Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria.

Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.

Investigation of the co-metabolic transformation of 4-chlorostyrene into 4-chlorophenylacetic acid in Pseudomonas fluorescens ST.

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.

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.

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.

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 (440 U mg-1protein) which allows the production of pure phenylacetaldehydes (>70 mmol L-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 1.25 mmol L-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 8.4 U mg-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.

Sphingopyxis fribergensis sp. nov., a soil bacterium with the ability to degrade styrene and phenylacetic acid.

Strain Kp5.2(T) is an aerobic, Gram-negative soil bacterium that was isolated in Freiberg, Saxony, Germany. The cells were motile and rod-shaped. Optimal growth was observed at 20-30 °C. The fatty acids of strain Kp5.2(T) comprised mainly C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). The major respiratory quinone was Q-10. The major polar lipids of strain Kp5.2(T) were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and sphingoglycolipid. The G+C content of the genomic DNA was 63.7%. Sequencing of the 16S rRNA gene of strain Kp5.2(T) allowed its classification into the family Sphingomonadaceae, and the sequence showed the highest similarity to those of members of the genus Sphingopyxis, with Sphingopyxis italica SC13E-S71(T) (99.15% similarity), Sphingopyxis panaciterrae Gsoil 124(T) (98.96%), Sphingopyxis chilensis S37(T) (98.90%) and Sphingopyxis bauzanensis BZ30(T) (98.51%) as the nearest neighbours. DNA-DNA hybridization and further characterization revealed that strain Kp5.2(T) can be considered to represent a novel species of the genus Sphingopyxis. Hence, the name Sphingopyxis fribergensis sp. nov. is proposed, with the type strain Kp5.2(T) ( = DSM 28731(T) = LMG 28478(T)).

Co-metabolic formation of substituted phenylacetic acids by styrene-degrading bacteria.

Some soil bacteria are able to metabolize styrene via initial side-chain oxygenation. This catabolic route is of potential biotechnological relevance due to the occurrence of phenylacetic acid as a central metabolite. The styrene-degrading strains Rhodococcus opacus 1CP, Pseudomonas fluorescens ST, and the novel isolates Sphingopyxis sp. Kp5.2 and Gordonia sp. CWB2 were investigated with respect to their applicability to co-metabolically produce substituted phenylacetic acids. Isolates were found to differ significantly in substrate tolerance and biotransformation yields. Especially, P. fluorescens ST was identified as a promising candidate for the production of several phenylacetic acids. The biotransformation of 4-chlorostyrene with cells of strain ST was shown to be stable over a period of more than 200 days and yielded about 38 mmolproduct gcelldryweight-1 after nearly 350 days. Moreover, 4-chloro-α-methylstyrene was predominantly converted to the (S)-enantiomer of the acid with 40% enantiomeric excess.

Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids.

The styrene oxide isomerase (SOI) represents a membrane-bound enzyme of the microbial styrene degradation pathway and has been discussed as promising biocatalyst. It catalyzes the isomerization of styrene oxide to phenylacetaldehyde. In this study a styC gene, which encodes the SOI of Rhodococcus opacus 1CP, was optimized for optimal expression in Escherichia coli BL21(DE3) pLysS. The expression of this synthetic styC was investigated and subsequently optimized. Highly active biomass was obtained yielding an SOI activity of 44.5 ± 8.7 U mg-1 after 10 h. This represents the highest SOI activity reported for crude cell extracts of SOI-containing bacterial strains. Remarkably, this biomass can be applied as whole cell biocatalyst for the production of phenylacetic acids from styrene oxides. In the case of non-substituted styrene oxide, nearly 730 mg l-1 phenylacetic acid (∼85% yield) was formed over a period of 20 days.

Styrene oxide isomerase of Sphingopyxis sp. Kp5.2.

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.

Immobilization of an integral membrane protein for biotechnological phenylacetaldehyde production.

Styrene oxide isomerase (SOI) has previously been shown to be an integral membrane protein performing a highly selective, hydrolytic ring opening reaction of epoxides to yield pure aldehydes. Earlier studies had also shown a high sensitivity of SOIs toward their product phenylacetaldehyde which caused an irreversible inhibition and finally complete loss of activity at higher aldehyde concentrations. Here we report on the covalent immobilization of a styrene oxide isomerase (SOI) on SBA-15 silica carriers. The production of the SOI from a Rhodococcus strain was optimized, the enzyme was enriched and immobilized, and finally the biocatalyst was applied in aqueous as well as in two-phase systems. Linkage of the protein to epoxide or amino groups on the SBA-based carriers led to relatively poor stabilization of the enzyme in an aqueous system. But, improved stability was observed toward organic phases like the non-toxic phthalate-related 1,2-cyclohexane dicarboxylic acid diisononyl ester (Hexamol DINCH) which here to our knowledge was used for the first time in a biotechnological application. With this two-phase system and the immobilized SOI, 1.6-2.0× higher product yields were reached and the lifetime of the biocatalyst was tremendously increased.

Styrene oxide isomerase of Rhodococcus opacus 1CP, a highly stable and considerably active enzyme.

Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg(-1) represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 µmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.