Abstract
BackgroundThe yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive S. cerevisiae invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound.ResultsBy searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by S. cerevisiae. To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography–tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g−1 glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L−1) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source.ConclusionsWe demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive S. cerevisiae. High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by S. cerevisiae.
Highlights
The yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals
ILV2 and ILV2c genes (Fig. 1a, b) were used as positive controls to assess the capabilities of the acetolactate synthase (ALS) enzymes
In this study, we demonstrated that a strategy for tugging carbon flux can increase 2,3-BDO production and reduce ethanol subgeneration in Crabtree-positive S. cerevisiae
Summary
The yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound. The metabolic engineering of S. cerevisiae to produce various chemicals generally faces a common challenge: how to reduce dominant ethanol production [19]. The mechanism of this glycolytic overflow metabolism is not completely understood, it is assumed to involve the redox homeostasis of NADH between the glycolytic and fermentative (ethanol) pathways [20, 21], in which a net of two NADH molecules are generated from one glucose molecule via glycolysis and subsequently oxidized in the ethanol formation reaction [22]) (Fig. 1a). An in silico metabolic simulation suggested that the greater difficulty in metabolically engineering the eukaryote S. cerevisiae to produce higher alcohol concentrations compared to the prokaryote Escherichia coli is due to the structurally limited flexibility of the central metabolism and mitochondrial compartmentation of eukaryotes [24]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call