Reiko Miyata

Fermentative production of pyruvate from glucose by Torulopsis glabrata

Abstract Novel strains selected from various genera of yeasts were investigated with regard to pyruvate productivity from glucose. The ability to produce pyruvate was found in yeasts belonging to the genera Saccharomyces, Toluropsis, Pichia, Candida and Trichosporon with a new resting cell method using the thiamine analogue, oxythiamine. In this first screening, strains belonging to the genus Saccharomyces showed the highest pyruvate productivity. In secondary screening using a fermentative method, strains belonging to the genus Torulopsis showed higher pyruvate productivity, with Torulopsis glabrata exhibiting the highest fermentative pyruvate productivity. These pyruvate producing yeast strains were divided into two types. The first type of yeast showed a high accumulation of pyruvate with the resting cell method, but a low accumulation with the fermentative method. The second type of yeast showed a high accumulation of pyruvate with the fermentative method, but a low accumulation with the resting cell method. The first type did not commonly possess thiamine-auxotrophy, while the second type exhibited at least double auxotrophy for both biotin and thiamine. All six strains belonging to T. glabrata required four vitamins; nicotinic acid, thiamine, pyridoxine and biotin for their growth and accumulated a high conversion of pyruvate (53.7 mM–300 mM) under the fermentative screening conditions. T. glabrata IFO 0005 was selected as the best strain among the T. glabrata. The optimum concentrations of these vitamins for the highest fermentative pyruvate productivity by T. glabrata IFO 0005 were determined. Thiamine and nicotinate were more effective towards productivity than other vitamins. The accumulation of pyruvic acid reached a final concentration of 648 mM (57.0 g/l as free pyruvic acid) at 59 h from an initial glucose concentration of 100 g/l by T. glabrata IFO 0005 under the optimal vitamin concentrations determined in this study. Consequently, the auxotrophy of multiple vitamins which are involved in the metabolism of pyruvate proved to be highly effective in the fermentative production of pyruvate from glucose.

Improvement of fermentative production of pyruvate from glucose by Torulopsis glabrata IFO 0005

Efficient production of pyruvate from glucose by Torulopsis glabrata IFO 0005 was investigated. A large amount of pyruvate was accumulated in a practical semisynthetic fermentation medium which contained both ammonium sulfate and soybean hydrolyzate as nitrogen sources, instead of the Polypepton used in the previous study. A significant increase in the maximum amount of pyruvate accumulated was noted in a fed-batch culture in a jar fermentor. The fermentative conditions were optimized in an Erlenmeyer flask, and then in a jar fermentor in this study. Of the fermentative conditions, the concentrations of four vitamins (nicotinic acid, thiamine, pyridoxine and biotin) and aeration affected the pyruvate fermentative productivity. The optimum concentration of nicotinic acid was 8 mg/l, which was 20 times higher than that required for full growth. The optimum concentration of thiamine was 0.03 mg/l, which is 40% lower than that for full growth. Pyruvate production was markedly dependent on aeration. The maximum final accumulation of pyruvic acid was achieved under anaerobic conditions, but a greater yield of pyruvic acid was obtained under aerobic conditions. Based on the optimum conditions in an Erlenmeyer flask, the optimal initial and feeding conditions in a jar fermentor were determined. Furthermore, we observed that cells grown under aerobic conditions developed mitochondria well compared with ones grown under anaerobic conditions, and investigated the effects of inhibitors, such as rotenone, of the electron transport chain, which exists in mitochondria, on pyruvate fermentation. The amount of pyruvate accumulated grown in the presence of 1.0 mM rotenone was 20% less than that accumulated grown in its absence by T. glabrata IFO 0005. Consequently, fed-batch fermentation with successive additions of glucose resulted in a maximum accumulation of 770 mM (67.8 g/l) pyruvic acid in 63 h (yield, 49.4%). The by-products were found to be 4 g/l of ethanol, 5 g/l of glycerol and 2 g/l of para-pyruvic acid.

Breeding of high-pyruvate-producing Torulopsis glabrata with acquired reduced pyruvate decarboxylase

An efficient method for isolation of pyruvate-producing mutants of Torulopsis glabrata IFO 0005 was established. On mutagenesis of the parent strain, mutants requiring acetate for complete growth were isolated. The acetate-leaky auxotrophic mutants demonstrated higher fermentative yields of pyruvate from glucose than the parent strain. Thus, the pyruvate decarboxylase (PDC) activity of mutants requiring acetate for complete growth was lower than that of the parent strain. A decrease in the PDC activity would therefore block the formation of acetate via acetaldehyde and thus increase pyruvate production. Among the mutants, T. glabrata ACII-33 had accumulated free pyruvic acid (60.3 g/l) in 47 h (yield, 67.3%; conversion of pyruvic acid from glucose consumed) in a 3 l jar-fermentor. This yield by strain ACII-33 represented at 15% increase compared to that with the parent strain.

Breeding of high-pyruvate-producing Torulopsis glabrata and amino acid auxotrophic mutants

An efficient method for the isolation of pyruvate-producing mutants of Torulopsis glabrata IFO 0005 was established. On mutagenesis of the parent strain, mutants requiring polypeptone for complete growth were isolated. Seven mutants among one hundred auxotrophs produced higher concentrations of pyruvate than the parent strain. Among them, arginine (L-Arg) auxotrophic mutants and an isoleucine (L-Ile) and valine (L-Val) double auxotrophic mutant exhibited higher fermentative production of pyruvate from glucose than the parent strain. T. glabrata X-15 and X-17 required L-Arg for complete growth. T. glabrata X-68 absolutely required L-Ile and L-Val for complete growth. These three strains (X-15, X-17 and X-68) have more than 10% higher yields of pyruvate than the parent strain. Among them, the best strain regarding pyruvate productivity, T. glabrata X-15, accumulated 59.5 g/l free pyruvic acid (yield, 60.1%; conversion to pyruvic acid of added glucose in 43 h) on a 3-l jar-fermentor scale. This yield with strain X-15 represented a 12% increase compared to that obtained with the parent strain.