Katsuji Murakami

Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae.

In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates.

Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

BackgroundBioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.ResultsWe compared A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and β-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield) and rice straw (43% yield). Although the commercial enzymes Acremonium cellulase (derived from A. cellulolyticus, Meiji Seika Co.) demonstrated a slightly lower cellulase specific activity than Accellerase 1000 (derived from T. reesei, Genencor), the glucose yield (over 65%) from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%). In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient.ConclusionWe investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We found that glucan-hydrolyzing activity of the culture supernatant from A. cellulolyticus was superior to that from T. reesei, while the xylan-hydrolyzing activity was superior for the cellulase from T. reesei. Moreover, Acremonium cellulase exhibited a greater glucan and mannan-hydrolyzing activity than Accellerase 1000.

Production of free and organic iodine by Roseovarius spp.

Two strains of iodine-producing bacteria were isolated from marine samples. 16S rRNA gene sequences indicated the strains were most closely related to Roseovarius tolerans, and phylogenetic analysis indicated both belong to the same genus. 5 mM iodide inhibited the growth of strain 2S5-2 almost completely, and of strain S6V slightly. Both strains produced free iodine and organic iodine from iodide. CH2I2, CHI3 and CH2ClI were the main organic iodines produced by strain 2S5-2, and CHI3 and CH2I2 by strain S6V. Experiments using cells and spent media suggested that the organic iodines were produced from the compounds released or contained in the media and cells were necessary for the considerable production of CH2I2 and CH2ClI, though CHI3 was produced by spent media with H2O2 or free iodine.

Degradation of triphenyltin by a fluorescent pseudomonad.

ABSTRACT Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3′,4′,7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 μM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 μM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads,P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosaATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.

Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis

ABSTRACT The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu2+ and Sn4+, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.

Utilization of Dimethyl Sulfide as a Sulfur Source with the Aid of Light by Marinobacterium sp. Strain DMS-S1

ABSTRACT Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related toMarinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.

Isolation of uracil auxotrophs of the fungus Acremonium cellulolyticus and the development of a transformation system with the pyrF gene.

Acremonium cellulolyticus CF-2612 is a cellulase hyper-producing mutant that originated from A. cellulolyticus Y-94. In this study, we isolated a uracil auxotroph (strain CFP3) derived from CF-2612, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicate that our transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successful.

Cellulase Hyperproducing Mutants Derived from the Fungus Trichoderma reesei QM9414 Produced Large Amounts of Cellulase at the Enzymatic and Transcriptional Levels

Cellulase hyperproducing mutants derived from the fungus Trichoderma reesei QM9414 were analyzed. They exhibited higher filter-paper degrading activity and a lower growth rate than the wild-type QM9414 strain. Transcription of the cellobiohydrolase I and endoglucanase I genes in the mutants was also greater than that of QM9414, suggesting that cellulase hyperproduction by these mutants was regulated at the transcriptional level.

Isolation of Thermophilic Acetogens and Transformation of Them with the pyrF and kanr Genes

The application of microbial catalysts to syngas from the gasification of lignocellulosic biomass is gaining interest. Acetogens, a group of anaerobic bacteria, can grow autotrophically on gaseous substrates such as hydrogen and carbon dioxide or syngas and produce acetate via the acetyl-CoA pathway. Here, we report the isolation from a soil sample of two thermophilic acetogen strains, Y72 and Y73, that are closely related to Moorella sp. HUC22-1 and M. thermoacetica ATCC39073. The optimal growth temperature and pH for the strains were 60 °C and 6.0-6.5. Uracil auxotrophy was induced in them by replacing the orotate monophosphate decarboxylase gene (pyrF) with the kanamycin resistant marker (kan(r)). The transformants were isolated by supplementation of the basal medium with 300 mg/L of kanamycin. The transformation efficiency of strains Y72 and Y73 was 20-fold higher than that of strain ATCC39073. Hence these strains are considered possible hosts for thermophilic syngas fermentation.

Ethanol Production from Xylo-oligosaccharides by Xylose-Fermenting Saccharomyces cerevisiae Expressing β-Xylosidase

Construction of xylose- and xylo-oligosaccharide-fermenting Saccharomyces cerevisiae strains is important, because hydrolysates derived from lignocellulosic biomass contain significant amounts of these sugars. We have obtained recombinant S. cerevisiae strain MA-D4 (D-XKXDHXR), expressing xylose reductase, xylitol dehydrogenase and xylulokinase. In the present study, we generated recombinant strain D-XSD/XKXDHXR by transforming MA-D4 with a β-xylosidase gene cloned from the filamentous fungus Trichoderma reesei. The intracellular β-xylosidase-specific activity of D-XSD/XKXDHXR was high, while that of the control strain was under the limit of detection. D-XSD/XKXDHXR produced ethanol, and xylose accumulated in the culture supernatant under fermentation in a medium containing xylo-oligosaccharides as sole carbon source. β-Xylosidase-specific activity in D-XSD/XKXDHXR declined due to xylose both in vivo and in vitro. D-XSD/XKXDHXR converted xylo-oligosaccharides in an enzymatic hydrolysate of eucalyptus to ethanol. These results indicate that D-XSD/XKXDHXR efficiently converted xylo-oligosaccharides to xylose and subsequently to ethanol.