saccharomyces cerevisiae has played a major role in harnessing this model organism for the synthesis of various high-value materials including isoprenoids and polyketides. Specifically, these biobased materials are poised to serve as platform chemicals, as is the case for triacetic acid lactone (TAL) and its relevance in synthesizing food preservatives, plastics, and flavoring agents. The yeast acetyl-CoA pool acts as precursor material for TAL biosynthesis, although tightly regulated enzymes make it advantageous to engineer heterologous machinery coupled to pathway interventions. To circumvent the pyruvate dehydrogenase bypass pathway and major source of cytosolic acetyl-CoA, a modified Escherichia coli pyruvate dehydrogenase (PDH) producing NADPH was overexpressed. This variant PDH was combined with the ZWF1 deletion to block native NADPH biosynthesis and create a driving force through cofactor regeneration. Overexpressing E. coli PDH alone increased NADPH/NADP+ levels 1.7-fold, whereas the engineered PDH increased the cofactor ratio 3.7-fold, compared to the base strain. Both enzymes increased acetyl-CoA during the early phase of batch growth; however, only the variant PDH coupled to Δzwf1 increased NADPH/NADP+ and sustained acetyl-CoA levels up to 12-fold and 3-fold, respectively. These changes correlated with TAL increases. While the unmodified PDH achieved a 30% boost, 3.0-fold and 4.4-fold increases were obtained for the engineered PDH alone or coupled to the ZWF1 disruption, respectively. To ensure efficient utilization of the increased acetyl-CoA pool for TAL production, disruptions of genes shuttling pyruvate and acetyl-CoA into the mitochondria were evaluated, leading to a 2.5-fold increase in TAL titer. The coupling of all strategies into a single platform strain resulted in 1.6 g/L TAL at the highest reported yield of 0.16 g/g (36% of theoretical yield from glucose). This combined approach illustrates a promising step toward improving strain development for synthesis of acetyl-CoA derived natural products.

Pathway manipulations enhance acetyl-CoA supply for natural product synthesis

251st National Meeting (2016)

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