Sadaharu Ui

Production of l‐2,3‐butanediol by a new pathway constructed in Escherichia coli

Aims:  A metabolic pathway for l‐2,3‐butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce l‐BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and l‐2,3‐butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD‐comb.

Molecular generation of an Escherichia coli strain producing only the meso-isomer of 2,3-butanediol

Abstract Klebsiella pneumoniae IAM 1063 forms 2,3-butanediol consisting of a mixture of the meso- and l -isomers from glucose. A gene fragment including genes coding for three enzymes (ALS, ALDC, and meso-BDH (D-AC forming)) involved in the formation of meso-BD of K. pneumoniae IAM 1063 was cloned in Escherichia coli JM109 after its insertion into pUC118. The resulting E. coli JM109 pBDO118 was cultured in LB medium containing glucose and formed only a meso-isomer with no contamination of the L-form. Various culture conditions for the production of meso-BD were tested. The highest meso-BD yield of 27% (w/w, 10.7 g/l) against initially added glucose was obtained in a LB culture shaken for 24 h at 30°C in the presence of 4% glucose. However, re-conversion of meso-BD to AC occurred with time. The highest productivity of meso-BD was 17.7 g/l, which was obtained using an initial glucose concentration of 10%.

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

ABSTRACT The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.

Hydrogen Production by Termite Gut Protists: Characterization of Iron Hydrogenases of Parabasalian Symbionts of the Termite Coptotermes formosanus

ABSTRACT Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H2) that is emitted by the termites; however, little is known about the physiology and biochemistry of H2 production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H2 production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H2 evolution rather than H2 uptake. H2 evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H2 evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H2 uptake activity of the endosymbiotic bacteria compensated for the strong H2 evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H2 production in termite guts has been largely underestimated.

Sequence Analysis of the Gene for and Characterization of D-Acetoin Forming meso-2,3-Butanediol Dehydrogenase of Klebsiella pneumoniae Expressed in Escherichia coli

Analysis of the nucleotide sequence of the meso-2,3-butanediol dehydrogenase (d-acetoin forming) (BDH) gene of Klebsiella pneumoniae IAM1063 revealed that it contains an open reading frame of 768 bp encoding a polypeptide of 256 amino acid residues with a molecular weight of 26,591 Da. The amino acid sequence deduced from the nucleotide sequence of the BDH gene is consistent with the amino-terminal and carboxyl-terminal amino acid sequences, amino acid composition, and molecular weight of purified BDH. The amino acid sequences of BDHs from K. pneumoniae IAM1063 and Klebsiella terrigena VTT-E-74023 showed 92.9% homology in the first 196 amino acid residues, but no homology elsewhere. Moreover, the BDH of K. pneumoniae IAM1063 was 15 amino acid residues longer than that of K. terrigena VTT-E-74023. The characteristics of the BDH from Escherichia coli JM109pBUD119 (containing the meso-BDH gene) coincided with that of the BDH from the gene source strain (K. pneumoniae IAM1063). Furthermore, the BDHs from K. pneumoniae IAM1063 and K. terrigena VTT-E-74023 showed similar catalytic properties. This suggests that the downstream of Gln at position 196 in the amino acid sequence is unrelated to BDH activity.

Production of large multienzyme complex by aerobic thermophilic fungus Chaetomium sp. nov. MS-017 grown on palm oil mill fibre.

Aims:  A novel xylanolytic multienzyme complex of the aerobic thermophilic fungus Chaetomium sp. nov. MS‐017 was produced on palm oil mill fibre (POMF) and partially characterized.

Cloning, expression and nucleotide sequence of the l-2,3-butanediol dehydrogenase gene from Brevibacterium saccharolyticum C-1012

Abstract A 3-kbp DNA fragment including the l -2,3-butanediol dehydrogenase ( l -BDH) gene ( bud C) from the chromosomal DNA of Brevibacterium saccharolyticum C-1012 was cloned in Escherichia coli JM109 after its insertion into pBluescript II SK + , and the resulting plasmid was named pLBD-SK. The bud C had an open reading frame consisting of 774 bp and encoded 258 amino acids. It was not included in a 2,3-butanediol operon such as is seen in the case of the meso -BDH gene ( bud C) of Klebsiella pneumoniae . For the expression of the bud C, the deletion plasmid pLBD2-119 was prepared from pLBD-SK. E. coli JM109/pLBD2-119 had higher l -BDH activity than that of Br. saccharolyticum C-1012. The l -BDH appeared as two bands on disc-PAGE. Isopropyl-β- d -thiogalacto-pyranoside (IPTG) influenced the quantity radio of the electrophoretic isoenzymes of l -BDH from E. coli JM109/pLBD2-119 that is, a higher relative mobility band with weak substrate specificity was abundantly produced by IPTG. The BDH was considered to belong to the short-chain dehydrogenase/reductase (SDR) family on the basis of the following distinctive features: it possessed two conservative sequences GXXXGXG and YXXXK, and it consisted of about 250 amino acids. As a result of a phylogenetic analysis of SDR family enzymes, the BDHs were considered to comprise a cluster independent from the other SDR enzymes.

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3‐Butanediol dehydrogenase (BDH) catalyzes the NAD‐dependent redox reaction between acetoin and 2,3‐butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l‐BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso‐BDH highlights the role of a hydrogen‐bond from a conserved Trp residue192. Site‐directed mutagenesis of three active site residues of meso‐BDH, including Trp190, which corresponds to Trp192 of l‐BDH, converted its stereospecificity to that of l‐BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.

Characterization of the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase gene from Bacillus cereus YUF-4

A 1.4-kbp DNA fragment, including the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase (AACRII/BDH) gene from the chromosomal DNA of Bacillus cereus YUF-4, was cloned in Escherichia coli DH5alpha after its insertion into pUC119, and the resulting plasmid was named pAACRII119. The AACRII/BDH gene had an open reading frame consisting of 1047 bp encoding 349 amino acids. The enzyme exhibited not only AACR activity, but also BDH activity. However, the gene was not located in a 2,3-butanediol (BD) operon, as is the case in the BDH gene of Klebsiella pneumoniae and that of K. terrigena. In addition, there was no BD-cycle-related enzyme gene in the region surrounding the AACRII/BDH gene. The AACR and BDH activities in E. coli DH5alpha/pAACRII119 were 200-fold higher than those in the original B. cereus YUF-4. The characteristics of the AACRII/BDH from E. coli DH 5alpha/pAACRII119 are similar to those of the AACRII/BDH from B. cereus YUF-4. The AACRII/BDH was considered to belong to the NAD(P)- and zinc-dependent long-chain alcohol dehydrogenase (group I ADH) family on the basis of the following distinctive characteristics: it possessed 14 strictly conserved residues of microbial group I ADH and consisted of about 350 amino acids. The enzymatic and genetic characteristics of AACRII/BDH were completely different from those of BDHs belonging to the short-chain dehydrogenase/reductase family. These findings indicated that the AACRII/BDH could be considered a new type of BDH.

Discovery of a New Mechanism of 2, 3-Butanediol Stereoisomer Formation in Bacillus cereus YUF-4

Abstract A mechanism of 2,3-butanediol (BD) stereoisomer formation was examined with respect to the BD cycle. The enzymes acetylacetoin synthase, acetylacetoin reductase (AACR), and acetylbutanediol hydrolase (ABDH), which are part of the BD cycle, were found to be present in the cell-free extract of the bacterial strain Bacillus cereus YUF-4. Two kinds of acetylbutanediol (ABD) stereoisomers were produced in the reduction of acetylacetoin (AAC) by AACR, which were identified as having 3R,4R and 3S,4R configurations by NMR spectroscopy and an enzymic method. The two ABD formations were found to be catalyzed independently by two respective enzymes: the former was catalyzed by a NADPH-dependent AACR ( 3R,4R -ABD forming) and the latter by a NADH-dependent AACR ( 3S,4R -ABD forming). The 3R,4R -ABD was converted into R,R -BD and the 3S,4R -ABD into R,S -BD by intracellular ABDH. These findings demonstrated the existence of a new BD isomer formation mechanism derived from the BD cycle.