Microbiology (2014), 160, 2481–2491

DOI 10.1099/mic.0.080259-0

Styrene oxide isomerase of Sphingopyxis sp. Kp5.2 Michel Oelschla¨gel, Juliane Zimmerling, Michael Schlo¨mann and Dirk Tischler Correspondence Michel Oelschla¨gel

Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Strasse 29, 09599 Freiberg, Germany

[email protected]

Received 22 April 2014 Accepted 1 September 2014

Styrene oxide isomerase (SOI) catalyses the isomerization of styrene oxide to phenylacetaldehyde. The enzyme is involved in the aerobic styrene catabolism via side-chain oxidation and allows the biotechnological production of flavours. Here, we reported the isolation of new styrene-degrading bacteria that allowed us to identify novel SOIs. Out of an initial pool of 87 strains potentially utilizing styrene as the sole carbon source, just 14 were found to possess SOI activity. Selected strains were classified phylogenetically based on 16S rRNA genes, screened for SOI genes and styrene-catabolic gene clusters, as well as assayed for SOI production and activity. Genome sequencing allowed bioinformatic analysis of several SOI gene clusters. The isolate Sphingopyxis sp. Kp5.2 was most interesting in that regard because to our knowledge this is the first time it was shown that a member of the family Sphingomonadaceae utilized styrene as the sole carbon source by side-chain oxidation. The corresponding SOI showed a considerable activity of 3.1 U (mg protein)–1. Most importantly, a higher resistance toward product inhibition in comparison with other SOIs was determined. A phylogenetic analysis of SOIs allowed classification of these biocatalysts from various bacteria and showed the exceptional position of SOI from strain Kp5.2.

INTRODUCTION Strains of the genera Pseudomonas, Xanthobacter, Corynebacterium, Exophiala and Rhodococcus have previously been reported to degrade styrene under aerobic conditions by the pathway of side-chain oxidation (Fig. 1b) (Mooney et al., 2006; Tischler & Kaschabek, 2012). It has been supposed that this pathway represented the major route to mineralize styrene by micro-organisms (Mooney et al., 2006). The upper pathway of styrene degradation commonly comprises a styrene monooxygenase (SMO, encoded by styA/styB), a styrene oxide isomerase (SOI, encoded by styC) and a phenylacetaldehyde dehydrogenase (PAD, encoded by styD). The respective enzymes convert styrene via styrene oxide and phenylacetaldehyde to phenylacetic acid as a central metabolite that can be further degraded (Beltrametti et al., 1997; Bestetti et al., 2004; Hartmans et al., 1990; Itoh et al., 1997; O’Connor et al., 1995; Teufel et al., 2010).

Abbreviations: DSMZ, German Collection of Microorganisms and Cell Cultures; PAD, phenylacetaldehyde dehydrogenase; SOI, styrene oxide isomerase; SMO, styrene monooxygenase. The GenBank/EMBL/DDBJ accession numbers for the sequences of the genes styC1 of 1CP, styC2 of 1CP, sty cluster of 1CP, styC of 5.3_2_1, styC of Kp5.2, sty cluster of Kp5.2, 16S rRNA of 5.3_2_1 and 16S rRNA of Kp5.2 are KF540254–KF540261, respectively. One supplementary table and three supplementary figures are available with the online Supplementary Material.

080259 G 2014 The Authors

Printed in Great Britain

Genes encoding the relevant enzymes for the upper pathway are usually clustered and ordered as sty(SR)ABCD (Beltrametti et al., 1997; O’Leary et al., 2001; Panke et al., 1998; Toda & Itoh, 2012; Velasco et al., 1998). The genes styS and styR encode a two-component regulatory element of the sty operon (O’Leary et al., 2001), and have so far been found only in pseudomonads (O’Leary et al., 2001; Panke et al., 1998; Tischler & Kaschabek, 2012; Velasco et al., 1998). Considering the enzymology of styrene degradation, substantial investigations have only been performed with SMOs (Huijbers et al., 2014; Mooney et al., 2006). In contrast, previous studies on SOIs were limited to the actinobacteria Corynebacterium sp. AC-5 (Itoh et al., 1997) and Rhodococcus opacus 1CP (Oelschla¨gel et al., 2012), to the alphaproteobacterium Xanthobacter sp. 124X (Hartmans et al., 1989), and the gammaproteobacterium Pseudomonas putida S12 (Miyamoto et al., 2007). SOI (EC 5.3.99.7) has been identified as a stable, integral membrane protein that catalyses the isomerization of styrene oxide into phenylacetaldehyde independent of cofactors (Itoh et al., 1997; Oelschla¨gel et al., 2012). Promising biotechnological applications of SOIs and a rather simple enrichment yielding activities up to 370 U (mg protein)–1 have been demonstrated (Itoh et al., 1997; Miyamoto et al., 2007; Oelschla¨gel et al., 2012, 2014). Phenylacetaldehydes represent important building blocks for the healthcare and pharmaceutical industries, and can serve as flavours in the 2481

M. Oelschla¨gel and others

Styrene CH2

CH2

Evaporation adaptor

(a)

SMO

O

CH2

CH2

CH2

O (b)

Styrene

SOI Styrene oxide

H

O PAD Phenylacetaldehyde

OH ... Phenylacetic acid degradation

Fig. 1. Principle of liquid strain cultivation for initial SOI activity screening. Biomass obtained from the isolation on solid mineral medium plates as described in Methods was used to inoculate 100 ml liquid mineral medium (pH 7) containing 0.05–0.1 % yeast extract and styrene. (a) The latter was added via an evaporation adaptor over 2 weeks in 8.7 mmol aliquots (total 50 mmol). (b) Vaporized styrene served as the carbon source and was converted by bacteria metabolizing styrene via side-chain oxygenation by the following steps: epoxidation of styrene to styrene oxide by SMO, isomerization of styrene oxide to phenylacetaldehyde by SOI and oxidation of phenylacetaldehyde to phenylacetic acid by PAD. Screening for SOI activity was performed to identify putative isolates metabolizing styrene by the side-chain oxidation described.

perfume industry (Ho¨lderich & Barsnick, 2001). SOIs can also serve in racemic resolution to produce enantiopure styrene oxides, whilst converting the opposite enantiomer to phenylacetaldehyde (Itoh et al., 1997). A disadvantage of SOIs with respect to their applications is an inhibition by the phenylacetaldehyde formed at higher product concentrations occurring during biotransformation (Oelschla¨gel et al., 2012). The inhibition is irreversible and can be overcome only to a limited extent by applying immobilized enzyme in two-phase systems (Oelschla¨gel et al., 2014). So far, SOI seems to be the most active enzyme of the upper styrene degradative pathway. Non-enriched SOIs in crude extracts obtained from Corynebacterium sp. AC-5, Pseudomonas fluorescens ST and R. opacus 1CP have been reported to have styrene oxide isomerization activities of ~7.6–11.0 U mg21 (Itoh et al., 1997; Oelschla¨gel et al., 2012). Therefore, screening procedures for SOI activity should easily provide access to the respective sty gene clusters and to interesting biocatalysts, such as SMOs and SOIs (Tischler & Kaschabek, 2012). Based on these observations, the present study aimed at identifying new styrene degraders harbouring sty gene clusters and thus at discovering novel SOIs. To achieve this, the distribution and organization of the corresponding catabolic clusters amongst bacteria was highlighted. For micro-organisms harbouring a SOI, phylogeny, activity, substrate specificity and stability of this enzyme were investigated. SOI of the isolate Sphingopyxis sp. Kp5.2 turned out to fulfil criteria which distinguished this enzyme from others reported previously. 2482

METHODS Chemicals and enzymes. Standard chemicals, 4-chloro-, 4-fluoro-

and non-substituted styrene oxide, styrene oxide enantiomers, phenylacetaldehyde, and substituted phenylacetic acids were purchased from Sigma Aldrich, AppliChem, Merck, Riedel-de Haen, Thermo Fisher Scientific, VWR International, Bio-Rad or Carl Roth at the highest purity available. 4-Methylstyrene oxide was synthesized freshly (Tischler et al., 2009). Bacterial strains and culture conditions. R. opacus 1CP (Gorlatov

et al., 1989) was available from the strain collection of the Institute of Biosciences (TU Bergakademie Freiberg, Freiberg, Germany) and represented a positive control for SOI activity (Oelschla¨gel et al., 2012). Strain 1CP was kept on mineral medium plates (Dorn et al., 1974) in the presence of 5 mM benzoate for preservation. P. fluorescens ST (DSM-6290; Baggi et al., 1983; Beltrametti et al., 1997; Bestetti et al., 2004) and Xanthobacter sp. 124X (DSM-6696; Hartmans et al., 1989) were additionally used as reference strains from the German Collection of Microorganisms and Cell Cultures (DSMZ). These strains were streaked out on solid mineral medium (Dorn et al. 1974) containing 20 g glucose l21 and incubated at 30 uC. Colonies obtained were used to inoculate liquid media. Further representatives of the genera Arthrobacter, Gordonia and Rhodococcus (Table S1, available in the online Supplementary Material) from the strain collection of the Institute of Biosciences were investigated for their ability to grow on styrene and cultured as described for the isolates below. Isolation of styrene-degrading bacteria. For isolation of styrene-

degrading bacteria, 0.2–0.4 g soil (from meadows, non-contaminated or contaminated with aliphatic and aromatic hydrocarbons, sampled in Freiberg or Hoyerswerda, Saxony, Germany) was suspended in 1 ml water, and incubated at 37 uC and 150 r.p.m. for 2–3 h. Samples were then stored without shaking for 20 min for solids to settle, and Microbiology 160

Styrene oxide isomerases of soil bacteria the supernatant was diluted and streaked out onto solid mineral medium without a carbon source. These plates were incubated at room temperature in a 5 l desiccator in which an evaporating aliquot of 50 ml styrene was provided as the sole source of carbon and energy (Oelschla¨gel et al., 2012). Grown colonies were picked and separated by streaking on solid mineral medium plates with styrene as the sole carbon source supplied via the gas phase. The procedure was repeated until pure isolates were obtained that were able to rapidly metabolize styrene (first visible colony formation after 48–72 h). Screening for SOI activity. Isolates were grown in 100 ml mineral medium in baffled 500 ml Erlenmeyer flasks at 30 uC under constant shaking (120 r.p.m.). Initially, 0.05–0.1 % yeast extract was added in a few cases to accelerate biomass formation and yield, respectively. Growth and SOI induction were stimulated by means of repeated additions of styrene (total 50 mmol, 8.7 mmol aliquots of styrene added through an evaporation adaptor, Fig. 1a). After 2 weeks, cultures were harvested by centrifugation for 20–30 min at 5000 g and 4 uC. Cells were suspended in 1–1.5 ml 25 mM phosphate buffer (pH 7.3), and to 0.5–1.0 ml of the cell suspension 600 mg zirconia beads and 8 U DNase I were added. For cell disruption, the batches were shaken vigorously for 1 h at 30 s21 and 4 uC in a swing mill (MM-200; Retsch). They were then investigated for SOI activity. In addition, 500 ml harvested cells was mixed with 500 ml glycerol and stored at 280 uC as a stock culture. Phylogenetic assignment of the SOI-positive isolates. If not

stated otherwise, all primers, cloning kits and PCR reagents were obtained from Eurofins MWG Operon or Thermo Scientific. Isolates were characterized phylogenetically by amplification and sequencing of a large part of the 16S rRNA gene. A 1495 bp fragment was amplified by using the primers 27f 59-AGAGTTTGATCCTGGCTCAG-39 (Lane, 1991, modified; Edwards et al., 1989) and 1522r 59-AAGGAGGTGATCCANCCGCA-39 (Edwards et al., 1989, modified). Colonies of putative styrene-degrading bacteria were picked from solid mineral medium plates containing 20 g glucose l21 and added directly to 50 ml PCR mixture. The PCR mixture contained 16 PCR buffer with 2.0 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate and 0.8 mM each primer (Jena Bioscience). Cells were disrupted during an initial incubation of the PCR mixture at 98 uC for 15 min. Afterwards, 2.5 U DreamTaq polymerase was added. DNA was denatured for 4.5 min at 95 uC followed by 30 cycles of amplification (95 uC for 30 s, 56 uC for 30 s and 72 uC for 90 s). In a final step, PCR mixtures were incubated at 72 uC for 5 min. PCR products obtained were purified by gel electrophoresis (1 % agarose, 90 V) and stained with SYBR Gold (Invitrogen). Bands with the correct size were isolated from the gel by a DNA Isolation Spin kit (AppliChem) or by an innuPREP Gel Extraction kit (Analytik Jena). DNA fragments purified were sequenced directly with 27f or 1522r primers by Eurofins MWG Operon or previously ligated into pJET1.2 (CloneJET PCR cloning kit) and transferred into Escherichia coli DH5a via heat-shock transformation for propagation. Plasmid DNA was purified by means of a Gene JET Plasmid Miniprep kit. Inserts of pJET1.2 derivatives obtained were sequenced independently with the primers pJET1.2fw or pJET1.2rev by Eurofins MWG Operon. Sequencing results obtained were aligned with the Staden Package (Bonfield et al., 1995) and the consensus sequence was compared with the National Center for Biotechnology Information databases by BLASTN (Altschul et al., 1990, 1997). Identification of SOI genes from selected isolates and reference strains. A SOI-specific PCR was performed with the

newly designed primers screenSOI-fw 59-GGNCAYGGNRTNYTNATGAT-39 and screenSOI-rev 59-TGNGCYTTNGCCCANCCYTC39. An aliquot of 40 ml PCR mixture contained 16 PCR buffer, 8.0 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate, 0.8 mM http://mic.sgmjournals.org

each primer, 0.030 mg BSA ml21 and 2.0 U DreamTaq polymerase. Colonies grown on solid media were picked and suspended in water. Cells were disrupted by incubation at 98 uC for 15 min. Afterwards, 2.8 ml sample was added to the PCR mixture. DNA was denatured initially for 4.5 min at 95 uC. After 36 cycles of amplification (95 uC for 30 s, 56–59 uC for 30 s and 72 uC for 45 s), PCR mixtures were incubated at 72 uC for 5 min. Products obtained were purified by gel electrophoresis (3 % agarose, 90 V) and stained with SYBR Gold or ethidium bromide. The bands of the expected size (~140 bp) were purified from the gel as described for 16S rDNA. Products obtained were ligated into pJET1.2 (CloneJET PCR Cloning kit), transferred into E. coli DH5a, purified, sequenced and aligned as described above. A 291 bp large genomic fragment of some strains was obtained by PCRs performed with the newly designed primer pair styB-SOI-fw 59CCKCTKMTGTTYTTYGCSAG-39 binding in styB and screenSOI-rev binding in styC. Another fragment of 1034 bp could be amplified with the designed styC-specific primers styC-GSPII 59-TGGATGHAYYTSRTBGGYGG-39 and styD-SOI-rev 59-ATCAKCAGYGGGRWRTTCCA39 that binds in styD. Verification of the complete styC genes obtained from the amplified fragments was performed with the primers SOIcompfw 59-AACACCTGAGATACACGGAG-39 and SOIcomprev 59-GAGTTCTTTCTGGGTAGGTC-39. These primers allowed the amplification of a 605 bp PCR product. These reactions were performed in 20–40 ml PCR mixtures containing 2–6 mM MgCl2 in addition to the ingredients described above for the SOI-specific PCR. Template DNA was added after cell disruption just by heating or in final concentrations of 0.5–1.0 ng ml21 after purification of genomic DNA by phenol/chloroform extraction (Wilson, 2001, modified). PCRs were performed as described for the SOI-specific PCR with annealing temperatures of 49–65 uC and adapted elongation time. PCR products formed were treated, stained, purified, cloned, sequenced and aligned as described above. Phylogenetic analysis of SOI-related genes. The styC-encoded protein sequences were aligned by means of CLUSTAL_X or CLUSTAL_W

and phylogenetic trees were reconstructed with the maximumlikelihood, neighbour-joining, minimum evolution, UPGMA and Fitch–Margoliash algorithms as described previously (Tischler et al., 2009, 2012). PHYLIP package 3.66 (http://evolution.genetics.washington. edu/phylip.html) and MEGA5 (Tamura et al., 2011) were utilized. SOI induction and production in selected strains for initial characterization. To determine SOI induction by various inducers,

biomass was cultivated for 7 days in 500 ml Erlenmeyer flasks with 100 ml 0.05 % (w/v) yeast-extract-containing mineral medium in the presence of one of the following carbon sources: styrene (total 35 mmol, 8.7 mmol aliquots added through an evaporation adaptor), styrene oxide (total 25 mmol, 6.1 mmol aliquots added through an evaporation adaptor), phenylacetaldehyde (total 0.35–0.5 mmol, added in 0.05–0.2 mmol portions directly to the medium) or phenylacetic acid (total 0.8 mmol, added in 0.1–0.5 mmol portions). Cells were then harvested, disrupted and assayed for SOI activity. To determine SOI substrate specificity and product inhibition, the respective SOIs were produced by a two-step cultivation approach. This procedure for SOI production was performed to improve SOI yields as described previously (Oelschla¨gel et al., 2012, 2014). It comprised a biomass production step utilizing glucose as the carbon source and a SOI production step applying a suitable inducer compound. In most experiments, a defined amount of yeast extract (0.05– 0.1 %, w/v) was added to overcome otherwise prolonged lag phases. First, precultures were cultivated in 1 l baffled Erlenmeyer flasks at 30 uC under constant shaking (120 r.p.m.) in 100 ml mineral medium with 0.05 % yeast extract and in the presence of glucose 2483

M. Oelschla¨gel and others (total 2.0–2.6 mmol, 0.3–0.5 mmol aliquots) to yield OD546.1. The total volume of mineral medium was then extended to 200 ml. After further addition of glucose (total 8.0 mmol, 1 mmol aliquots) up to OD546.1, SOI was induced with overall 182 mmol suitable inducer (8.7–26.1 mmol aliquots added through an evaporation adaptor) over 10 days. Cells were harvested and treated as described above. During the biomass production step utilizing glucose as the carbon source, doubling times were also determined for the selected strains. Therefore, cultures containing 50 ml mineral medium with 3 mM glucose in 500 ml baffled Erlenmeyer flasks were inoculated with precultures mentioned above to OD546 0.4. These flasks were incubated as described for precultures and doubling times were determined by repeated OD546 measurement. As mentioned above, SOIs were identified as integral membrane proteins in a previous study (Oelschla¨gel et al., 2012). This allowed a simple enrichment of the membrane-bound enzyme from crude extract, thus avoiding side-reactions of other enzymes during enzyme assays. Partial purification of the SOI from the disrupted cells obtained by two-step cultivation was achieved by an initial centrifugation step at 10 000 g for 20 min at 4 uC to separate whole cells and large cell debris. In a second step at 50 000 g for 24 h at 4 uC, the insoluble SOI was separated from cytosolic proteins as described previously (Oelschla¨gel et al., 2012). The SOI-containing pellet was suspended in 25 mM phosphate buffer (pH 7.3) and used for activity measurements or stored at 220 uC. Enzyme assays and protein quantification. Activity of SOIs was

determined as described previously with GC or reversed-phase HPLC by quantification of the reaction product phenylacetaldehyde formed from the substrate styrene oxide over time (Oelschla¨gel et al., 2012). HPLC was performed with 50 % (v/v) methanol containing 0.1 % (w/v) phosphoric acid as mobile phase at a flow rate of 0.7 ml min21. The net retention volume (difference of retention times and dead time multiplied by flow rate of the mobile phase) of the analysed product phenylacetaldehyde under these conditions was 3.1 ml. For the determination of the enantioselectivity of SOIs, the assay was used with pure (S)- and (R)-styrene oxide instead of racemic epoxide. SOI activity toward substituted styrene oxides was determined indirectly by quantification of the corresponding phenylacetic acids that were formed by chemical oxidation from the phenylacetaldehydes (Oelschla¨gel et al., 2012). The oxidized products were analysed by HPLC. The following net retention volumes were obtained: phenylacetic acid, 2.8 ml; 4-chlorophenylacetic acid, 7.2 ml; 4fluorophenylacetic acid, 3.4 ml; 4-methylphenylacetic acid, 5.5 ml. Peaks obtained were compared with authentic reference compounds with respect to retention volume and UV spectrum. For investigation of enzyme stability with respect to product inhibition, 30 mg enzyme preparation was suspended in 400 ml 25 mM phosphate buffer (pH 7.3) containing 5 % (v/v) methanol. Then, 0 or 50 mM phenylacetaldehyde (1 M stock solution in methanol) was added and the batches were incubated for 15 min at room temperature under constant shaking. Samples were diluted by the addition of 1 ml 25 mM phosphate buffer (pH 7.3) and centrifuged (13 000 g, 5 min, 20 uC). The SOI-containing pellet obtained was washed with 25 mM phosphate buffer (pH 7.3), centrifuged (13 000 g, 5 min, 20 uC) and resuspended in 40 ml fresh 25 mM phosphate buffer (pH 7.3). Determination of residual activity was performed as described above. Determination of protein concentrations was performed by the method of Bradford (Bradford, 1976) as described previously (Oelschla¨gel et al., 2012). 2484

RESULTS AND DISCUSSION Isolates with the ability to degrade styrene via side-chain oxidation In order to identify new strains that degraded styrene via side-chain oxidation, various actinobacterial strains of the strain collection of the Institute of Biosciences were cultivated initially on solid mineral medium under a styrene atmosphere, thus providing styrene as the sole carbon source. In total, 11 Gordonia, two Arthrobacter and 18 Rhodococcus strains could grow under these conditions, and were further cultivated in liquid cultures with styrene and subsequently screened for SOI activity (Table S1). Of these strains, only R. opacus 1CP showed measurable SOI activity. It was thus included in the study as the reference strain (Oelschla¨gel et al., 2012) beside P. fluorescens ST and Xanthobacter sp. 124X, which were obtained from the DSMZ and treated similarly. Additionally, 54 putative styrene-utilizing strains were isolated from soil samples and cultivated as described above. During subsequent assays for SOI activity, 11 of the 54 isolates tested positive (Table S1, Figs S1 and S2). Only these 11 were further investigated to identify possibly novel SOIs. The 16S rDNA amplification and sequencing of these 11 isolates allowed classification into the genera Rhodococcus (n55), Sphingopyxis (n51) and Sphingobium (n55) (data not shown). Thus, out of 87 styrene-utilizing strains subjected to SOI activity screening only 14 showed SOI activity. It cannot be ruled out completely that in some cases the activity determination of the SOI may have been inhibited by uncommonly high PAD activity, but the low abundance of SOIs amongst the soil bacteria tested indicated that alternative or modified pathways for styrene degradation may have been dominant. A few studies have revealed the possibility of styrene degradation via alternative routes (Patrauchan et al., 2008; Toda & Itoh, 2012; Warhurst et al., 1994). In addition to representatives of the genus Rhodococcus, five Sphingobium isolates and one strain related to Sphingopyxis showed SOI activity (Tables 1 and S1). To our knowledge, styrene degradation via side-chain oxygenation has not been shown previously for members of the family Sphingomonadaceae and corresponding SOIs have so far not been characterized. Therefore, genetic and biochemical studies were focused on the isolates Rhodococcus sp. 5.3_2_1, Sphingobium sp. Kp5.1_1 and Sp8.3_1b, and Sphingopyxis sp. Kp5.2. Identification and characterization of styC genes and sty gene clusters Based on conserved regions of available SOI sequences of Rhodococcus sp. ST-5 (Toda & Itoh, 2012) and various Pseudomonas strains (Beltrametti et al., 1997; Lin et al., 2010; Panke et al., 1998; Park et al., 2006; Velasco et al., 1998), the primers screenSOI-fw and screenSOI-rev were Microbiology 160

Styrene oxide isomerases of soil bacteria

Table 1. SOI activity of strains after growth on various substrates Strain

Highest specific SOI activity [U (mg protein)”1]*

P. fluorescens ST Rhodococcus sp. 5.3_2_1 R. opacus 1CP Sphingobium sp. Kp5.1_1 Sphingobium sp. Sp8.3_1b Sphingopyxis sp. Kp5.2 Xanthobacter sp. 124X

Relative specific SOI activity (%) in crude cell extract after growth on

20.7±1.4 1.76±0.18 10.0±4.5 4.66±0.44 7.58±0.57 3.09±0.39 8.65±0.23

Styrene

Styrene oxide

Phenylacetaldehyde

Phenylacetic acid

85.9±8.5 53.0±16.8 100±45* 32.3±3.5 72.3±7.2 100±13* 50.4±6.2

100±7* 100±10* 17.9±3.0 100±9* 100±8* 70.8±7.4 33.1±7.6

0.73±0.42 76.9±18.5 1.51±0.73 30.3±4.0 59.3±2.5 43.9±4.8 100±3*

,0.5 ,0.5 ,0.5 11.7±0.2 15.3±2.7 10.0±0.9 0.54±0.02

Data represent the mean±SD of four independent measurements. *The highest specific activity found on one of the substrates is given and was set as 100 %.

designed. This SOI-specific PCR was applied to the reference strains P. fluorescens ST, R. opacus 1CP and Xanthobacter sp. 124X as well as to Rhodococcus sp. 5.3_2_1, Sphingobium sp. Sp8.3_1b and Sphingopyxis sp. Kp5.2. To our knowledge, of all strains tested, only the styC sequence of P. fluorescens ST had been known previously.

R. opacus 1CP

PCR products of the expected size of ~140 bp were obtained for almost all strains (Fig. S3) encoding sequences related to SOI genes (sequence data not shown). In the case of Xanthobacter sp. 124X, amplification of such a fragment was not possible, indicating larger variations in the primer binding regions.

1281 bp 534 bp 507 bp 1489/1573 bp*

styA Rhodococcus sp. ST-5

1284 bp

styA Rhodococcus sp. ST-10

1287 bp

styA

styB

styC

/8450 bp/ 501 bp

styD

styC2

537 bp 507 bp 1491/1509 bp*

styB

styC

styD

546 bp 681 bp

styB

hypothetical protein

Pseudomonas sp. Y2 1314 bp

2928/949 bp*

624 bp

1248 bp

paaK

styS

styR

styA

1503 bp

969 bp

1269 bp

Sphingopyxis sp. Kp5.2 306 bp 1296 bp

paaK

hypothetical protein

2076 bp

enoyl-CoA hydratase

feaB (similar to styD)

LysR transcriptional regulator

styA

513 bp 510 bp 1491/509 bp*

styB

styC

styD

1302 bp

outer membrane protein

498 bp 561 bp 663 bp 408 bp

styB

styC outer histidine kinase membrane sensor protein protein

Fig. 2. Localization and organization of sty genes. The organization of sty catabolic gene clusters is illustrated for R. opacus 1CP (GenBank accession number KF540256), Rhodococcus sp. ST-5 and ST-10 (Toda & Itoh, 2012), Pseudomonas sp. Y2 (Velasco et al., 1998), and Sphingopyxis sp. Kp5.2 (GenBank accession number KF540259). Genes involved in styrene metabolism were: styA/styB of SMO, styC of SOI, styC2 of a StyC-similar protein (59 % identical positions on protein level), styD/feaB of PAD with different sizes depending on two potential start codons (indicated by an asterisk), and styS and styR of sensor and regulator proteins, respectively. paaK encoded a phenylacetyl-CoA ligase. http://mic.sgmjournals.org

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*

P. chlororaphis subsp. aureofaciens AAC27439 ABE29428 B. xenovorans LB400 AEO27340 Pseudomonas sp. 19-rlim StyC_1CP R. opacus 1CP Rhodococcus sp. 5.3_2_1 StyC_5.3_2_1 Rhodococcus sp. ST-5 AB594507 R. wratislaviensis IFP 2016 ELB93553 AAC23720 Pseudomonas sp. VLB120 CAA04002 Pseudomonas sp. Y2 P. putida SN1 ABB03729 P. fluorescens ST CAB06825 Pseudomonas sp. LQ26 ADE62392 H. effusa AP103 EIT71605 Gammaproteobacterium BDW918 EIF44117 StyC2_1CP R. opacus 1CP ELB89314 R. wratislaviensis IFP 2016 GAC06213 G. agarilytica NO2 Meth. petroleiphilum PM1 ABM93818 StyC_Kp5.2 Sphingopyxis sp. Kp5.2 WP_018251007 S. melonis EFY94429 Meta. anisopliae ARSEF 23 Gammaproteobacterium HTCC2143 EAW31154 EAQ96818 C. litoralis KT71

100

*

40

Transmembrane helix 3

membrane helix 2

Microbiology 160

AAC27439 ABE29428 AEO27340 StyC_1CP StyC_5.3_2_1 AB594507 ELB93553 AAC23720 CAA04002 ABB03729 CAB06825 ADE62392 EIT71605 EIF44117 StyC2_1CP ELB89314 GAC06213 ABM93818 StyC_Kp5.2 WP_018251007 EFY94429 EAW31154 EAQ96818

20

*

120

*

*

Trans*

60

80

*

71 64 64 64 64 64 64 65 65 65 65 65 57 65 64 57 64 65 87 57 78 64 64

Transmembrane helix 4 140

*

160

*

180

*

200

164 167 157 168 168 168 168 169 169 169 169 169 151 160 166 170 157 161 186 151 190 156 156

M. Oelschla¨gel and others

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Transmembrane helix 1

Styrene oxide isomerases of soil bacteria

Fig. 3. Multiple sequence alignment of SOIs. SOIs and related proteins were aligned using CLUSTAL_X version 1.8 (Higgins & Sharp, 1988; Thompson et al., 1997) and GeneDoc version 2.6.003 (Nicholas et al., 1997). Shading indicates different degrees of conserved amino acids and motifs (black, 100 % identity in all sequences; dark grey, 80 %; grey, 60 %). Transmembrane helices were predicted with the TMHMM server 2.0 (Krogh et al., 2001). Sequences obtained in this study are underlined and GenBank accession numbers of proteins with proven SOI activity are shaded grey. Protein sequences from the following organisms were considered: Burkholderia xenovorans LB400, Congregibacter litoralis KT71, gammaproteobacterium BDW918, gammaproteobacterium HTCC2143, Glaciecola agarilytica NO2, Hydrocarboniphaga effusa AP103, Metarhizium anisopliae ARSEF 23, Methylibium petroleiphilum PM1, R. opacus 1CP [this study, StyC_1CP (GenBank accession number KF540254) and StyC2_1CP (GenBank accession number KF540255)], Rhodococcus sp. 5.3_2_1 [this study, StyC_5.3_2_1 (GenBank accession number KF540257)], Rhodococcus sp. ST-5, Rhodococcus wratislaviensis IFP 2016, Pseudomonas chlororaphis subsp. aureofaciens, P. fluorescens ST, P. putida SN1, Pseudomonas sp. 19-rlim, Pseudomonas sp. LQ26, Pseudomonas sp. VLB120, Pseudomonas sp. Y2, Sphingomonas melonis and Sphingopyxis sp. Kp5.2 [this study, StyC_Kp5.2 (GenBank accession number KF540258)].

The sequences of the complete SOI genes from R. opacus 1CP (GenBank accession number KF540254) and strain 5.3_2_1 (GenBank accession number KF540257) were obtained after further PCRs as described in Methods based on PCR products obtained by SOI-specific PCR. Later genome sequencing of strain 1CP (unpublished) verified the styC sequence obtained and provided insights into the clustering of sty genes in strain 1CP (GenBank accession number KF540256) (Fig. 2). The styrene catabolic genes styA/styB, styC and styD were clustered similarly to those of pseudomonads (Velasco et al., 1998) and Rhodococcus sp. ST-5 (Toda & Itoh, 2012). Regulatory genes, such as styS and styR, could neither be identified in nor neighboured to the sty cluster of strain 1CP. The SOI amino acid sequences of strain 1CP and of isolate 5.3_2_1 (both 168 aa) showed 100 % identity, whereas the 16S rDNA results obtained allowed us to distinguish between both strains. Strain 1CP had previously been classified as R. opacus (Gorlatov et al., 1989; Tsitko et al., 1999) and isolate 5.3_2_1 can be assigned to Rhodococcus jostii. The SOI match could be a consequence of horizontal gene transfer, which has often been reported for rhodococci (Larkin et al., 1998). The SOIs of 1CP and 5.3_2_1 were 64 and 86 % identical on the amino acid level to SOIs of Pseudomonas sp. Y2 [GenBank accession number CAA04002 (Velasco et al., 1998)] and Rhodococcus sp. ST-5 [GenBank accession number AB594507 (Toda & Itoh, 2012)], respectively (Figs 3 and 4). Interestingly, Rhodococcus strains 1CP and IFP 2016 (genome sequence GCA_000325625) harboured a further styC-like gene in addition to styC. Whilst styC was part of a sty gene cluster, the other styC-like gene is localized somewhere else in the genomes (Fig. 2). The second hypothetical SOI shares ~60 % identical amino acids (166 aa) with those encoded by the sty clusters (Figs 3 and 4). These findings raise questions about the function of these SOI-like proteins which might be solved in future investigations. For the Sphingobium and Sphingopyxis isolates, we attempted to amplify complete styC genes by additional PCRs as described in Methods. However, no PCR products were obtained thus indicating a different organization of http://mic.sgmjournals.org

the sty clusters in both strains. Finally, the styC gene (GenBank accession number KF540258) of Sphingopyxis sp. Kp5.2 was obtained from a genome-sequencing project with the CeBiTec (Bielefeld, Germany; unpublished). The rest of the relevant styrene-catabolic cluster was also predicted (GenBank accession number KF540259) (Fig. 2). Indeed, clustering of the genes styA/styB, styC and styD in isolate Kp5.2 differed from known sty operons of pseudomonads and Rhodococcus strains (Toda & Itoh, 2012; Velasco et al., 1998). The styD (feaB) in Sphingopyxis was not directly adjacent to the sty genes, but was located ~1100 bp upstream of styABC. This fact explained the unsuccessful attempts to obtain the complete styC gene by PCRs mentioned above. The regulatory elements styS and styR known from pseudomonads were not identified in the respective cluster of Sphingopyxis. However, a hypothetical outer membrane protein and a hypothetical histidine kinase sensor protein were found to be encoded downstream and a LysR transcriptional regulator upstream of styABC. The presence of a StyS-like histidine kinase sensor protein and a LysR regulator may indicate a regulatory function of the respective genes for styrene metabolism in strain Kp5.2. In contrast to 1CP, no further SOI-related gene was identified on the genome of strain Kp5.2. Evidence for a novel SOI from Sphingopyxis sp. Kp5.2 In strain Kp5.2, styC was found to have a size of 561 bp encoding a product of 186 aa. Compared with known SOIs, SOI from Sphingopyxis sp. Kp5.2 seems to be 26–27 aa longer at the N-terminal side, but 10 aa shorter at the C terminus (Fig. 3). One additional isoleucine was also incorporated in the first transmembrane helix of the protein (Fig. 3, position 64). Like the other membrane-bound SOIs it also contained four predicted transmembrane helices (Fig. 3; Krogh et al., 2001). At the protein level, the SOI from strain Kp5.2 shared 62 % identical positions with SOIs from Pseudomonas sp. Y2 (GenBank accession number CAA04002 (Velasco et al., 1998)] and Rhodococcus sp. ST-5 (GenBank accession number AB594507 (Toda & Itoh, 2012)]. Conclusively, the SOI of the alphaproteobacterium Sphingopyxis sp. Kp5.2 (SOI-4; GenBank accession number 2487

M. Oelschla¨gel and others

Rhodococcus sp. ST-5 (AB594507)

R. opacus 1CP (SOI-1; KF540254) 100 H. effusa AP103 (EIT71605)

R. wratislaviensis IFP 2016 (ELB93553) Methylibium petroleiphilum PM1 (ABM93818) Sphingopyxis sp. Kp5.2 (SOI-4; KF540258)

Rhodococcus sp. 5.3_2_1 (SOI-3; KF540257)

100

Gammaproteobacterium BDW918 (EIF44117) G. agarilytica NO2 (GAC06213)

99

30 Pseudomonas sp. LQ26 (ADE62392) 3130 26 39 99 P. fluorescens ST (CAB06825) 30 61 42 58 48 P. putida SN1 (ABB03729) Pseudomonas sp. Y2 (CAA04002)

100

S. melonis (WP_018251007) 99 Metarhizium anisopliae ARSEF 23 (EFY94429) R. opacus 1CP (SOI-2; KF540255)

Pseudomonas sp. VLB120 (AAC23720)

R. wratislaviensis IFP 2016 (ELB89314)

100

96

99

100

B. xenovorans LB400 (ABE29428) P. chlororaphis subsp. aureofaciens (AAC27439)

Pseudomonas sp. 19-rlim (AEO27340)

0.1

Gammaproteobacterium HTCC2143 (EAW31154)

C. litoralis KT71 (EAQ96818)

Fig. 4. Dendrogram of StyCs and similar proteins. The dendrogram was generated using MEGA5 (Tamura et al., 2011) with available protein sequences based on the alignment of Fig. 3. The result of the procedure with the neighbour-joining algorithm is shown. In addition, bootstrap values (1000 iterations) were calculated to indicate reliability of the branches given as percentages. The p-cymene methyl hydroxylase from P. chlororaphis subsp. aureofaciens [GenBank accession number AF012555 (Dutta et al., 2010)] and highly related proteins (GenBank accession numbers EAW31154, EAQ96818, ABE29428 and AEO27340) served as outgroups. Sequences obtained during this work (SOI-1 to -4) are underlined and enzymes with proven SOI activity are shaded grey.

KF540258) in the dendrogram (Fig. 4) represented a novel branch amongst styrene oxide isomerases that was more related to the SOI-inactive outgroup proteins [GenBank accession numbers AF012555 (Dutta et al., 2010), EAW31154, EAQ96818, ABE29428 and AEO27340] than other functional SOIs. Induction and production of the WT SOIs for initial characterization With the aim of revealing conditions under which SOI was expressed, induction of SOI with the potential inducers styrene, styrene oxide, phenylacetaldehyde and phenylacetic acid as the sole carbon source was investigated (Table 1). Only these substrates were investigated initially because previous investigations had shown a high specificity of the induction of styC in R. opacus 1CP by substrates and intermediates of styrene side-chain oxidation, and no or 2488

only a minor effect on SOI expression by other structurally similar compounds (Oelschla¨gel et al., 2014). Further studies (Hartmans et al., 1989; O’Connor et al., 1995) had also shown the highest enzyme activities of proteins encoded by sty genes after growth on substrates involved in the side-chain oxidation. For P. fluorescens ST, styrene oxide was proven to be the most efficient inducer (Table 1). Amongst the rhodococci, the highest SOI activity in strain 5.2_3_1 was achieved with styrene oxide and in strain 1CP with styrene. With a relative SOI activity of 1.5 % compared with styreneinduced biomass, phenylacetaldehyde does not seem to be an efficient inducer for strain 1CP based on this study, whilst a relative SOI activity of 16 % had been determined during previous investigations (Oelschla¨gel et al., 2012). In contrast to the present investigation, biomass in the previous study had been cultivated with 0.8 instead of Microbiology 160

Styrene oxide isomerases of soil bacteria

0.35 mmol substrate and for 14 instead of 7 days. This suggested that the induction of the SOI is also dependent on incubation time and inducer concentration. For Xanthobacter sp. 124X, phenylacetaldehyde was identified to be the best inducer (Table 1). The two Sphingobium strains showed similar expression patterns after induction with various substrates, of which styrene oxide yielded the highest isomerase activities. For Sphingopyxis sp. Kp5.2, styrene was the best substrate for efficient styC expression. Interestingly, Sphingopyxis sp. Kp5.2 showed styC expression also after growth on glucose alone (1.1±0.3 U mg21). This again indicated a different regulation compared with pseudomonads and rhodococci because in the case of P. putida CA-3, glucose had been reported to be a sty operon repressor (O’Leary et al., 2001). With respect to total SOI activities, the highest activities were reached by P. fluorescens ST (20.7 U mg21), R. opacus 1CP (10 U mg21) and Xanthobacter sp. 124X (8.7 U mg21), whereas Sphingopyxis sp. Kp5.2 yielded a SOI activity of only 3.1 U mg21 (Table 1). Similar activities had been determined previously for Corynebacterium sp. AC-5 (7.6 U mg21) after induction with styrene (Itoh et al., 1997). As it had been found previously that a two-step cultivation may be beneficial for the production of SOI, such a cultivation with glucose for biomass production and a suitable inducer such as styrene for SOI expression was investigated for the new isolates and reference strains (Table 2). Styrene was chosen for the second step because it induced SOI activity in all strains investigated (Table 1) and its toxic effect on the biomass was lower than that of styrene oxide (Oelschla¨gel et al., 2014). The SOI enrichment procedure, which is described in Methods, worked well for R. opacus 1CP, P. fluorescens ST and also for isolate 5.3_2_1, but not for the alphaproteobacteria. This was also indicated by a comparison of SOI activity in crude extracts of biomass induced with a total of 350 mmol styrene l21 (assuming complete dissolution in aqueous phase) over 7 days (Table 1) with the activity in the membrane fraction from biomass obtained after

two-step cultivation with glucose and subsequent induction with 910 mmol styrene l21 over 10 days (Table 2). In the latter membrane fraction, a drastic loss of specific activity was observed for the alphaproteobacteria, which might have been due to a different form of anchoring in the membrane(s) and which might make an adjustment of the enrichment protocol necessary. This feature clearly distinguishes SOIs of alphaproteobacteria including strain Kp5.2 from known epoxide isomerases. Substrate specificity and stability of SOIs The enzymes enriched in the membrane fractions by centrifugation as described above were used for biotransformation studies (Table 2, Fig. 5). Owing to identical styC genes of Rhodococcus strains 1CP and 5.3_2_1 (Figs 3 and 4), these studies were only performed with the enzyme of strain 1CP. All enzymes tested showed the highest activity with styrene oxide as substrate (Fig. 5). Sphingobium sp. Sp8.3_1b showed remarkably high relative SOI activities for 4-chlorostyrene oxide and 4-fluorostyrene oxide of 16.1±2.2 and 36.1± 12.7 % compared with styrene oxide, respectively. The lowest activity with 4-fluorostyrene oxide was observed for Sphingopyxis sp. Kp5.2 at 8.52±4.40 %. For the SOIs obtained from the two Sphingobium strains and from Sphingopyxis sp. Kp5.2, the relative activity for 4-methyl-styrene oxide was only 17–28 % compared with styrene oxide, whilst the other strains tested showed relative values of 66–81 %. With respect to enantioselectivity, all enzymes investigated showed a similar preference for (S)-styrene oxide and only ~36–64 % relative activity toward the (R)-enantiomer (Table 2, Fig. 5). Amongst all enzymes investigated so far, SOI of Sphingopyxis sp. Kp5.2 was found to possess the highest selectivity for the (S)-epoxide. It therefore may be a valuable candidate for racemic resolution of styrene oxides as previously investigated for other SOIs by Itoh et al. (1997). The stability of 30 mg enriched protein toward the previously described irreversible product inhibition

Table 2. SOI activity of strains after growth on glucose and styrene induction Strain

Doubling time on glucose (h)

Specific SOI activity from enriched membrane fraction Racemic styrene oxide

P. fluorescens ST Rhodococcus sp. 5.3_2_1 R. opacus 1CP Sphingobium sp. Kp5.1_1 Sphingobium sp. Sp8.3_1b Sphingopyxis sp. Kp5.2 Xanthobacter sp. 124X

1.62±0.01 1.69±0.28 2.68±0.03 3.27±0.26 3.51±0.03 3.86±0.01 10.61±0.11

10.1±2.3

(S)-styrene oxide 30.0±3.9

ND

ND

6.99±0.95 0.056±0.012 0.0015±0.0004 0.063±0.014 0.14±0.04

20.4±2.5 0.068±0.016 0.0033±0.0002 0.15±0.01 0.23±0.03

Data represent the mean±SD of three to four independent measurements. ND, Not determined. http://mic.sgmjournals.org

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M. Oelschla¨gel and others

Relative specific activity (%)

100

4-Fluorostyrene oxidea 4-Methylstyrene oxidea

4-Chlorostyrene oxidea (R)-Styrene oxideb

80 60 40 20

Xanthobacter sp. 124X

Sphingopyxis sp. Kp5.2

Sphingobium sp.Sp8.3_1b

Sphingobium sp. Kp5.1_1

R. opacus 1CP

P. fluorescens ST

0

Fig. 5. Substrate specificity of investigated SOIs. Activity measurements were performed as described in Methods. Data represent the mean±SD of four independent measurements. aRelative activities presented refer to specific SOI activity toward racemic styrene oxide (Table 2). bRelative specific SOI activity in the presence of (R)-styrene oxide refers to the activity toward the (S)-enantiomer (Table 2).

(Oelschla¨gel et al., 2012) was investigated. A 15 min incubation of the enzyme preparation in 50 mM phenylacetaldehyde resulted in considerable inactivation of almost all SOIs investigated. However, the SOIs of Sphingopyxis sp. Kp5.2 and Xanthobacter sp. 124X showed remarkable residual activities of 11–12 %, indicating a higher stability.

Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new

The results obtained by the initial enzymic characterization presented here indicated that SOIs from Sphingopyxis and Sphingobium strains behave significantly differently compared with the isomerases known from pseudomonads and rhodococci. In particular, the more stable SOI of strain Kp5.2 seemed to have a higher suitability for (S)-styrene oxide conversion despite a narrower substrate acceptance in general. This also indicated an outstanding position of the enzyme – a conclusion also derived from phylogenetic calculations (Fig. 4). These facts lead us to conclude that SOI of strain Kp5.2 is a novel SOI representative possibly useful for biotechnological applications.

Beltrametti, F., Marconi, A. M., Bestetti, G., Colombo, C., Galli, E., Ruzzi, M. & Zennaro, E. (1997). Sequencing and functional analysis of

ACKNOWLEDGEMENTS We thank Mr Tang Youbo for assistance in the isolation of styrenedegrading organisms, and the team at CeBiTec (Bielefeld, Germany) with special regard to Dr Jo¨rn Kalinowski and Dr Christian Ru¨ckert for Sphingopyxis sp. Kp5.2 genome sequencing. M. O. was supported by a fellowship from the Deutsche Bundesstiftung Umwelt, and D. T. was supported by a grant from the European Social Fund and the Saxonian Government (GETGEOWEB: 100101363).

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