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Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst. [ABSTRACT FROM AUTHOR]

(−)-vibo-Quercitol is a deoxyinositol (1L-1,2,4/3,5-cyclohexanepentol) that occurs naturally in low concentrations in oak species, honeydew honey, and Gymnema sylvestre. The author’s research group recently reported that (−)-vibo-quercitol and scyllo-quercitol (2-deoxy-myo-inositol, 1,3,5/2,4-cyclohexanepentol), a stereoisomer of (−)-vibo-quercitol, are stereoselectively synthesized from 2-deoxy-scyllo-inosose by the reductive reaction of a novel (−)-vibo-quercitol 1-dehydrogenase in Burkholderia terrae and of a known scyllo-inositol dehydrogenase in Bacillus subtilis, respectively. The author’s research group therefore identified two enzymes capable of producing both stereoisomers of deoxyinositols, which are rare in nature. (−)-vibo-Quercitol and scyllo-quercitol are potential intermediates for pharmaceuticals. In this review, the author describes the biosynthesis and enzymatic production of quercitols and myo-inositol stereoisomers and their application in the production of potential pharmaceuticals. [ABSTRACT FROM AUTHOR]

(−)- vibo-Quercitol is a deoxyinositol (1 l-1,2,4/3,5-cyclohexanepentol) that naturally occurs in oak species, honeydew honey, wines aged in oak barrels, and Gymnema sylvestre and is a potential intermediate for pharmaceuticals. We found that (−)- vibo-quercitol is stereoselectively synthesized from 2-deoxy- scyllo-inosose by the reductive reaction of a novel (−)- vibo-quercitol 1-dehydrogenase found in the proteomes of Burkholderia, Pseudomonas, and Arthrobacter. Among them, Burkholderia terrae sp. AKC-020 (40-1) produced a (−)- vibo-quercitol 1-dehydrogenase appropriate for synthesizing (−)- vibo-quercitol with a high diastereomeric excess. The enzyme was strongly induced in Bu. terrae cells when quercitol or 2-deoxy- scyllo-inosose was used as carbon source in the culture medium. The enzyme is NAD(H)-dependent and shows highly specific activity for (−)- vibo-quercitol and myo-inositol among the substrates tested. The enzyme gene ( qudh) was obtained by PCR using degenerate primers based on the N-terminal and internal amino acid sequences of the purified enzyme, followed by thermal asymmetric interlaced PCR. The qudh gene showed homology with inositol 2-dehydrogenase (sharing 49.5% amino acid identity with IdhA from Sinorhizobium meliloti 1021). We successfully produced several recombinant (−)- vibo-quercitol 1-dehydrogenases and related enzymes identified by genome database mining using an Escherichia coli expression system. This revealed that scyllo-inositol dehydrogenase (IolX) in Bacillus subtilis can catalyze the reduction of 2-deoxy- scyllo-inosose to yield scyllo-quercitol, a stereoisomer of (−)- vibo-quercitol. Thus, we successfully identified two enzymes to produce both stereoisomers of deoxyinositols that are rare in nature. [ABSTRACT FROM AUTHOR]

The published online version contains mistake in the chemical structure of scyllo-inosose in Fig. 5 and Fig. 7. The correct configuration of 1-hydroxyl group in scyllo-inosose should have been the same to myo-inositol. [ABSTRACT FROM AUTHOR]

We describe the enantioselective epoxidation of straight-chain aliphatic alkenes using a biocatalytic system containing styrene monooxygenase from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749. The biocatalyzed enantiomeric epoxidation of 1-hexene to ( S)-1,2-epoxyhexane (>44.6 mM) using 2-propanol as the hydrogen donor was achieved under optimized conditions. The biocatalyst had broad substrate specificity for various aliphatic alkenes, including terminal, internal, unfunctionalized, and di- and tri-substituted alkenes. Here, we demonstrate that this biocatalytic system is suitable for the efficient production of enantioenriched ( S)-epoxyalkanes. [ABSTRACT FROM AUTHOR]

Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride ( m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved. [ABSTRACT FROM AUTHOR]

The asymmetric reduction of ketones is one of the most promising processes for producing chiral alcohols. However, dehydrogenases or reductases that can catalyze the reduction of ketones to give anti-Prelog chiral alcohols have been limited to some NADP/NADPH-dependent enzymes. Recently, we reported a novel NAD/NADH-dependent alcohol dehydrogenase (ADH) from Leifsonia sp. and Pseudomonas ADH homologs from soil metagenomes. Moreover, we have established an efficient hydrogen-transfer bioreduction process with 2-propanol as a hydrogen donor using Leifsonia ADH. This review focuses on the recent development of novel ADHs for producing industrially useful anti-Prelog chiral alcohols from various ketones. [ABSTRACT FROM AUTHOR]

Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced ( S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding ( S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration. [ABSTRACT FROM AUTHOR]

We found two NADH-dependent reductases (QNR and bacC) in Microbacterium luteolum JCM 9174 (M. luteolum JCM 9174) that can reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Alcohol dehydrogenase from Leifsonia sp. (LSADH) was combined with these reductases to regenerate NAD⁺ to NADH in situ in the presence of 2-propanol as a hydrogen donor. The reductase and LSADH genes were efficiently expressed in E. coli cells. A number of constructed E. coli biocatalysts (intact or immobilized) were applied to the resting cell reaction and optimized. Under the optimized conditions, (R)-(-)-3-quinuclidinol was synthesized from 3-quinuclidinone (15% w/v, 939 mM) giving a conversion yield of 100% for immobilized QNR. The optical purity of the (R)-(-)-3-quinuclidinol produced by the enzymatic reactions was >99.9%. Thus, E. coli biocatalysis should be useful for the practical production of the pharmaceutically important intermediate, (R)-(-)-3-quinuclidinol. [ABSTRACT FROM AUTHOR]

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K and V values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium. [ABSTRACT FROM AUTHOR]

The enzymatic production of ( S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with ( S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl ( S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of ( S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of ( S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds. [ABSTRACT FROM AUTHOR]

The article offers information on a research that uses a recombinant futalosine hydrolase (TTHA0556) of Thermus thermophilus (T. thermophilus) in functional analyses. It cites an alternative pathway known as the futalosine pathway, which operates in bacteria such as the Helicobacter pylori, Thermus thermophilus, Streptomyces coelicolor, and the Campylobacter jejuni. Findings show that recombinant TTHA0556 is highly specific to futalosine while it rejects other nucleotides and nucleosides.

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl ( S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system. [ABSTRACT FROM AUTHOR]

Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereoselectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride ( m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production. [ABSTRACT FROM AUTHOR]

The article describes the cloning of a KS505a biosynthetic gene cluster. According to the authors, a putative KS-505a biosynthetic gene cluster consisting of 24 open reading frames (ORFs) was judged to be present on a 28-kb DNA fragment. They add that a gene disruption experiment was also used to confirm that the cluster indeed participated in KS-505a biosynthesis.

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to ( S)/( R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced ( S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h−1 ml−1 catalyst or ( R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h−1 ml−1 catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced ( R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% ( w/ v) 4-hydroxy-2-butanoate over 500 h. [ABSTRACT FROM AUTHOR]

We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35°C. The Km value was calculated to be 13.7 ± 1.0 μM for GGDP and the kcat value was 3.3 × 10−2/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30°C. The Km value for ent-CDP was estimated to be 2.6 ± 0.2 μM and the kcat value was 1.4 × 10−3/sec. [ABSTRACT FROM PUBLISHER]

The article discusses the gene encryption of Leifsonia alcohol dehydrogenase (LSADH) which is a valuable biocatalyst for developing (R)-chiral alcohols, was cloned from the genomic Deoxyribonucleic Acid (DNA) of Leifsonia. The recombinant Escherichia coli which overexpressed ISADH acquired (R)-form chiral alcohols from ketones applying 2-propanol as a hydrogen donor with the highest level of productivity ever accounted and enantiomeric excess.

Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) Biosci. Biotechnol. Biochem. 65, 1627–1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the –50th, –8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity. [ABSTRACT FROM AUTHOR]

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and β-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R )-2-chloro-1-(3-chlorophenyl)ethanol (28 mg·mL-1 ) from m -chlorophenacyl chloride, ethyl (R )-4-chloro-3-hydroxy butanoate) (28 mg·mL-1 ) from ethyl 4-chloro-3-oxobutanoate and (S )-N-tert -butoxycarbonyl(Boc)-3-pyrrolidinol from N -Boc-3-pyrrolidinone (51 mg·mL-1 ), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3–7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported. [ABSTRACT FROM AUTHOR]

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non-hemetype bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1-methoxynaphthalene and thiophene were converted to o and p-bromoanisoles, 1-methoxy-4-bromonaphthalene and 2-bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans-cinnamic acid, trans-cinnamyl alcohol and cis-propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates. [ABSTRACT FROM AUTHOR]

The ltaE gene encoding for a thermostable low-specificity L-threonine aldolase, which catalyzes the cleavage of L-threonine/L-allo-threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ltaE gene under the control of the lac promoter, resulted in a 227-fold increase in the specific activity above the level detected in E. coli cells containing the control vector. The enzyme is thermostable : it retained its full activity upon heating at 60 +C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed. Knockout of the ltaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ltaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert. [ABSTRACT FROM AUTHOR]

A correction to the article "Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol-water medium" that was published in the February 9, 2012 issue is presented.