Tetsu Yonehara

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.

Abstract 519: Identification of a novel target for antibody therapy of breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Identification of breast cancer-specific antigens may promote development of safe and successful immunotherapy of breast cancer. Caprin-1 has been known to be an intracellular protein related to cell proliferation as a function, but it has not been reported to be a target for the treatment of cancer. Here we report that this molecule is a novel breast cancer antigen which can be targeted for antibody therapy. Caprin-1 is highly expressed in at least 70% of breast cancers based on the results of immunohistochemical staining of human several breast cancer tissues, but hardly expressed in human normal tissues. Importantly, a part of this molecule is highly expressed in extracellular region of many breast cancer cells. A mouse monoclonal antibody raised against the extracellular domain of Caprin-1 showed anti-tumor effects in all tumor-bearing mouse models in which various cancer cells expressing Caprin-1 were implanted. The monoclonal antibody induced complete regression in one of the tumor-bearing models. Humanized antibody has been prepared and preclinical studies are currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 519. doi:1538-7445.AM2012-519

Membrane-Integrated Fermentation System for Improving the Optical Purity of D-Lactic Acid Produced during Continuous Fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.

A membrane-integrated fermentation reactor system: its effects in reducing the amount of sub-raw materials for D-lactic acid continuous fermentation by Sporolactobacillus laevolacticus.

Continuous fermentation by retaining cells with a membrane-integrated fermentation reactor (MFR) system was found to reduce the amount of supplied sub-raw material. If the amount of sub-raw material can be reduced, continuous fermentation with the MFR system should become a more attractive process for industrialization, due to decreased material costs and loads during the refinement process. Our findings indicate that the production rate decreased when the amount of the sub-raw material was reduced in batch fermentation, but did not decrease during continuous fermentation with Sporolactobacillus laevolacticus. Moreover, continuous fermentation with a reduced amount of sub-raw material resulted in a productivity of 11.2 g/L/h over 800 h. In addition, the index of industrial process applicability used in the MFR system increased by 6.3-fold as compared with the conventional membrane-based fermentation reactor previously reported, suggesting a potential for the industrialization of this D-lactic acid continuous fermentation process.

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.

ATP production by an AMP deaminase-impaired mutant of a methanol yeast, Candida boidinii

Abstract A mutant strain, which was impaired with respect to its ability to grow on AMP as the sole nitrogen source, was derived from Candida boidinii ( Kloeckera sp.) no. 2201. AMP deaminase of the mutant strain exhibited the same allosteric patterns as those of the wild type strain. The specific activity in the cell-free extract of the mutant strain, however, was lower than that of the wild type strain. Sorbitol-treated cells of the mutant strain produced ATP from AMP at a high conversion rate (95 mol%).