Michel Oelschlägel

Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

ABSTRACT 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.

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.

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

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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.

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)).

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

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

ABSTRACT 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.

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.

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

Highlights • Co-metabolic turnover of 4-Cl-styrene was investigated in a Pseudomonas strain.• Important parameters to optimize this co-metabolism were investigated.• Inhibition by product and under some conditions by the substrate were revealed.• Influence of salt and trace elements on the inhibition were proved.• 1.4-fold more product in a 18.5-fold shorter reaction time were achieved.