Thiamine-Starved Lactococcus lactis for Producing Food-Grade Pyruvate

Lactococcus lactis is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, L. lactis can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of L. lactis into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient L. lactis strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and alpha-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an alpha-acetolactate decarboxylase deficient L. lactis strain, pyruvate could be converted to alpha-acetolactate and diacetyl. Summing up, by starving L. lactis for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.