Styrene Oxide Isomerase Research Articles

Engineering Styrene Oxide Isomerase Towards Enhanced Chemoenzymatic Synthesis of (S)‐Flurbiprofen

AbstractThe conventional synthesis of (S)‐flurbiprofen, a valuable non‐steroidal anti‐inflammatory drug (NSAID), involves inefficient chiral resolution with low‐yielding multiple reaction steps, or asymmetric synthesis with low enantioselectivity and/or regioselectivity. Here, we developed high‐yielding, enantioselective, and sustainable synthesis of (S)‐flurbiprofen via two chemoenzymatic approaches from a simple alkene: (A) enzymatic epoxidation‐isomerization‐oxidation cascade and Pd‐catalyzed arylation; and (B) chemical epoxidation, enzymatic isomerization‐oxidation cascade, and Pd‐catalyzed arylation. Styrene oxide isomerase (SOI)‐catalyzed enantio‐retentive Meinwald rearrangement was the key step in the two pathways, but SOI suffers from low activity. SOI was engineered via two rounds of iterative saturation mutagenesis and screening with a three‐enzyme cascade assay, generating a double‐mutant SOIDM (N99A/D95A) with 5.5‐fold improvement in catalytic efficiency (kcat/KM). In Pathway A, cascade biotransformation of 3‐fluoro‐4‐bromo‐α‐methylstyrene with engineered E. coli strain expressing SOIDM, styrene monooxygenase (SMO) and aldehyde dehydrogenase (ALDH), and subsequent Pd‐catalyzed arylation afforded (S)‐flurbiprofen in 98% yield and 91% ee. In Pathway B, chemical epoxidation (Sharpless dihydroxylation and ring‐closing), cascade conversion using an engineered E. coli strain expressing SOIDM, ALDH and NADH oxidase (NOX), and Pd‐catalyzed arylation produced (S)‐flurbiprofen in 83% yield and 98% ee. Compared to conventional synthesis, the developed methods reduced reaction steps, minimized waste generation and improved enantioselectivity or overall yield.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement—a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound—and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

Styrene Oxide Isomerase-Catalyzed Meinwald Rearrangement in Cascade Biotransformations: Synthesis of Chiral and/or Natural Chemicals.

Styrene oxide isomerase (SOI) catalyzes the Meinwald rearrangement of aryl epoxides to carbonyl compounds via a 1,2-trans-shift in a stereospecific manner. A number of cascade biotransformations with SOI-catalyzed epoxide isomerization as a key step have been developed to convert readily available substrates into valuable chiral chemicals. Cascade conversion of terminal or internal alkenes into chiral acids, alcohols or amines was achieved, which involved SOI-catalyzed enantio-retentive isomerization of terminal epoxides via 1,2-H shift, or internal epoxides via 1,2-methyl shift. SOI-involved cascades were also developed to convert racemic epoxides into chiral acids or amines via dynamic kinetic resolution. Additionally, combining SOI-catalyzed isomerization with enantioselective C-C bond forming enzymes enabled the synthesis of chiral amino acids or amino alcohols from racemic epoxides. Finally, integration of SOI-involved cascades with biosynthesis pathways allowed for the direct utilization of renewable substrates for the sustainable synthesis of high-value natural chemicals such as alcohols, acids, and esters.

Biocatalytic Cascade Conversion of Racemic Epoxides to (S)‐2‐Arylpropionic Acids, (R)‐ and (S)‐2‐Arylpropyl Amines

AbstractNew types of one‐pot multi‐step enzymatic cascade transformation involving dynamic kinetic resolution to convert racemic substrates to chiral products in high ee and yield were developed. Unique cascade reactions of easily accessible racemic trans‐β‐methyl or α‐methyl epoxides to produce (S)‐2‐arylpropionic acids, (R)‐ and (S)‐2‐arylpropyl amines were demonstrated via styrene oxide isomerase (SOI)‐catalyzed Meinwald epoxide rearrangement to generate 2‐arylpropanal in situ, which was followed by spontaneous racemization, and alcohol dehydrogenase (ADH)‐catalyzed (S)‐enantioselective oxidation or transaminase (TA)‐catalyzed (R)‐ or (S)‐enantioselective amination, respectively. Cascade reactions performed with isolated enzymes or whole‐cell biocatalysts produced pharmaceutically relevant (S)‐2‐arylpropionic acids, (R)‐ and (S)‐2‐arylpropyl amines with high enantioselectivity and yield. The cascades starting with in situ aldehyde generation from epoxides effectively minimized side reactions related to aldehyde instability and displayed a wide substrate scope.magnified image

Understanding the in silico aspects of bacterial catabolic cascade for styrene degradation.

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.

Polystyrene Bioremediation: A Perspective on Microbial and Environmental Constrained Interventions

The polystyrene is the specific kind of plastics based upon petroleum which is composed of monomers of vinyl benzene (styrene). Regardless of the attraction of polystyrene, multiple organizations and municipalities are enduring an emerging challenge during disposal of polystyrene products and packaging. It is appraised that the products of polystyrene accounts for not more than 1% of the overall burden of landfill resources. It could be a greater health threat for humans also who are on the topmost level of food chain and farsighted styrene monomers of the plastics used in engineering polystyrene has been categorized as a potential humanoid carcinogen by the International Agency for Research on Cancer (IARC) and the US National Institutes of Health (NIH). As a few researchers have been published on the degradation of polystyrene plastic but the key point addressed in this review is the recognition of microbial and fungal enzymes which are known at present to be involved in polystyrene monomer plastic degradation. The major bacterial and fungal enzymes involved within the degradation reaction of vinyl side chain include styrene monooxygenase, styrene oxide isomerase, styrene monooxygenase, flavin adenine dinucleotide (FAD) reductase, styrene isomerase and phenyl acetaldehyde dehydrogenase. Omics”-based approaches revitalized the study of PAH catabolism by permitting for an integrative assessment of the biochemical mechanism in charge to degrade PAH including polystyrene on the polluted locations. The applications of such enzymes in procedures that would permit the degradation of polystyrene plastics contaminating niches is a dare for future cohorts of microbiology experts

Efficient chemoenzymatic synthesis of α-aryl aldehydes as intermediates in C-C bond forming biocatalytic cascades.

Multi-enzyme biocatalytic cascades are emerging as practical routes for the synthesis of complex bioactive molecules. However, the relative sparsity of water-stable carbon electrophiles limits the synthetic complexity of molecules made from such cascades. Here, we develop a chemoenzymatic platform that leverages styrene oxide isomerase (SOI) to covert readily accessible aryl epoxides into α-aryl aldehydes through a Meinwald rearrangement. These unstable aldehyde intermediates are then intercepted with a C-C bond forming enzyme, ObiH, that catalyzes a transaldolase reaction with l-threonine to yield synthetically challenging β-hydroxy-α-amino acids. Co-expression of both enzymes in E. coli yields a whole cell biocatalyst capable of synthesizing a variety of stereopure non-standard amino acids (nsAA) and can be produced on gram-scale. We used isotopically labelled substrates to probe the mechanism of SOI, which we show catalyzes a concerted isomerization featuring a stereospecific 1,2-hydride shift. The viability of in situ generated α-aryl aldehydes was further established by intercepting them with a recently engineered decarboxylative aldolase to yield γ-hydroxy nsAAs. Together, these data establish a versatile method of producing α-aryl aldehydes in simple, whole cell conditions and show that these intermediates are useful synthons in C‒C bond forming cascades.

Enzyme-Catalyzed Meinwald Rearrangement with an Unusual Regioselective and Stereospecific 1,2-Methyl Shift.

The Meinwald rearrangement is a synthetically useful reaction but often lacks regioselectivity and stereocontrol. A significant challenge in the Meinwald rearrangement of internal epoxides is the non-regioselective migration of different substituents to give a mixture of products. Herein, an enzyme-catalyzed regioselective and stereospecific 1,2-methyl shift in the Meinwald rearrangement of internal epoxides is reported. Styrene oxide isomerase (SOI) catalyzed the unique isomerization of internal epoxides through 1,2-methyl shift without 1,2-hydride shift to give the corresponding aldehydes and a cyclic ketone as the sole product. SOI-catalyzed isomerization showed high stereospecificity, fully retaining the stereoconfiguration. The synthetic utility of this enzymatic Meinwald rearrangement was demonstrated by its incorporation into three new types of enantioselective cascades, to convert trans-β-methyl styrenes into the corresponding R-configured alcohols, acids, or amines in high ee and yield.

Enzyme‐Catalyzed Meinwald Rearrangement with an Unusual Regioselective and Stereospecific 1,2‐Methyl Shift

AbstractThe Meinwald rearrangement is a synthetically useful reaction but often lacks regioselectivity and stereocontrol. A significant challenge in the Meinwald rearrangement of internal epoxides is the non‐regioselective migration of different substituents to give a mixture of products. Herein, an enzyme‐catalyzed regioselective and stereospecific 1,2‐methyl shift in the Meinwald rearrangement of internal epoxides is reported. Styrene oxide isomerase (SOI) catalyzed the unique isomerization of internal epoxides through 1,2‐methyl shift without 1,2‐hydride shift to give the corresponding aldehydes and a cyclic ketone as the sole product. SOI‐catalyzed isomerization showed high stereospecificity, fully retaining the stereoconfiguration. The synthetic utility of this enzymatic Meinwald rearrangement was demonstrated by its incorporation into three new types of enantioselective cascades, to convert trans‐β‐methyl styrenes into the corresponding R‐configured alcohols, acids, or amines in high ee and yield.

Styrene Oxide Isomerase Catalyzed Meinwald Rearrangement Reaction: Discovery and Application in Single-Step and One-Pot Cascade Reactions

Styrene oxide isomerase (SOI) is a unique enzyme that catalyzes the Meinwald rearrangement of aryl epoxides into carbonyl compounds. In this review, we first summarize the scientific literature on the discovery and characterization of SOI’s catalytic properties such as activity, substrate scope, and enantioselectivity. We then discuss its potential use for biocatalytic synthesis of aldehydes via single-step reactions using cell-free extracts and whole cells. Emerging technologies, such as the use of a stability tag, could enhance SOI biotransformation to reach product concentrations as high as 3.37 M. SOI has also been utilized in the engineering of biocatalytic cascades to produce valuable compounds such as chiral acids, alcohols, and amines from styrene. The best systems developed to date could reach product concentrations up to 100 mM. Additionally, recent efforts are focused on the metabolic engineering of the “styrene-derived” pathway involving SOI for microbial production of the aroma compound 2-phenylethanol (2-PE) from renewable sources such as l-phenylalanine, glucose, or glycerol, achieving concentrations up to 85 mM. The application of two-phase aqueous–organic systems in practical biotransformation reactions involving SOI is also notable. Finally, some perspectives and recommendations for future work are given to set the stage for development of SOI research toward applications in sustainable industrial biocatalysis.

Engineering a Synthetic Pathway for Tyrosol Synthesis in Escherichia coli.

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.

Utilization of a styrene-derived pathway for 2-phenylethanol production in budding yeast.

2-Phenylethanol (2-PE) is an important flavor ingredient and is widely applied in the fields of food, cosmetics, and pharmaceuticals. Despite that Saccharomyces cerevisiae has the ability to naturally synthesize 2-PE via the Ehrlich pathway, de novo synthesis of 2-PE in high titer still remains a huge challenge. In this study, a non-native styrene degradation pathway was introduced into S. cerevisiae, which represents the first time to demonstrate the functional expression of "styrene-derived" 2-PE synthesis in yeast. Using a host strain engineered with L-phenylalanine (L-Phe) overproduction, the heterologous 2-PE pathway coupled with endogenous Ehrlich pathway produced 233 mg/L 2-PE under shake flasks. Additionally, we further engineered the permease transporters to improve the intracellular L-Phe availability, and further improved the 2-PE titer to 680 mg/L. Taken together, our work represents one of the pioneering reports to explore "styrene-derived" pathway in S. cerevisiae. The synthetic yeast described here might be used as a platform for the future development of next-generation high-yielding 2-PE yeast strains.Key Points• A styrene-derived pathway was established in yeast for 2-phenylethanol productions; membrane-associated styrene oxide isomerase was functional in yeast.• Transporter engineering to improve the L-phenylalanine importation with enhanced 2-phenylethanol productions.

High‐Level Production of Phenylacetaldehyde using Fusion‐Tagged Styrene Oxide Isomerase

AbstractAn order‐of‐magnitude improvement in the production of phenylacetaldehyde to 3.37 M (405 g L−1) from the enzymatic isomerisation of styrene oxide was achieved. A small ubiquitin‐related modifier (SUMO) tag increases the productivity of the whole‐cell biocatalytic system by enhancing the expression of active membrane‐bound styrene oxide isomerase (SOI) while retaining enzyme catalytic efficiency and broad natural substrate scope. The isomerisation was performed by using Escherichia coli expressing SUMO‐tagged SOI in an organic‐aqueous biphasic system to yield 96% of phenylacetaldehyde.magnified image

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2.

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.

A Review: The Styrene Metabolizing Cascade of Side-Chain Oxygenation as Biotechnological Basis to Gain Various Valuable Compounds.

Styrene is one of the most produced and processed chemicals worldwide and is released into the environment during widespread processing. But, it is also produced from plants and microorganisms. The natural occurrence of styrene led to several microbiological strategies to form and also to degrade styrene. One pathway designated as side-chain oxygenation has been reported as a specific route for the styrene degradation among microorganisms. It comprises the following enzymes: styrene monooxygenase (SMO; NADH-consuming and FAD-dependent, two-component system), styrene oxide isomerase (SOI; cofactor independent, membrane-bound protein) and phenylacetaldehyde dehydrogenase (PAD; NAD+-consuming) and allows an intrinsic cofactor regeneration. This specific way harbors a high potential for biotechnological use. Based on the enzymatic steps involved in this degradation route, important reactions can be realized from a large number of substrates which gain access to different interesting precursors for further applications. Furthermore, stereochemical transformations are possible, offering chiral products at high enantiomeric excess. This review provides an actual view on the microbiological styrene degradation followed by a detailed discussion on the enzymes of the side-chain oxygenation. Furthermore, the potential of the single enzyme reactions as well as the respective multi-step syntheses using the complete enzyme cascade are discussed in order to gain styrene oxides, phenylacetaldehydes, or phenylacetic acids (e.g., ibuprofen). Altered routes combining these putative biocatalysts with other enzymes are additionally described. Thus, the substrates spectrum can be enhanced and additional products as phenylethanols or phenylethylamines are reachable. Finally, additional enzymes with similar activities toward styrene and its metabolic intermediates are shown in order to modify the cascade described above or to use these enzyme independently for biotechnological application.

Biocatalytic Formal Anti-Markovnikov Hydroamination and Hydration of Aryl Alkenes

Biocatalytic anti-Markovnikov alkene hydroamination and hydration were achieved based on two concepts involving enzyme cascades: epoxidation–isomerization–amination for hydroamination and epoxidation–isomerization–reduction for hydration. An Escherichia coli strain coexpressing styrene monooxygenase (SMO), styrene oxide isomerase (SOI), ω-transaminase (CvTA), and alanine dehydrogenase (AlaDH) catalyzed the hydroamination of 12 aryl alkenes to give the corresponding valuable terminal amines in high conversion (many ≥86%) and exclusive anti-Markovnikov selectivity (>99:1). Another E. coli strain coexpressing SMO, SOI, and phenylacetaldehyde reductase (PAR) catalyzed the hydration of 12 aryl alkenes to the corresponding useful terminal alcohols in high conversion (many ≥80%) and very high anti-Markovnikov selectivity (>99:1). Importantly, SOI was discovered for stereoselective isomerization of a chiral epoxide to a chiral aldehyde, providing some insights on enzymatic epoxide rearrangement. Harnessing this s...

Regio‐ and Stereoselective Oxidation of Styrene Derivatives to Arylalkanoic Acids via One‐Pot Cascade Biotransformations

AbstractGreen and selective oxidation methods are highly desired in chemical synthesis and manufacturing. In this work, we have developed a biocatalytic method for the regio‐ and stereoselective oxidation of styrene derivatives into arylacetic and (S)‐2‐arylpropionic acids via a one‐pot epoxidation–isomerization–oxidation sequence. This was done via the engineering of Escherichia coli (StyABC‐EcALDH) coexpressing styrene monooxygenase (SMO), styrene oxide isomerase (SOI) and aldehyde dehydrogenase (EcALDH) as an active and easily available whole‐cell catalyst. Regioselective oxidation of styrene and 11 substituted styrenes using the E. coli cells was performed in a one‐pot set‐up, producing 12 phenylacetic acids in both high conversion and high yield. Engineering of E. coli (StyABC‐ADH9v1) coexpressing SMO, SOI and ADH9v1 (a mutated alcohol dehydrogenase) led to biocatalysts capable of regio‐ and stereoselective oxidation of α‐methylstyrene derivatives to the corresponding chiral acids. One‐pot asymmetric synthesis of 4 (S)‐2‐arylpropionic acids was achieved in good conversion and excellent ee with the E. coli cells. This is a new type of asymmetric alkene oxidation to give chiral acids with no chemical counterpart thus far. The cascade bio‐oxidation operates under mild conditions, uses molecular oxygen, exhibits very high regio‐ and enantioselectivity, and gives high conversion, thus providing a green and efficient method for the synthesis of arylacetic acids and (S)‐2‐arylpropionic acids directly from easily available styrenes.magnified image

Heterologous production of different styrene oxide isomerases for the highly efficient synthesis of phenylacetaldehyde

The styrene oxide isomerase (SOI, StyC) represents a key enzyme of the styrene-degrading pathway and has been discussed as promising biocatalyst during recent studies. The enzyme enables the synthesis of pure phenylacetaldehyde from styrene oxide. In this study the native as well as the corresponding codon-optimized genes of three different SOIs from Rhodococcus opacus 1CP (StyC-1CP), Sphingopyxis fribergensis Kp5.2 (StyC-Kp5.2), and Pseudomonas fluorescens ST (StyC-ST) were investigated for the expression in Escherichia coli BL21(DE3)pLysS. Specific enzyme activities of 61.9±7.5Umg−1, 23.2±2.8Umg−1, and 10.9±1.2Umg−1 were achieved after 6–9h for the codon-optimized gene of strain 1CP and the native genes of Kp5.2 and ST, respectively. Afterwards, these enzymes were enriched and applied for biotransformation studies. A complete conversion of 150mM styrene oxide to phenylacetaldehyde was observed for the enzyme StyC-Kp5.2 indicating a significantly improved stability towards product inactivation. Remarkably, more than 300mM product (>36gL−1, yield of about 80%) were finally synthesized from 400mM substrate with 150U of this enzyme within 60-120min. This represents the highest product concentration which has been reached with this type of enzymes, so far.

Characterization of Aldehyde Dehydrogenases Applying an Enzyme Assay with In Situ Formation of Phenylacetaldehydes.

Herein, different dehydrogenases (DH) were characterized by applying a novel two-step enzyme assay. We focused on the NAD(P)+-dependent phenylacetaldehyde dehydrogenases because they produce industrially relevant phenylacetic acids, but they are not well studied due to limited substrate availability. The first assay step comprises a styrene oxide isomerase (440Umg-1protein) which allows the production of pure phenylacetaldehydes (>70mmolL-1) from commercially available styrene oxides. Thereafter, a DH of interest can be added to convert phenylacetaldehydes in a broad concentration range (0.05 to 1.25mmolL-1). DH activity can be determined spectrophotometrically by following cofactor reduction or alternatively by RP-HPLC. This assay allowed the comparison of four aldehyde dehydrogenases and even of an alcohol dehydrogenase with respect to the production of phenylacetic acids (up to 8.4Umg-1protein). FeaB derived from Escherichia coli K-12 was characterized in more detail, and for the first time, substituted phenylacetaldehydes had been converted. With this enzyme assay, characterization of dehydrogenases is possible although the substrates are not commercially available in sufficient quality but enzymatically producible. The advantages of this assay in comparison to the former one are discussed.

Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13mgL(-1) after 24h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas.