Kyoto Industrial Science and Technology Innovation Center

Kyoto, Japan

Kyoto Industrial Science and Technology Innovation Center

Kyoto, Japan
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Mikami Y.,Kyoto University | Yoneda H.,Kyoto University | Tatsukami Y.,Kyoto University | Tatsukami Y.,Japan Society for the Promotion of Science | And 4 more authors.
AMB Express | Year: 2017

The demand for ammonia is expected to increase in the future because of its importance in agriculture, industry, and hydrogen transportation. Although the Haber–Bosch process is known as an effective way to produce ammonia, the process is energy-intensive. Thus, an environmentally friendly ammonia production process is desired. In this study, we aimed to produce ammonia from amino acids and amino acid-based biomass-like resources by modifying the metabolism of Escherichia coli. By engineering metabolic flux to promote ammonia production using the overexpression of the ketoisovalerate decarboxylase gene (kivd), derived from Lactococcus lactis, ammonia production from amino acids was 351 mg/L (36.6% yield). Furthermore, we deleted the glnA gene, responsible for ammonia assimilation. Using yeast extract as the sole source of carbon and nitrogen, the resultant strain produced 458 mg/L of ammonia (47.8% yield) from an amino acid-based biomass-like material. The ammonia production yields obtained are the highest reported to date. This study suggests that it will be possible to produce ammonia from waste biomass in an environmentally friendly process. © 2017, The Author(s).

Matsui K.,Kyoto University | Bae J.,Kyoto University | Esaka K.,Kyoto University | Morisaka H.,Kyoto University | And 4 more authors.
Applied and Environmental Microbiology | Year: 2013

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates. © 2013, American Society for Microbiology.

Tatsukami Y.,Kyoto University | Nambu M.,Kyoto University | Morisaka H.,Kyoto University | Morisaka H.,Kyoto Industrial Science and Technology Innovation Center | And 3 more authors.
BMC Microbiology | Year: 2013

Background: Rhizobia are symbiotic nitrogen-fixing soil bacteria that show a symbiotic relationship with their host legume. Rhizobia have 2 different physiological conditions: a free-living condition in soil, and a symbiotic nitrogen-fixing condition in the nodule. The lifestyle of rhizobia remains largely unknown, although genome and transcriptome analyses have been carried out. To clarify the lifestyle of bacteria, proteome analysis is necessary because the protein profile directly reflects in vivo reactions of the organisms. In proteome analysis, high separation performance is required to analyze complex biological samples. Therefore, we used a liquid chromatography-tandem mass spectrometry system, equipped with a long monolithic silica capillary column, which is superior to conventional columns. In this study, we compared the protein profile of Mesorhizobium loti MAFF303099 under free-living condition to that of symbiotic conditions by using small amounts of crude extracts. Result: We identified 1,533 and 847 proteins for M. loti under free-living and symbiotic conditions, respectively. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many of the enzymes involved in the central carbon metabolic pathway were commonly detected under both conditions. The proteins encoded in the symbiosis island, the transmissible chromosomal region that includes the genes that are highly upregulated under the symbiotic condition, were uniquely detected under the symbiotic condition. The features of the symbiotic condition that have been reported by transcriptome analysis were confirmed at the protein level by proteome analysis. In addition, the genes of the proteins involved in cell surface structure were repressed under the symbiotic nitrogen-fixing condition. Furthermore, farnesyl pyrophosphate (FPP) was found to be biosynthesized only in rhizobia under the symbiotic condition. Conclusion: The obtained protein profile appeared to reflect the difference in phenotypes under the free-living and symbiotic conditions. In addition, KEGG pathway analysis revealed that the cell surface structure of rhizobia was largely different under each condition, and surprisingly, rhizobia might provided FPP to the host as a source of secondary metabolism. M. loti changed its metabolism and cell surface structure in accordance with the surrounding conditions. © 2013 Tatsukami et al.; licensee BioMed Central Ltd.

Nambu M.,Kyoto University | Tatsukami Y.,Kyoto University | Tatsukami Y.,Japan Society for the Promotion of Science | Morisaka H.,Kyoto University | And 4 more authors.
Journal of Proteomics | Year: 2015

Rhizobia are nitrogen-fixing bacteria that establish a symbiotic relationship with leguminous plants. To understand the mechanism by which rhizobia alter their metabolism to establish successful nitrogen-fixing symbiotic relationship with hosts, Lotus japonicus were inoculated with Mesorhizobium loti. Bacteroids were isolated from nodules harvested at 2. weeks (the early stage of nodule development), and at 3 and 4. weeks (the intermediate stage of nodule development) post-inoculation. Using a quantitative time-course proteome analysis, we quantified the variations in the production of 537 proteins in M. loti bacteroids during the course of nodule maturation. The results revealed significant changes in the carbon and amino acid metabolisms by M. loti upon differentiating into bacteroids. Furthermore, our findings suggested that M. loti enters a nitrogen-deficient condition during the early stages of nodule development, and then a nitrogen-rich condition during the intermediate stages of nodule development. In addition, our data indicated that M. loti assimilated ammonia during the intermediate stages of nodule development. Our results provide new insights into the course of physiological transitions undergone by M. loti during nodule maturation. © 2015 Elsevier B.V.

Shinohara M.,Kyoto University | Sakuragi H.,Kyoto University | Morisaka H.,Kyoto University | Morisaka H.,Kyoto Industrial Science and Technology Innovation Center | And 6 more authors.
AMB Express | Year: 2013

Clostridium cellulovorans has been one of promising microorganisms to use biomass efficiently; however the basic metabolic pathways have not been completely known. We carried out 13C-isotopomer-based target metabolome analysis, or carbohydrate conversion process analysis, for more profound understanding of metabolic pathways of the bacterium. Our findings that pyruvate + oxaloacetate, fumarate, and malate inside and outside cells exhibited 13C incorporation suggest that C. cellulovorans exactly fixed CO2 and partly operated the TCA cycle in a reductive manner. Accompanied with CO2 fixation, the microorganism was also found to produce and secrete lactate. Overall, our study demonstrates that a part of C. cellulovorans metabolic pathways related to glycolysis and the TCA cycle are involved in CO2 fixation. © 2013 Shinohara et al.

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