Toshihiko Ooi

Improvement of Poly(3-Hydroxybutyrate) [P(3HB)] Production in Corynebacterium glutamicum by Codon Optimization, Point Mutation and Gene Dosage of P(3HB) Biosynthetic Genes

In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB Re) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C. glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC Re) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC Re with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaC Re and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C. glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA Re) and acetoacetyl-CoA reductase (PhaB Re). This finding prompted us to carry out a gene dosage of phaAB Re for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB Re with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(3HB) was increased by more than 4-fold compared with the non-P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

SummaryWa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme

Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34mol%) than that grown on glucose (26mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60mol% LA from xylose, while glucose gave a 47mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3gl(-1)) was remarkably higher than that of P(3HB) (4.1gl(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization.

β-Mannanase and xylanase of Bacillus subtilis 5H active for bleaching of crude pulp

A bacterial strain, 5H was isolated as a utilizer of locust bean gum (LBG) from soil in Chiang Mai, Thailand, and identified as Bacillus subtilis. Strain 5H produced β-mannanase and xylanase, which were purified to homogeneity from the culture filtrate by ion exchange chromatography and gel filtration. The molecular weights of β-mannanase and xylanase were found to be 37,000 and 26,000 by sodium dodecyl sulfonate polyacrylamide gel electrophoresis, respectively. The enzymatic properties of both were determined. Mannanase hydrolyzed LBG and konjak mannan endwisely, to a final hydrolytic degree of 15% and 21%, respectively, and the main products of both were disaccharides. Xylanase hydrolyzed larch wood xylan and oat spelt xylan, to a final hydrolytic degree of 19% and 38%, respectively, and produced mainly disaccharides. This culture filtrate (4.9 units/ml β-mannanase and 3.2 units/ml xylanase), a mixture of purified β-mannanase (4.9 units/ml) and xylanase (3.2 units/ml), and purified xylanase (3.2 units/ml) bleached crude paper pulp from 30% to 38% brightness, and xylanase was found to be more effective for paper-bleaching than β-mannanase.

Purification and properties of mutanase from Bacillus circulans

Abstract Bacterial strain HU-M1 was isolated as a mutan-degrading microbe from soil and identified as Bacillus circulans . From an HU-M1 culture supernatant mutanase was purified 260-fold by ammonium sulfate fractionation and DEAE-Toyopearl 650M (twice), Toyopearl HW55-F, and Butyl-Toyopearl 650M column chromatography with an activity recovery of 10%. The M r of the mutanae is 160,000 (monomer). The optimal pH and temperature for the mutanase are pH 6.9 and 50°C for a 10-min reaction. The mutanase is stable at up to 40°C for 60-min and at pH 6.0 to 11.0 at 4°C for 48 h, and logarithmically inactivated at 54°C with a half life of 2.6 min. Mutant hydrolysates produced by the enzyme had di-, tri-, and tetramer of glucose and the final hydrolysis was 38%.

Production of lactobionic acid from whey by Pseudomonas sp. LS13-1.

Pseudomonas sp. LS13-1 was isolated as a producer of lactobionic acid from whey and when grown with 207 g whey l−1 (150 g lactose l−1 equivalent) and three intermittent additions of 69 g whey l−1 (50 g lactose l−1 equivalent) in a fed-batch culture at pH 5.5 in a 2-l jar fermenter, it produced 175 g lactobionic acid l−1 after 180 h. In a lactose medium it produced 240 lactobionic acid l−1 from a total of 300 g lactose l−1 after 155 h. With the addition of 20 CaCO3 l−1 instead of pH control, 290 g lactobionic acid l−1 was produced in the lactose medium after 155 h with a yield of higher than 90% (mon mol−1).

Polyhydroxyalkanoates production from cellulose hydrolysate in Escherichia coli LS5218 with superior resistance to 5-hydroxymethylfurfural

Poly[3-hydroxybutyrate-co-3-hydroxyvalerate(3HV)] was produced in recombinant Escherichia coli LS5218 from ruthenium-catalyzed cellulose hydrolysate and propionate. The strain was found to be resistant to 5-hydroxymethylfurfural (5-HMF), which is a major inhibitory byproduct generated in the cellulose hydrolysis reaction. The 3HV fraction was successfully regulated in the range of 5.6-40 mol%.

Secretive expression of the Aspergillus aculeatus cellulase (FI-CMCase) by Saccharomyces cerevisiae☆

A cDNA encoding FI-carboxymethylcellulase (FI-CMCase) of the fungus Aspergillus aculeatus was expressed in Saccharomyces cerevisiae under the control of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) promoter of S. cerevisiae. The transformed cells were able to secrete FI-CMCase efficiently into the culture medium as active enzyme. The recombinant FI-CMCases were observed to be two different enzymes of different molecular mass, one of which corresponded to native FI-CMCase (non-glycosylated FI-CMCase) and the other of which was an N-glycosylated protein (glycosylated FI-CMCase). The recombinant glycosylated FI-CMCase showed a higher thermostability than that of the native enzyme, although the former showed slightly lower activity toward the substrate than the latter.

An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.

The Escherichia coli outer‐membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2′. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon‐like peptide‐1 (7–37) in 4 M urea. The P1–P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The kcat/Km ratio increased with basic residues at P4 or P6, mainly due to a lower Km rather than an increase in kcat. On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli‐expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.

Degradation of synthetic water-soluble polymers by hydroquinone peroxidase

Abstract The hydroquinone peroxidase of the lignin decolorizing bacterium, Azotobacter beijerinckii HM121 degraded 1 g/ l of polyacrylamide ( M r 2,000,000) to small molecules ( M r less than one thousand) in 30 min in the presence of 5 mM hydrogen peroxide and 5 μM tetramethylhydroquinone. The two reaction products were purified, and identified as 1-dodecene-2, 4, 6, 8, 10-pentacarboxyamide and 1-hexadecene-2, 4, 6, 8, 10, 12, 14-heptacarboxyamide comprising 5 and 7 acrylamide units, respectively. The enzyme also degraded 1 g/ l of polyacrylic acid ( M r 450,000) in 1 h, 1 g/ l of polyethylene glycol ( M r 4,000,000) in 1 h, and 1 g/ l of polyvinyl alcohol ( M r 88,000) for 20 h. The degradation rate decreased with a decrease in the degree of polymerization of the polyethylene glycol from M r 20,000 to 400. In addition 1 g/ l of polyethylene glycol ( M r 4,000,000) was degraded in the absence of tetramethylhydroquinone, although it took 20 h.