Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase.

By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.     

Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X

Styrene oxide and 2-phenylethanol metabolism in the styrene-degrading Xanthobacter sp. strain 124X was shown to proceed via phenylacetaldehyde and phenylacetic acid. In cell extracts 2-phenylethanol was oxidized by a phenazine methosulfate-dependent enzyme, probably a pyrroloquinoline quinone enzyme. Xanthobacter sp. strain 124X also contains a novel enzymatic activity designated as styrene oxide isomerase. Styrene oxide isomerase catalyzes the isomerization of styrene oxide to phenylacetaldehyde. The enzyme was partially purified and shown to have a very high substrate specificity. Of the epoxides tested, styrene oxide was the only substrate transformed. The initial step in styrene metabolism in Xanthobacter sp. strain 124X is oxygen dependent and probably involves oxidation of the aromatic nucleus.     

Possible regulatory role for nonaromatic carbon sources in styrene degradation by Pseudomonas putida CA-3.

Styrene metabolism in styrene-degrading Pseudomonas putida CA-3 cells has been shown to proceed via styrene oxide, phenylacetaldehyde, and phenylacetic acid. The initial step in styrene degradation by strain CA-3 is oxygen-dependent epoxidation of styrene to styrene oxide, which is subsequently isomerized to phenylacetaldehyde. Phenylacetaldehyde is then oxidized to phenylacetic acid. Styrene, styrene oxide, and phenylacetaldehyde induce the enzymes involved in the degradation of styrene to phenylacetic acid by P. putida CA-3. Phenylacetic acid-induced cells do not oxidize styrene or styrene oxide. Thus, styrene degradation by P. putida CA-3 can be subdivided further into an upper pathway which consists of styrene, styrene oxide, and phenylacetaldehyde and a lower pathway which begins with phenylacetic acid. Studies of the repression of styrene degradation by P. putida CA-3 show that glucose has no effect on the activity of styrene-degrading enzymes. However, both glutamate and citrate repress styrene degradation and phenylacetic acid degradation, showing a common control mechanism on upper pathway and lower pathway intermediates.     

Construction of a bacterial biosensor for styrene

A new bacterial biosensor for styrene has been developed and characterized. A translational fusion of the lacZ gene to the sty promoter of Pseudomonas sp. strain Y2 has been inserted into miniTn5. Transposition of the recombinant transposon to the chromosome of Pseudomonas sp. strain Y2 resulted in a whole-cell biosensor able to detect and degrade styrene. In this biosensor, the endogenous StyS/StyR system detects the presence of styrene and turns on the expression of the exogenous reporter gene from the transferred construction. Other compounds such as toluene, epoxystyrene, phenylacetaldehyde and 2-phenylethanol also induced expression of beta-galactosidase although quantitative differences in their effect are clearly detected. Non-inducing compounds affect differently the sensitivity to inducing compounds when present in a mixture.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase.

The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid. The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase. The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase. Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption.     

Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase

A whole cell biocatalytic process was developed to enable the efficient oxidation of styrene to chiral (S)-styrene oxide with an enantiomeric excess better than 99%. Recombinant Escherichia coli cells were employed to express the genes styAB encoding the styrene monooxygenase of Pseudomonas sp. strain VLB120 from an expression plasmid utilizing the alk regulatory system of P. oleovorans GPo1. The strains reached specific activities of up to 70 U* (g cell dry weight)(-1) in shake-flask experiments with glucose as the carbon source. An efficient two-liquid phase fed-batch process was established for the production of (S)-styrene oxide with hexadecane as an apolar carrier solvent and a nutrient feed consisting of glucose, magnesium sulfate, and yeast extract. Engineering of the phase fraction and the composition of organic phase and feed led to a 2-L scale process with maximal volumetric productivities of 2.2 g (S)-styrene oxide per liter liquid volume per hour. This optimized process was based completely on defined medium and used bis(2-ethylhexyl)phthalate as the apolar carrier solvent, which together with substrate and inducer consisted of 50% of the total liquid volume. Using this system, we were able to produce per liter liquid volume 11 g of enantiopure (S)-styrene oxide in 10 h.     

Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides

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.     

The effect of nutrient limitation on styrene metabolism in Pseudomonas putida CA-3.

Styrene degradation in Pseudomonas putida CA-3 has previously been shown to be subject to catabolite repression in batch culture. We report here on the catabolite-repressing effects of succinate and glutamate and the effects of a limiting inorganic-nutrient concentration on the styrene degradation pathway of P. putida CA-3 in a chemostat culture at low growth rates (0.05 h-1). Oxidation of styrene and the presence of styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities were used as a measure of the expression of the styrene degradation pathway. Both glutamate and succinate failed to repress the styrene degradation ability under growth conditions of carbon and energy limitation. Lower levels of enzyme activities of the styrene degradation pathway were seen in cells grown on styrene or phenylacetic acid (PAA) under conditions of both ammonia and sulfate limitation than were seen under carbon and energy limitation. Cells grown on PAA under continuous culture oxidize styrene and styrene oxide and possess styrene oxide isomerase and NAD(+)-dependent phenylacetaldehyde dehydrogenase activities. Catabolite repression of styrene metabolism was observed in cells grown on styrene or PAA in the presence of growth-saturating (nonlimiting) concentrations of succinate or glutamate under sulfate limitation.     

Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.     

Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase

Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.     

Mutagenicity of styrene on metabolizing D7 strain of saccharomyces cerevisiae

The level of cyt.p-450 in the D7 strain of the yeast S.cerevisiae depended on the substrate supporting the growth, on its concentration, on the starting inoculated number of cells. (1) In the yeast grown on D-mannose where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. It was detected a maximal concentration during the logarithmic phase when in the cultures there are about 50 . 10(6) cell/ml. We use cells harvested at this moment of the growth for mutagenesis tests. The tested substances were dimethylnitrosamine and styrene. DMNA to probe the sensibility of our cells and styrene that has always given contrasting results but from which the formation is known of genetically active metabolite: styrene oxide(6-7). Styrene gave positive results with our metabolizing yeast cells.     

Purification and characterization of phenylacetaldehyde reductase from a styrene-assimilating Corynebacterium strain, ST-10.

A novel phenylacetaldehyde reductase was purified about 50-fold to homogeneity from Corynebacterium sp. strain ST-10, which can assimilate gaseous styrene as the sole carbon and energy source. The enzyme was inductively synthesized when grown on gaseous styrene and had an important role in styrene metabolism in vivo. The enzyme had a molecular weight of 155,000 and was composed of four identical subunits (molecular weight, 42,000). The enzyme catalyzed the reduction of not only phenylacetaldehyde but also various aldehydes and ketones; however, it did not catalyze the reverse reaction, the dehydrogenation of 2-phenylethanol. The enzyme required NADH as a cofactor and showed no activity with NADPH; therefore, it was defined as an NADH-dependent phenylacetaldehyde reductase. The enzyme stereospecifically produced (S)-(-)-1-phenylethanol from acetophenone; therefore, it would be useful as a biocatalyst.     

Indigo formation by microorganisms expressing styrene monooxygenase activity.

The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole.     

手性苯基环氧乙烷的生物不对称合成

以苯乙烯为唯一碳源和能源,从不同来源的土壤样品中初筛分离出12株好氧细菌和2株真菌,经复筛,对液体培养物进行手性气相色谱分析,得到一株产生手性苯基环氧乙烷活力较高的菌种PS-1206,并对其发酵、产酶及苯乙烯的全细胞转化进行了研究,利用微生物细胞在30℃,pH 7.0,10mmol/L磷酸缓冲液中转化0.5%苯乙烯10h,获得?苯基环氧乙烷,e.e%值为80%,转化产率为35%。     

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee >98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee >98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.     

Diauxic Growth of Fusarium oxysporum during Aerobic Culture in the Presence of Nitrate/Nitrite

Styrene oxide isomerase (SOI) [EC 5.3.99.7], most probably located in the cell wall, was partially purified from Coiynebacterium sp. AC-5 cells grown in a styrene gas atmospheres. The enzyme catalyzed the isomerization reaction to give phenylacetaldehyde, but did not catalyze its reverse reaction. The optimum pH of the reaction was around 7.0, and the enzyme was unstable below pH 6.0. The K_m toward styrene oxide was very low (7.7 × 10^?5 m), indicating its high affinity for styrene oxide. The enzyme showed strict substrate specificity, and epoxide compounds other than styrene oxide did not serve as substrates. (S)-Styrene oxide was preferentially converted by the enzyme, compared with the (R)-isomer. The possible application of SOI as a biocatalyst is also discussed.     

Development of recombinantPseudomonas putida containing homologous styrene monooxygenase genes for the production of (S)-styrene oxide

Recently isolated,Pseudomonas putida SN1 grows on styrene as its sole carbon and energy source through successive oxidation of styrene by styrene monooxygenase (SMO), styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase. For the production of (S)-styrene oxide, two knockout mutants of SN1 were constructed, one lacking SOI and another lacking both SMO and SOI. These mutants were developed into whole-cell biocatalysts by transformation with a multicopy plasmid vector containing SMO genes (styAB) of the SN1. Neither of these self-cloned recombinants could grow on styrene, but both converted styrene into an enantiopure (S)-styrene oxide (e.e.>99%). Whole-cell SMO activity was higher in the recombinant constructed from the SOI-deleted mutant (130 U/g cdw) than in the other one (35 U/g cdw). However, the SMO activity of the former was about the same as that of the SOI-deleted SN1 possessing a single copy of thestyAB gene that was used as host. This indicates that the copy number ofstyAB genes is not rate-limiting on SMO catalysis by whole-cell SN1.     

Serogrouping of oral Streptococcus intermedius

Employing twenty fresh oral isolates of Streptococcus intermedius, studies were carried out to characterize serological relations among the isolates and also between the isolates and the strains of bacterial species closely related to S. intermedius. The Rantz-Randall extracts from the cells were used as antigens. The anti-rabbit serum raised against S. intermedius ATCC 27335T reacted with the cell extracts from only three strains of the isolates, which were designated serogroup I strains. The other isolates were classified into four serogroups, I, III, IV, and V, which specifically reacted with the cell extracts from the homologous serogroup strains. However, the serogroup II antiserum formed in immunodiffusion a common precipitin line between the extracts from the cells of serogroups II and I. The serogroups I, III, IV, and V antisera reacted with none of the extracts from the bacterial cells closely related to S. intermedius, which included Streptococcus anginosus ATCC 33397T, Streptococcus constellatus ATCC 27823T, three NCTC strains of "Streptococcus milleri," and three ATCC strains of Streptococcus MG. The precipitin line formed by the homologous reaction of the serogroup II antiserum was found to be a reaction of identity with that formed by the extract from "S. milleri" NCTC 10708. Conversely, the antiserum against NCTC 10708 strain did not react with the cell extracts of serogroup II.     

Epoxide hydrolase activity of Streptomyces strains

The discovery of epoxide hydrolases within a Streptomyces sp. strain collection is described. Screening was performed in 96 well microtiter plates using a modified 4-(p-nitrobenzyl)pyridine assay with styrene oxide, 1,2-epoxy-hexane or 3-phenyl ethylglycidate (3-PEG) as substrates. Out of 120 strains investigated, S. antibioticus Tü4, S. arenae Tü495 and S. fradiae Tü27 exhibited epoxide hydrolase activity. These strains were further investigated by performing laboratory-scale biotransformations utilizing styrene oxide, 1,2-epoxy-hexane and 3-PEG followed by subsequent quantitative analysis employing chiral gas chromatography. The highest conversions were achieved with whole cells from S. antibioticus Tü4 in the presence of 10% (v/v) DMSO. However, enantioselectivity was only satisfying (E = 31) in the presence of 5% (v/v) acetone, which allowed isolation of optically pure non-hydrolyzed (R)-styrene oxide (99% enantiomeric excess (ee)) and (S)-phenyl-1,2-ethandiol (72% ee) at 55% conversion after 24 h. The resolution of 3-PEG proceeded with slightly lower enantioselectivity albeit higher reaction rates. With S. fradiae Tü27 and S. arenae Tü495 enantioselectivity towards styrene oxide was only E = 3-4.