A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

Microbial production of the aromatic building-blocks (S)-styrene oxide and (R)-1,2-phenylethanediol from renewable resources

(S)-Styrene oxide and (R)-1,2-phenylethanediol are chiral aromatic molecular building blocks used commonly as precursors to pharmaceuticals and other specialty chemicals. Two pathways have been engineered in Escherichia coli for their individual biosynthesis directly from glucose. The novel pathways each constitute extensions of the previously engineered styrene pathway, developed by co-expressing either styrene monooxygenase (SMO) or styrene dioxygenase (SDO) to convert styrene to (S)-styrene oxide and (R)-1,2-phenylethanediol, respectively. StyAB from Pseudomonas putida S12 was determined to be the most effective SMO. SDO activity was achieved using NahAaAbAcAd of Pseudomonas sp. NCIB 9816-4, a naphthalene dioxygenase with known broad substrate specificity. Production of phenylalanine, the precursor to both pathways, was systematically enhanced through a number of mutations, most notably via deletion of tyrA and over-expression of tktA. As a result, (R)-1,2-phenylethanediol reached titers as high as 1.23 g/L, and at 1.32 g/L (S)-styrene oxide titers already approach their toxicity limit. As with other aromatics, product toxicity was strongly correlated with a model of membrane accumulation and disruption. This study additionally demonstrates that greater flux through the styrene pathway can be achieved if its toxicity is addressed, as achieved in this case by reacting styrene to less toxic products. See accompanying commentary by Brian F. Pfleger DOI: 10.1002/biot.201300251     

Protein engineering of hydrogenase 3 to enhance hydrogen production

The large subunit (HycE, 569 amino acids) of Escherichia coli hydrogenase 3 produces hydrogen from formate via its Ni–Fe-binding site. In this paper, we engineered HycE for enhanced hydrogen production by an error-prone polymerase chain reaction (epPCR) using a host that lacked hydrogenase activity via the hyaB hybC hycE mutations. Seven enhanced HycE variants were obtained with a novel chemochromic membrane screen that directly detected hydrogen from individual colonies. The best epPCR variant contained eight mutations (S2T, Y50F, I171T, A291V, T366S, V433L, M444I, and L523Q) and had 17-fold higher hydrogen-producing activity than wild-type HycE. In addition, this variant had eightfold higher hydrogen yield from formate compared to wild-type HycE. Deoxyribonucleic acid shuffling using the three most-active HycE variants created a variant that has 23-fold higher hydrogen production and ninefold higher yield on formate due to a 74-amino acid carboxy-terminal truncation. Saturation mutagenesis at T366 of HycE also led to increased hydrogen production via a truncation at this position; hence, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30-fold. This is the first random protein engineering of a hydrogenase.     

Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system

ABSTRACT

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL^?1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol^?1) styrene, respectively.     

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.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

Stabilization of mesophilic Allochromatium vinosum cytochrome c′ through specific mutations modeled by a thermophilic homologue

AVCP cytochrome c′ from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c′ (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability.     

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.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2‐bromo phenyl acetic acid esters

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S‐enantioselectivity (E‐value = 5) during the hydrolytic kinetic resolution of 2‐bromo‐phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R‐selective enzyme (E‐value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low‐hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site‐directed mutagenesis. This work led to the isolation of one double substituted variant (D97A‐V232F), which displays a total preference for the R‐enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5‐fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R‐ and S‐enantiomers in the wild‐type enzyme and the D97A‐V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R‐enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity. Biotechnol. Bioeng. 2010;106: 852–859. © 2010 Wiley Periodicals, Inc.     

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

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.     

Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 +/- 0.09 versus 1.04 +/- 0.07 micromol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 +/- 2 versus 2.4 +/- 0.3 micromol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 +/- 0.5 versus 2.6 +/- 0.0 micromol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 +/- 0.3 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 +/- 0.8 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP)

To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of k_cat/K_m value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.     

Directed evolution of a beta-galactosidase from <Emphasis Type="Italic">Pyrococcus woesei</Emphasis> resulting in increased thermostable beta-glucuronidase activity

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24

Rhodococcus strain I24 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from I24 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalene-type dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain I24. Received: 8 December 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999