Hideki Sawai

Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation

Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.

Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l-lactate production by Saccharomyces cerevisiae

In order to determine whether transcriptome data obtained by DNA microarray analysis could be used to identify the genes involved in target metabolite production, we tried to identify the genes involved in l-lactate production by l-lactate-producing recombinant Saccharomyces cerevisiae strains. We obtained DNA microarray data for these strains. Plasmids carrying lactic acid bacteria, bovine, and human l-lactate dehydrogenase (LDH) genes were introduced into PDC1-disrupted S. cerevisiae strains. l-Lactate productivity of the strains harboring the human and bovine LDH genes was higher than that of the strains harboring lactic acid bacteria LDH genes. DNA microarray analysis revealed that the expression of 388 genes was significantly altered in the strains with the human and bovine LDH genes. Of these, the l-lactate productivity of human LDH-harboring deletion strains of 289 genes was compared with that of the standard and 56 randomly selected deletion strains containing the same LDH gene to validate the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production in the recombinant strains. Only deletion strains of the genes selected on the basis of the DNA microarray data showed significantly altered l-lactate production as compared to the standard and the randomly selected deletion strains. Our results indicated that the genes related to l-lactate production could be successfully identified by selecting the genes that exhibited significantly altered expression on DNA microarray analysis, and the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production was discussed.

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.

A novel membrane-integrated fermentation reactor system: application to pyruvic acid production in continuous culture by Torulopsis glabrata

This paper describes the performance of a novel bio-reactor system, the membrane-integrated fermentation reactor (MFR), for efficient continuous fermentation. The MFR, equipped with an autoclavable polyvinylidene difluoride membrane, has normally been used for biological wastewater treatment. The productivity of the MFR system, applied to the continuous production of pyruvic acid by the yeast Torulopsis glabrata, was remarkably high. The volumetric productivity of pyruvic acid increased up to 4.2xa0g/l/h, about four times higher than that of batch fermentation. Moreover, the membrane was able to filter fermentation broth for more than 300xa0h without fouling even though the cell density of the fermentation broth reached 600 as OD660. Transmembrane pressure, used as an indicator of membrane fouling, remained below 5xa0kPa throughout the continuous fermentation. These results clearly indicate that the MFR system is a simple and highly efficient system that is applicable to the fermentative production of a range of biochemicals.