ABSTRACT
Two vectors, pT7NScamAB and pRED, have been used for the functional expression of bacterial class I cytochrome P450 (P450) genes in Escherichia coli, which utilize putidaredoxin reductase (CamA) and putidaredoxin (CamB), and the reductase domain of a self-sufficient P450RhF respectively, for electron transfer from NAD(P)H to a P450 protein. We here compared the efficiency of bioconversion with the two vectors towards n-octane, cyclohexane, n-butylbenzene, and 2-n-butylbenzofuran using two well-characterized CYP153A genes, aciA and CYP153A13a (P450balk). As for n-octane bioconversion, aciA and pT7camAB was the best combination for the production of 1-octanol and 1,8-octanediol. As for the bioconversion of cyclohexane, n-butylbenzene and 2-n-butylbenzofuran, CYP153A13a with pRED achieved the most efficient bioconversion, as compared by conversion ratio per active CYP153A protein content. It was also found that 2-n-butylbenzofuran is biotransformed into 4-benzofuran-2-yl-butyric acid via 4-benzofuran-2-yl-butan-1-ol with E. coli cells expressing CYP153A.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Genes, Bacterial , Genetic Vectors/genetics , Amino Acid Sequence , Benzofurans/metabolism , Cell Survival , Cyclohexanes/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Gene Expression , Molecular Sequence Data , Octanes/metabolismABSTRACT
Our biotransformation using Escherichia coli expressing a cytochrome P450 (CYP) belonging to the CYP153A family from Acinetobacter sp. OC4 produced a great amount of 1-octanol (2,250 mg per liter) from n-octane after 24 h of incubation. This level of production is equivalent to the maximum level previously achieved in biotransformation experiments of alkanes. In addition, the initial production rate of 1-octanol was maintained throughout the entire incubation period. These results indicate that we have achieved the functional and stable expression of a CYP in E. coli for the first time. Further, our biotransformation system showed alpha,omega-diterminal oxidation activity of n-alkanes, and a large amount of 1,8-octanediol (722 mg per liter) was produced from 1-octanol after 24 h of incubation. This is the first report on the bioproduction of alpha,omega-alkanediols from n-alkanes or 1-alkanols.
Subject(s)
Acinetobacter/classification , Acinetobacter/enzymology , Alkanes/chemistry , Alkanes/metabolism , Cytochrome P-450 Enzyme System/metabolism , Escherichia coli/metabolism , Mixed Function Oxygenases/metabolism , Acinetobacter/genetics , Cell Extracts , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/isolation & purification , Enzyme Stability , Escherichia coli/genetics , Gene Expression , Gene Expression Regulation, Enzymologic , Isomerism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/isolation & purification , Octanes/metabolismABSTRACT
A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested. The occurrence of GFL was studied at three stages during fruiting body formation. The largest quantity of hemagglutinating activity was found in the fruiting body, and lesser amounts in the mycelial mat and the primordium. The 24-kDa band of GFL was found at all three stages, and the band-intensity corresponded to the level of activity in each sample. By cloning and sequencing the GFL-cDNA, the primary structure of this lectin was determined. GFL is composed of 181 amino acids, having no signal peptide. The amino acid sequence was found to be homologous to those of so-called jacalin-related plant lectins, suggesting that GFL is the first example of a jacalin-related lectin of fungal origin.
Subject(s)
Basidiomycota/metabolism , Grifola/chemistry , Lectins/chemistry , Plant Lectins/metabolism , Amino Acid Sequence , Basidiomycota/chemistry , Carbohydrate Metabolism , Chromatography, Ion Exchange , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , Databases, Genetic , Electrophoresis, Polyacrylamide Gel , Hemagglutination Tests , Lectins/biosynthesis , Lectins/genetics , Molecular Sequence Data , Molecular Weight , Sequence HomologyABSTRACT
cDNA of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was expressed in Pichia pastoris, and the expression product was purified and characterized. The product was functional, and the hemagglutinating activity was inhibited most strongly by the addition of N-acetyl-D-galactosamine as was the native lectin. The native lectin is a glycoprotein having five glycosylation recognition signals, and the expression product showed slightly larger molecular mass than that of the native one due to further glycosylation.
Subject(s)
Lectins/genetics , Lectins/metabolism , Pichia/genetics , Pleurotus/chemistry , Carbohydrate Metabolism , DNA, Complementary/genetics , Glycosylation , Hemagglutination Inhibition Tests , Lectins/chemistry , Lectins/isolation & purification , Pleurotus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
By cloning and sequencing cDNA, the primary structure of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was determined. The amino acid sequence was novel and elucidated unique properties of this lectin: It was composed of 373 amino acids, 33 of which constitute a signal sequence. The sequence of the mature lectin consisted of two homologous regions having five glycosylation recognition signals and six cysteine residues. However, the distribution of these elements in the two regions was biased. Expression of cDNA in Escherichia coli and Pichia pastoris revealed the requirement of glycosylation to produce the functional lectin. Gel filtration followed by gel electrophoretic analyses of the purified lectin showed that the active component moved faster than the bulk of the protein, suggesting that the most active lectin formed an oligomer of subunits through disulfide bonds. From these observations, a model for the structure of the active form of this lectin is proposed. Southern hybridization using the cDNA as a probe revealed the presence of several genes. The lectin gene was composed of five exons and five introns.
