Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst.

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.

Correction to: Biosynthesis and production of quercitols and their application in the production of pharmaceuticals: current status and prospects.

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.

Biosynthesis and production of quercitols and their application in the production of pharmaceuticals: current status and prospects.

(-)-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.

Development of a Novel Escherichia coli-Kocuria Shuttle Vector Using the Cryptic pKPAL3 Plasmid from K. palustris IPUFS-1 and Its Utilization in Producing Enantiopure (S)-Styrene Oxide.

The novel cryptic pKPAL3 plasmid was isolated from the Gram-positive microorganism Kocuria palustris IPUFS-1 and characterized in detail. pKPAL3 is a circular plasmid that is 4,443 bp in length. Open reading frame (ORF) and homology search analyses indicated that pKPAL3 possesses four ORFs; however, there were no replication protein coding genes predicted in the plasmid. Instead, there were two nucleotide sequence regions that showed significant identities with untranslated regions of K. rhizophila DC2201 (NBRC 103217) genomic sequences, and these sequences were essential for autonomous replication of pKPAL3 in Kocuria cells. Based on these findings, we constructed the novel Escherichia coli-Kocuria shuttle vectors pKITE301 (kanamycin resistant) and pKITE303 (thiostrepton resistant) from pKPAL3. The copy numbers of the constructed shuttle vectors were estimated to be 20 per cell, and they exhibited low segregation stability in Kocuria transformant cells in the absence of antibiotics. Moreover, constructed vectors showed compatibility with the other K. rhizophila shuttle vector pKITE103. We successfully expressed multiple heterologous genes, including the styrene monooxygenase gene from Rhodococcus sp. ST-10 (rhsmo) and alcohol dehydrogenase gene from Leifsonia sp. S749 (lsadh), in K. rhizophila DC2201 using the pKITE301P and pKITE103P vectors under the control of the glyceraldehyde 3-phosphate dehydrogenase (gapdh) promotor. The RhSMO-LSADH co-expressing K. rhizophila was used as a biocatalyst in an organic solvent-water biphasic reaction system to efficiently convert styrene into (S)-styrene oxide with 99% ee in the presence of 2-propanol as a hydrogen donor. The product concentration of the reaction in the organic solvent reached 235 mM after 30 h under optimum conditions. Thus, we demonstrated that this novel shuttle vector is useful for developing biocatalysts based on organic solvent-tolerant Kocuria cells.

Functional Characterization of Epitheaflagallin 3-O-Gallate Generated in Laccase-Treated Green Tea Extracts in the Presence of Gallic Acid.

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.

Identification and characterization of a novel (-)-vibo-quercitol 1-dehydrogenase from Burkholderia terrae suitable for production of (-)-vibo-quercitol from 2-deoxy-scyllo-inosose.

(-)-vibo-Quercitol is a deoxyinositol (1L-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.

Characterization of two cryptic plasmids from Kocuria palustris IPUFS-1 and construction of novel Escherichia coli-Kocuria shuttle vector for biocatalysis.

Two cryptic plasmids, designated pKPAL1 and pKPAL2, were identified from the gram-positive bacterium Kocuria palustris IPUFS-1, which was isolated from a fish source. The 2251-bp and 2488-bp circular genomes of pKPAL1 and pKPAL2, respectively, were sequenced. Subsequent open reading frame (ORF) and homology search analyses suggested that pKPAL1 and pKPAL2 possess two and three ORFs, respectively, and encode the putative replication proteins, RepA and RepB, like the genomes of several plasmids in gram-positive bacteria. Thus, pKPAL1 and pKPAL2 were inferred to belong to the ColE2 plasmid family. We constructed novel Escherichia coli-Kocuria shuttle vectors pKITE101-103 based on pKPAL1. The constructed shuttle vector was stably maintained in Kocuria transformant cells, and vector copy number was estimated to be about 60 per cell. Leifsonia sp. S749 alcohol dehydrogenase (LSADH) was efficiently expressed in Kocuria rhizophila DC2201 using the pKITE103P vector under the control of the promoter of glyceraldehyde 3-phosphate dehydrogenase (gapdh). Herein, we demonstrate that the novel shuttle vector is a useful tool for developing biocatalysts based on organic solvent-tolerant Kocuria cells.

Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa.

BACKGROUND: Flavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis. RESULTS: In this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg(2+)-independent O-methyltransferases and showed high amino acid sequence similarity (60-95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-L-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3',5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids. CONCLUSIONS: Five FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3',5,7-tetramethylated quercetin was obtained as the final product.

Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.

Screening gene-specific amplicons from metagenomes (S-GAM) is a highly promising technique for the isolation of genes encoding enzymes for biochemical and industrial applications. From metagenomes, we isolated phenylacetaldehyde reductase (par) genes, which code for an enzyme that catalyzes the production of various Prelog's chiral alcohols. Nearly full-length par genes were amplified by PCR from metagenomic DNA, the products of which were fused with engineered par sequences at both terminal regions of the expression vector to ensure proper expression and then used to construct Escherichia coli plasmid libraries. Sequence- and activity-based screening of these libraries identified different homologous par genes, Hpar-001 to -036, which shared more than 97% amino acid sequence identity with PAR. Comparative characterization of these active homologs revealed a wide variety of enzymatic properties including activity, substrate specificity, and thermal stability. Moreover, amino acid substitutions in these genes coincided with those of Sar268 and Har1 genes, which were independently engineered by error-prone PCR to exhibit increased activity in the presence of concentrated 2-propanol. The comparative data from both approaches suggest that sequence information from homologs isolated from metagenomes is quite useful for enzyme engineering. Furthermore, by examining the GAM-based sequence dataset derived from soil metagenomes, we easily found amino acid substitutions that increase the thermal stability of PAR/PAR homologs. Thus, GAM-based approaches can provide not only useful homologous enzymes but also an alternative to directed evolution methodologies.

Characterization and cloning of laccase gene from Hericium coralloides NBRC 7716 suitable for production of epitheaflagallin 3-O-gallate.

Epitheaflagallin 3-O-gallate (ETFGg) is a minor polyphenol found in black tea extract, which has good physiological functions. It is synthesized from epigallocatechin gallate (EGCg) with gallic acid via laccase oxidation. Various basidiomycetes and fungi were screened to find a suitable laccase for the production of ETFGg. A basidiomycete, Hericium coralloides NBRC 7716, produced an appropriate extracellular laccase. The purified laccase produced twice the level of ETFGg compared with commercially available laccase from Trametes sp. The enzyme, termed Lcc2, is a monomeric protein with an apparent molecular mass of 67.2 kDa. The N-terminal amino acid sequence of Lcc2 is quite different from laccase isolated from the fruiting bodies of Hericium. Lcc2 showed similar substrate specificity to known laccases and could oxidize various phenolic substrates, including pyrogallol, gallic acid, and 2,6-dimethoxyphenol. The full-length lcc2 gene was obtained by PCR using degenerate primers, which were designed based on the N-terminal amino acid sequence of Lcc2 and conserved copper-binding sites of laccases, and 5'-, and 3'-RACE PCR with mRNA. The Lcc2 gene showed homology with Lentinula edodes laccase (sharing 77% amino acid identity with Lcc6). We successfully produced extracellular Lcc2 using a heterologous expression system with Saccharomyces cerevisiae. Moreover, it was confirmed that the recombinant laccase generates similar levels of ETFGg as the native enzyme.

Microbial production of aliphatic (S)-epoxyalkanes by using Rhodococcus sp. strain ST-10 styrene monooxygenase expressed in organic-solvent-tolerant Kocuria rhizophila DC2201.

We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes.

Efficient PCR-based amplification of diverse alcohol dehydrogenase genes from metagenomes for improving biocatalysis: screening of gene-specific amplicons from metagenomes.

Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.

Use of the anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols.

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.

PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.

The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols.

Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure.

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.

Gene cloning and characterization of two NADH-dependent 3-quinuclidinone reductases from Microbacterium luteolum JCM 9174.

We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(-)-3-quinuclidinol.

Production of (R)-3-quinuclidinol by E. coli biocatalysts possessing NADH-dependent 3-quinuclidinone reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH).

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.

Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides.

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.

Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp. ST-5 and ST-10.

Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography-mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10.