A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

作者：Ishii Jun^*; Morita Keisuke; Ida Kengo; Kato Hiroko; Kinoshita Shohei; Hataya Shoko; Shimizu Hiroshi; Kondo Akihiko; Matsuda Fumio^*

来源：Biotechnology for Biofuels, 2018, 11(1): 180.

DOI：10.1186/s13068-018-1176-y

• Pyruvate flux
• Crabtree effect
• Acetolactate synthase
• Ethanol subgeneration
• 2,3-Butanediol production
• Pyruvate decarboxylase (PDC) deficient

Background: The 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.
Results: By 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 (PDC Delta) 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 PDC Delta strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDC Delta 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.
Conclusions: We 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.

• 出版日期2018-6-26