Tamotsu Hoshino

Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1

A 40-kbp DNA fragment was isolated from the cosmid library of Vibrio marinus strain MP-1. Among the 22 putative open reading frames (ORFs) in this fragment, ORFs 8, 9, 10 and 11 had high homology with ORFs 5, 6, 7 and 8 of the eicosapentaenoic acid biosynthesis gene cluster, respectively. Then, we speculate that these ORFs are responsible for docosahexaenoic acid biosynthesis in this bacterium.

Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature

A psychrotrophic bacterium producing a protease active at low temperatures was isolated from fish intestine and identified as a Pseudomonas species. Optimum growth and protease‐producing temperatures of this strain were 15°C and 10°C, respectively. The maximum temperature for proteolytic activity was 25°C, an unusually low temperature.

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis

Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold‐adapted organisms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus. We examined its biochemical properties and thermal hysteresis activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified as an extracellular protein of approximately 28 kDa, which halved in size following digestion with glycosidase. The A. psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity (for example thermal hysteresis = 0.42 °C for a 0.48 mm solution) similar to that of fish antifreeze proteins, while a unique rugged pattern was created on the facets of the ice bipyramid. The thermal hysteresis activity of the A. psychrotrophicus antifreeze protein was maximized under alkaline conditions, while that of the T. ishikariensis antifreeze protein was greatest under acidic conditions. The T. ishikariensis antifreeze protein exhibited a bursting ice growth normal to the c‐axis of the ice crystal and high thermal hysteresis activity (approximately 2 °C), as in the case of insect hyperactive antifreeze proteins. From these results, we speculate that the A. psychrotrophicus antifreeze protein is very different from the T. ishikariensis antifreeze protein, and that these two psychrophiles have evolved from different genes.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Microbial community structure, pigment composition, and nitrogen source of red snow in Antarctica.

Abstract“Red snow” refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly 15N-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.

Increasing n-3 polyunsaturated fatty acid content of fish oil by temperature control of lipase-catalyzed acidolysis

An attempt was made to further increase the content of n-3 polyunsaturated fatty acid (n-3 PUFA) of fish oil by lipase-catalyzed acidolysis (reaction between fish oil and n-3 PUFA-enriched free fatty acid) without solvent. A bioreactor system was constructed composed of a water-jacketed packed-bed column and a substrate reservoir with a circulation pipeline between the packed-bed column and the reservoir. By keeping the temperature of the reservoir at −10°C (for the first 20 h), followed by −20°C (for the subsequent 40 h) during the batch acidolysis, crystals of free fatty acid appeared, which were removed intermittently by a cotton plug packed in the tip of the outlet pipe in the reservoir. The n-3 PUFA content of the triacylglycerol fraction increased a further 10% by the reduced temperature of the reservoir.

Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance

ABSTRACT Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.

An Application of Wastewater Treatment in a Cold Environment and Stable Lipase Production of Antarctic Basidiomycetous Yeast Mrakia blollopis

Milk fat curdle in sewage is one of the refractory materials for active sludge treatment under low temperature conditions. For the purpose of solving this problem by using a bio-remediation agent, we screened Antarctic yeasts and isolated SK-4 strain from algal mat of sediments of Naga-ike, a lake in Skarvsnes, East Antarctica. The yeast strain showed high nucleotide sequence homologies (>99.6%) to Mrakia blollopis CBS8921T in ITS and D1/D2 sequences and had two unique characteristics when applied on an active sludge; i.e., it showed a potential to use various carbon sources and to grow under vitamin-free conditions. Indeed, it showed a biochemical oxygen demand (BOD) removal rate that was 1.25-fold higher than that of the control. We considered that the improved BOD removal rate by applying SK-4 strain was based on its lipase activity and characteristics. Finally, we purified the lipase from SK-4 and found that the enzyme was quite stable under wide ranges of temperatures and pH, even in the presence of various metal ions and organic solvents. SK-4, therefore, is a promising bio-remediation agent for cleaning up unwanted milk fat curdles from dairy milk wastewater under low temperature conditions.

Establishment of a novel gene expression method, BICES (biomass-inducible chromosome-based expression system), and its application to the production of 2,3-butanediol and acetoin.

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase-T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54gL(-1) [99.6% in (R,S)-form] and 31gL(-1), respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass.