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Optically pure sulfoxides are valuable organosulfur compounds extensively employed in medicinal and organic synthesis. In this study, we present a biocatalytic oxidation‐reduction cascade system designed for the preparation of enantiopure sulfoxides. The system involves the cooperation of a low‐enantioselective chimeric oxidase SMO (styrene monooxygenase) with a high‐enantioselective reductase MsrA (methionine sulfoxide reductase A), facilitating "non‐selective oxidation and selective reduction" cycles for prochiral sulfide oxidation. The regeneration of requisite cofactors for MsrA and SMO was achieved via a cascade catalysis process involving three auxiliary enzymes, sustained by cost‐effective D‐glucose. Under the optimal reaction conditions, a series of heteroaryl alkyl, aryl alkyl and dialkyl sulfoxides in R configuration were synthesized through this "one‐pot, one step" cascade reaction. The obtained compounds exhibited high yields of >90 % and demonstrated enantiomeric excess (ee) values exceeding 90 %. This study represents an unconventional and efficient biocatalytic way in utilizing the low‐enantioselective oxidase for the synthesis of enantiopure sulfoxides. [ABSTRACT FROM AUTHOR]

Styrene is a nonpolar organic compound used in very high volume for the industrial scale production of commercially important polymers such as polystyrene resins as well as copolymers like acrylonitrile butadiene styrene, latex, and rubber. These resins are widely used in the manufacturing of various products including single‐use plastics such as disposable cups and containers, protective packaging, heat insulation, and so forth. The large‐scale utilization leads to the over‐accumulation of styrene waste in the environment causing deleterious health risks including cancer, neurological impairment, dysbiosis of central nervous system, and respiratory problems. To eliminate the accumulating waste. Microbial enzyme‐based system represents the most environmental friendly and sustainable approach for elimination of styrene waste. However, comprehensive understanding of the enzyme–substrate interaction and associated pathways would be crucial for developing large‐scale disposal systems. This study aims to understand the molecular interaction between the protein‐ligand complexes of the styrene catabolic reactions by bacterial enzymes of sty operon. Molecular docking analysis for catalytic enzymes namely, styrene monooxygenase (SMO), styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase (PAD) of the bacterial sty operon was carried out with their individual substrates, that is, styrene, styrene oxide, and phenylacetic acid, respectively. The binding energy, amino acids forming binding cavity, and binding interactions between the protein‐ligand binding sites were calculated for each case. The obtained binding energies showed a stable association of these complexes indicating the future scope of their utilization for large‐scale bioremediation of styrene, and its commercially used polymers and copolymers. [ABSTRACT FROM AUTHOR]

Optically pure sulfoxides are noteworthy compounds applied in a wide range of industrial fields; however, the biocatalytic deracemization of racemic sulfoxides is challenging. Herein, a high‐enantioselective methionine sulfoxide reductase A (MsrA) was combined with a low‐enantioselective styrene monooxygenase (SMO) for the cyclic deracemization of sulfoxides. Enantiopure sulfoxides were obtained in >90 % yield and with >90 % enantiomeric excess (ee) through dynamic "selective reduction and non‐selective oxidation" cycles. The cofactors of MsrA and SMO were subsequently regenerated by the cascade catalysis of three auxiliary enzymes through the consumption of low‐cost D‐glucose. Moreover, this "one‐pot, one‐step" cyclic deracemization strategy exhibited a wide substrate scope toward various aromatic, heteroaromatic, alkyl and thio‐alkyl sulfoxides. This system proposed an efficient strategy for the green synthesis of chiral sulfoxide. [ABSTRACT FROM AUTHOR]

• Thermostabilized mutants of Na StyA were constructed for asymmetric epoxidation. • Mutant TM18 had a half-life 148-fold of the parental and enhanced solvent tolerance. • Mutant produced chiral epoxides up to 4-fold yield of the parental with >99 %ee. • Mutants displayed increased local rigidity and reduced residue cross-correlation motions. Chiral epoxides are one of the most versatile building blocks in organic synthesis, but the asymmetric epoxidation of unfunctionalized terminal olefins remains a challenge. Na StyA is a styrene monooxygenase from Nocardia altamirensis that functions as an enantioselective epoxygenase for terminal olefins. To improve its robustness, we investigated over 50 mutants designed via evolutionary- and energy-based approaches, and successfully identified 18 beneficial mutations, which then led to a combinatorial mutant of TM18 with drastically enhanced thermostability and solvent tolerance while retaining excellent activity and enantioselectivity. Its turnover frequency and half-life at 50 °C were increased to 5- and 148-fold of the parental. In the epoxidation of terminal olefins, TM18 achieved high product yields of over 4-fold of the parental, and excellent enantiomeric excess of 99 % to >99 % ee. The analysis of trajectory data from molecular dynamics simulations further validated that the mutations in TM18 increased its local rigidity, as inferred by RMSF and PCA analyses and through novel salt bridges formation. Furthermore, TM18 displayed reduced residue cross-correlation motions. The combined effects of these mutations result in an overall improvement in the structural and dynamic thermostability of TM18. [ABSTRACT FROM AUTHOR]

Abstract Background Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. Results In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. Conclusions This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme’s active site.

Asymmetric epoxidation catalyzed with styrene monooxygenase (SMO) is a powerful enzymatic process producing enantiopure styrene epoxide derivatives. To establish a more diversified reservoir of SMOs, a new SMO from Bradyrhizobium sp. ORS 375, named BrSMO, was mined from the database and characterized. BrSMO was constituted of an epoxygenase component of 415 amino acid residues and an NADH-dependent flavin reductase component of 175 residues. BrSMO catalyzed the epoxidation of styrene and 7 more styrene derivatives, yielding the corresponding (S)-epoxides with excellent enantiomeric excesses (95– > 99% ee), with the highest activity achieved for styrene. BrSMO also catalyzed the asymmetric sulfoxidation of 7 sulfides, producing the corresponding (R)-sulfoxides (20–90% ee) with good yields. [ABSTRACT FROM AUTHOR]

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH 2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FAD ox reduction intricacies (k red =110 s -1 for bound FAD ox ), identifying free FAD ox binding as the rate-determining step. The aerobic oxidation of FADH 2 (k ox =90 s -1 ) and subsequent decomposition to FAD ox and H 2 O 2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (k dec =0.2 s -1 ) compared to free cofactor (k dec =1.8 s -1 ). At varied styrene concentrations, k ox for FADH 2 ranged from 80 to 120 s -1 . Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH 2 channelling between subunits. This work contributes to comprehending FADH 2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Enantiopure organic compounds are of major importance for the chemical and pharmaceutical industry. Flavin‐dependent group E monooxygenases, composed of monooxygenase and reductase, are known to perform epoxidation of substituted alkenes as well as sulfoxidation in a regio‐ and enantioselective fashion. Group E is divided into styrene monooxygenases (SMO) and indole monooxygenases (IMO). Hitherto mainly SMOs have been characterized. In this study, we assayed 31 monooxygenases from both types, while 23 of which showed activity. They almost exclusively produced (S)‐styrene oxide at high enantiomeric excess with maximum activities of 0.73 μmol min−1 mg−1 (kcat=0.54 s−1). In case of sulfoxidation, we found that the enantioselectivity is contrary between both types. IMOs preferably produce the (S)‐enantiomer while SMOs have a tendency to produce the (R)‐enantiomer. Sequence analysis and molecular docking of substrates allowed identifying fingerprint motives: SMO N46‐V48‐H50‐Y73‐H76‐S96 and IMO S46‐Q48‐M50‐V/I73‐I76‐A96. These form an essential part of the active site while the loop (AS44‐51) interacts with the co‐substrate and other amino acids direct the substrate. The motives clearly distinguish group E monooxygenases and define the enantioselectivity and thus direct biotechnological applications. Two‐hour biotransformations with several sulfides in conjunction with upscale experiments (10 and 100 mg scale) resulted in the identification of promising candidates for the realization of biocatalytic processes. [ABSTRACT FROM AUTHOR]

Since ancient times, people have been attracted by dyes and they were a symbol of power. Some of the oldest dyes are indigo and its derivative Tyrian purple, which were extracted from plants and snails, respectively. These 'indigoid dyes' were and still are used for coloration of textiles and as a food additive. Traditional Chinese medicine also knows indigoid dyes as pharmacologically active compounds and several studies support their effects. Further, they are interesting for future technologies like organic electronics. In these cases, especially the indigo derivatives are of interest but unfortunately hardly accessible by chemical synthesis. In recent decades, more and more enzymes have been discovered that are able to produce these indigoid dyes and therefore have gained attention from the scientific community. In this study, group E monooxygenases (styrene monooxygenase and indole monooxygenase) were used for the selective oxygenation of indole (derivatives). It was possible for the first time to show that the product of the enzymatic reaction is an epoxide. Further, we synthesized and extracted indigoid dyes and could show that there is only minor by-product formation (e.g. indirubin or isoindigo). Thus, group E monooxygenase can be an alternative biocatalyst for the biosynthesis of indigoid dyes. [ABSTRACT FROM AUTHOR]

Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (˃95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO.

The side-chain oxygenation of styrene is able to yield substituted phenylacetic acids from corresponding styrenes by co-metabolic transformation. This co-metabolization was investigated in Pseudomonas fluorescens ST using 4-chlorostyrene as co-substrate. It was shown that non-substituted styrene is necessary to ensure the co-metabolic process. Furthermore, aspects affecting the co-transformation were studied, e.g. cell density, amount of inducer, pH, effects of co-substrate/co-product. It was demonstrated that 4-chlorophenylacetic acid and 4-chlorostyrene are able to inhibit the reaction. But, these inhibitions are influenced by salt and trace elements. Finally, a protocol was established which considers all findings. Therewith, about 6.7 g L−1 co-product were obtained after 451 h. Compared to previous studies, the co-product concentration was improved by the factor 1.4 while the reaction time was decreased by the factor 18.5. The study offers also aspects for prospective improvements in order to establish an efficient way to gain substituted acids without genetic manipulation.

Among bacteria, only a single styrene-specific degradation pathway has been reported so far. It comprises the activity of styrene monooxygenase, styrene oxide isomerase, and phenylacetaldehyde dehydrogenase, yielding phenylacetic acid as the central metabolite. The alternative route comprises ring-hydroxylating enzymes and yields vinyl catechol as central metabolite, which undergoes meta-cleavage. This was reported to be unspecific and also allows the degradation of benzene derivatives. However, some bacteria had been described to degrade styrene but do not employ one of those routes or only parts of them. Here, we describe a novel "hybrid" degradation pathway for styrene located on a plasmid of foreign origin. As putatively also unspecific, it allows metabolizing chemically analogous compounds (e.g., halogenated and/or alkylated styrene derivatives). Gordonia rubripertincta CWB2 was isolated with styrene as the sole source of carbon and energy. It employs an assembled route of the styrene side-chain degradation and isoprene degradation pathways that also funnels into phenylacetic acid as the central metabolite. Metabolites, enzyme activity, genome, transcriptome, and proteome data reinforce this observation and allow us to understand this biotechnologically relevant pathway, which can be used for the production of ibuprofen. IMPORTANCE The degradation of xenobiotics by bacteria is not only important for bioremediation but also because the involved enzymes are potential catalysts in biotechnological applications. This study reveals a novel degradation pathway for the hazardous organic compound styrene in Gordonia rubripertincta CWB2. This study provides an impressive illustration of horizontal gene transfer, which enables novel metabolic capabilities. This study presents glutathione-dependent styrene metabolization in an (actino-)bacterium. Further, the genomic background of the ability of strain CWB2 to produce ibuprofen is demonstrated. [ABSTRACT FROM AUTHOR]

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.

A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH2 dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL−1) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL−1, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH2 cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mgStyA−1) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mgStyA−1 by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.

Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s) and FAD (4.4 µM, 56 s). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 ( K = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases. [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]

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min g cell dry weight resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite ( S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[ b]thiophene and 2-methylbenzo[ b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[ b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[ b]thiophene and 2-methylbenzo[ b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group. [ABSTRACT FROM AUTHOR]

Abstract: The utilization of the cellular metabolism for cofactor regeneration is a common motivation for the application of whole cells in redox biocatalysis. Introduction of an active oxidoreductase into a microorganism has profound consequences on metabolism, potentially affecting metabolic and biotransformation efficiency. An ambitious goal of systems biotechnology is to design process-relevant and knowledge-based engineering strategies to improve biocatalyst performance. Metabolic flux analysis (MFA) has shown that the competition for NAD(P)H between redox biocatalysis and the energy metabolism becomes critical during asymmetric styrene epoxidation catalyzed by growing Escherichia coli containing recombinant styrene monooxygenase. Engineering TCA-cycle regulation allowed increased TCA-cycle activities, a delay of acetate formation, and enhanced NAD(P)H yields during batch cultivation. However, at low biomass and product concentrations, the cellular metabolism of both the mutants as well as the native host strains could cope with increased NADH demands during continuous two-liquid phase biotransformations, whereas elevated but still subtoxic product concentrations were found to cause a significantly increased NAD(P)H demand and a compromised efficiency of metabolic operation. In conclusion, operational conditions determine cellular energy and NAD(P)H demands and thus the biocatalytic efficiency of whole-cell redox biocatalysts. [Copyright &y& Elsevier]

Abstract: Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-β-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene. [Copyright &y& Elsevier]

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]

Abstract: 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. [Copyright &y& Elsevier]

A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH2 dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL-1) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL-1, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH2 cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mgStyA -1) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mgStyA -1 by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity. [ABSTRACT FROM AUTHOR]

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an α- or β-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of α-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by site-directed mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene. [ABSTRACT FROM AUTHOR]

Abstract: (S)-styrene oxide, a useful chiral intermediate, has been synthesized using an electroenzymatic method with direct electrochemical FADH2 regeneration. Low electroenzymatic efficiency arising from the fast FADH2 reoxidation could be overcome by employing a zinc oxide/carbon black composite electrode. The attractive interaction between zinc oxide and styrene monooxygenase kept the local enzyme concentration high near the electrode surface, thereby increasing the accessibility of FADH2 from the electrode surface to the enzyme. By adjusting the reaction conditions such as oxygen solubility, a high electroenzymatic efficiency of 65% was obtained. As a result, the reaction rate was increased while the amount of the side-products from the cofactor reoxidation process was decreased. The metal oxide/carbon black composite electrode can be efficiently used for electroenzymatic syntheses using diffusible-flavin dependent monooxygenases. [ABSTRACT FROM AUTHOR]

Abstract: A novel styrene monooxygenase (SMO) was isolated from Pseudomonas sp. LQ26, a styrene degrader from activated sludge. Sequence alignment demonstrated that it was the most distant member of all SMOs originating from the genus of Pseudomonas. The substrate spectrum of this enzyme extended beyond typical SMO substrates to 1-allylbenzene analogues, previously reported as non-substrates for the SMO from Pseudomonas fluorescens ST. The results demonstrate for the first time the asymmetric epoxidation of both conjugated and unconjugated alkenes catalyzed by SMO and suggest that a much broader substrate spectrum is expected for SMOs. [ABSTRACT FROM AUTHOR]

Abstract: A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180U/gCDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400mM in the organic phase within 6h, resulting in an average specific and volumetric productivity of 6.4mmol/gdrycells/h (106U/gdrycells) and 67mM/h (1110U/Laq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance. [Copyright &y& Elsevier]

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, ( S)-styrene oxide, was formed from styrene while ( 1S,2R)-indene oxide was the predominant enantiomer ( e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively. [ABSTRACT FROM AUTHOR]

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of ( S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes. [ABSTRACT FROM AUTHOR]

Abstract: A styrene-degrading strain, Pseudomonas putida SN1, was engineered to produce enantiopure (S)-styrene oxide. Among four catabolic genes, styABCD, which encode the enzymes responsible for styrene degradation, styC was disrupted by homologous recombination to remove styrene oxide isomerase activity that converts styrene oxide to phenylacetaldehyde. The styC-negative mutant could not grow on styrene or styrene oxide, and it accumulated enantiopure (S)-styrene when incubated with styrene. When the reaction was conducted with citrate as a carbon source, the mutant exhibited a high whole-cell activity of styrene oxide formation (170U/gdcw) that was similar to the styrene-degradation activity of the wild-type strain. In a 3L bioreactor experiment employing a water–bis(2-ethylhexyl)phthalate two-phase system, in 13h the mutant could accumulate approximately 150mM (S)-styrene oxide at an enantiomeric excess greater than 98% in the organic phase. [Copyright &y& Elsevier]

Abstract: Pseudomonas putida CA-3 is capable of consuming a number of aromatic and aliphatic hydrocarbons. With the exception of styrene none of the alkenes tested are capable of supporting the growth of P. putida CA-3 as sole sources of carbon and energy. The highest rate of alkene consumption was observed with styrene as the substrate. A 6.5- and 15.5-fold lower rate of substrate consumption was observed with indene and indole with the concomitant formation of 2-indanone and indigo, respectively. The presence of a sulphur (benzothiopene) or oxygen (benzofuran) in the cyclopentene ring resulted in further decreases in the rate of substrate consumption by whole cells of P. putida CA-3. P. putida CA-3 is incapable of consuming benzene and consumes toluene at a low rate. No detectable products were observed in supernatants of cultures incubated with benzothiopene, benzofuran or toluene. The aliphatic alkenes 1-octene and 1,7-octadiene were both consumed by whole cells of P. putida CA-3 at a rate equivalent to indene consumption. The consumption of (R) styrene oxide was 1.7- and 1.25-fold higher than that of the S isomer and the racemic mix, respectively. The rate of racemic indene oxide, 1,2-epoxyoctane and 1,2-epoxy-7-octene consumption was lower than their equivalent alkene and 55-, 11.8-, and 27.5-fold lower than the rate of racemic styrene oxide consumption. A transposon mutant incapable of growth with styrene or styrene oxide failed to transform indole to indigo. The ratio of styrene utilisation relative to other substrates changes in the mutant strain compared to the wild-type strain, e.g., Indene biotransformation by mutant AF5 is 1.9-fold higher than styrene consumption compared to the wild-type strain CA-3 where the rate of styrene consumption is 6.7-fold higher than indene consumption. This trend is also observed for other alkenes and epoxides. [Copyright &y& Elsevier]

The effects of various nutrient-limiting conditions on expression of the sty operon in Pseudomonas putida CA-3 were investigated. It was observed that limiting concentrations of the carbon source phenylacetic acid, resulted in high levels of phenylacetyl coenzyme A (CoA) ligase activity, this was accompanied also by upper pathway styrene monooxygenase enzyme activity. The introduction of inorganic nutrient limitations, (nitrate, sulfate and phosphate), caused a dramatic reduction in detectable levels of phenylacetyl CoA ligase activity, particularly in the presence of the primary carbon source, succinate. Under these conditions it was no longer possible to detect styrene monooxygenase activity. Reverse transcription PCR analysis of total RNA, isolated under each of the continuous culture conditions examined, revealed that variations in the levels of enzyme activity coincided with altered patterns of corresponding paaK (phenylacetyl CoA ligase) and styA (styrene monooxygenase) gene expression. Transcription of the upper pathway regulatory sensor kinase gene styS was also observed to be growth condition-dependent. These observations suggest that induction/repression of the sty operon in P. putida CA-3, during growth on phenylacetic acid under continuous culture conditions, involves regulatory mechanisms coordinately affecting both the upper and lower pathways and acting at the level of gene transcription. [Copyright &y& Elsevier]

Tyrosol is an aromatic compound with great value that is widely used in the food and pharmaceutical industry. In this study, we reported a synthetic pathway for converting p -coumaric acid ( p -CA) into tyrosol in Escherichia coli . We found that the enzyme cascade comprising ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae , styrene monooxygenase (SMO), styrene oxide isomerase (SOI) from Pseudomonas putida , and phenylacetaldehyde reductase (PAR) from Solanum lycopersicum could efficiently synthesize tyrosol from p -CA with a conversion rate over 90%. To further expand the range of substrates, we also introduced tyrosine ammonia-lyase (TAL) from Flavobacterium johnsoniae to connect the synthetic pathway with the endogenous l-tyrosine metabolism. We found that tyrosol could be efficiently produced from glycerol, reaching 545.51 mg/L tyrosol in a tyrosine-overproducing strain under shake flasks. In summary, we have established alternative routes for tyrosol synthesis from p -CA (a potential lignin-derived biomass), glucose, and glycerol.

Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (˃95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO. [ABSTRACT FROM AUTHOR]