Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120

Abstract

In order to design a biocatalyst for the production of optically pure styrene oxide, an important building block in organic synthesis, the metabolic pathway and molecular biology of styrene degradation in Pseudomonas sp. strain VLB120 was investigated. A 5.7-kb XhoI fragment, which contained on the same strand of DNA six genes involved in styrene degradation, was isolated from a gene library of this organism in Escherichia coli by screening for indigo formation. T7 RNA polymerase expression experiments indicated that this fragment coded for at least five complete polypeptides, StyRABCD, corresponding to five of the six genes. The first two genes encoded the potential carboxy-terminal part of a sensor, named StySc, and the complete response regulator StyR. Fusion of the putative styAp promoter to a lacZ reporter indicated that StySc and StyR together regulate expression of the structural genes at the transcriptional level. Expression of styScR also alleviated a block that prevented translation of styA mRNA when a heterologous promoter was used. The structural genes styA and styB produced a styrene monooxygenase that converted styrene to styrene oxide, which was then converted to phenylacetaldehyde by StyC. Sequence homology analysis of StyD indicated a probable function as a phenylacetaldehyde dehydrogenase. To assess the usefulness of the enzymes for the production of enantiomerically pure styrene oxide, we investigated the enantiospecificities of the reactions involved. Kinetic resolution of racemic styrene oxide by styrene oxide isomerase was studied with E. coli recombinants carrying styC, which converted styrene oxide at a very high rate but with only a slight preference for the S enantiomer. However, recombinants producing styrene monooxygenase catalyzed the formation of (S)-styrene oxide from inexpensive styrene with an excellent enantiomeric excess of more than 99% at rates up to 180 U g (dry weight) of cells-1.

FIG. 1

(A) Proposed reaction sequence mediated by enzymes encoded on the 5.7-kb XhoI fragment of Pseudomonas sp. strain VLB120. Compounds, from left to right: styrene, styrene oxide (the asterisk indicates the chiral carbon atom), phenylacetaldehyde, and phenylacetic acid. (B) Restriction site map and genetic structure of the XhoI fragment in pSPW1. Stippled boxes indicate vector pZero2.1-derived sequences. The lacZp promoter is provided by the vector. The putative sty promoter is denoted styAp. The restriction sites external to the two XhoI sites, and therefore external to the insert, were derived from the pZero2.1 polylinker. Arrows indicate annealing sites for PCR primers P1 to P4 (see Materials and Methods). (C) Analysis of the XhoI fragment with different deletion derivatives of pSPW1. The sites used for deletions are indicated (and also presented in panel B). The lacZp promoter remained unaffected by the deletions. Designations of the plasmids resulting from the deletions are indicated on the right. E. coli recombinant strains carrying such plasmids were analyzed for formation of styrene oxide (So) from styrene or of phenylacetaldehyde and 2-phenylethanol (Pl) from styrene oxide or production of indigo (In) after overnight growth on LB medium. Plus signs indicate quantitative conversion of the substrate or blue color for indigo; minus signs indicate no conversion or no blue color in the culture medium. The plus sign in parentheses indicates an at least 12-fold-lower styrene oxide specific activity than that indicated by a plus sign without parentheses (conversion was not complete). (D) pPT7T-derived constructs for T7 RNA polymerase-based labeling of translation products. The fragments were obtained by digestion with the indicated restriction enzymes and introduced into the pPT7T polylinker with either the same sites in the polylinker or sites indicated additionally. The position of the T7 promoter (T7p) and the T7 terminator (stem-loop structure) are given only for one construct but are present in all plasmids. The names of the resulting plasmids are given on the right. Restriction sites relevant for constructions: A, ApaI; Bg, BglII; C, ClaI; E, EcoRI; F, FspI; H, HincII; Nc, NcoI; Nr, NruI; P, PstI; Sl, SalI; Sc, SacI; Sm, SmaI; Sn, SnaBI; St, StuI; Xb, XbaI; Xh, XhoI.

FIG. 2

[³⁵S]methionine labeling of T7 RNA polymerase-based translation products. E. coli BL21(DE3) carrying pPT7T without the insert (lane 1), with the complete 5.7-kb XhoI fragment (lane 2), or with fragments containing only one complete ORF (lanes 3 to 8) was induced with IPTG; proteins synthesized under control of the T7 promoter were labeled with [³⁵S]methionine. Protein extracts were separated on an SDS–12% polyacrylamide gel. Lanes: 1, pPT7T; 2, pT7ST-ScD; 3, pT7ST-R; 4, pT7ST-A; 5, pT7ST-Am; 6, pT7ST-B; 7, pT7ST-C; 8, pT7ST-D.

FIG. 3

Effect of shortening or removing the 163-bp sequence upstream of styA in the presence or absence of the regulatory genes styScR. (A) Structure of the investigated plasmids. Drawing is not to scale. The stippled and hatched boxes represent the wild-type SD sequence and altered SD sequences of styA, respectively. The latter was inserted to provide translational signals. (B) The left graph shows styrene oxide formation in E. coli JM101 cells carrying one of the plasmids, 1 to 3, shown in panel A, measured 4 h after IPTG induction. The right graph shows styrene oxide formation in E. coli JM101 cells carrying pSTFull and pSTHalf in the presence of low-copy-number plasmid pCKST-ScR expressing styScR from lacZp, measured 4 h after IPTG induction.

FIG. 4

Production of (S)-styrene oxide by StyAB. (A) Separation of 1.5 mM racemic styrene oxide on a chiral cyclodextrin column. The peak at 18.7 min is phenylacetaldehyde, which can be formed due to the injector temperature. (B) (S)-Styrene oxide with an e.e. of 93% formed by E. coli JM101(pBG63), which produces xylene oxygenase and served as a reference. (C) (S)-Styrene oxide is formed with an e.e. of >99% by E. coli JM101(pSPW5), which produces styrene monooxygenase. Columns were intentionally overloaded to obtain a signal for the least abundant enantiomer in panels B and C.

FIG. 5

Involvement of stySc and styR in transcriptional regulation of sty structural genes. (A) Plasmids used for analysis. The basic replicon determining the plasmid copy number is indicated in brackets. The hatched elements represent the styAp promoter and the part of styA that has been fused to the ′lacZ gene of pUJ9. Drawing is not to scale. (B) Transcriptional activity of IPTG-induced E. coli CC118 carrying different plasmid combinations. Left panel, experiment with styScR present on one plasmid; right panel: experiments with stySc and styR present on two different plasmids. Minus signs indicate that the vector without the insert was present as a control; plus signs indicate the presence of the plasmid shown on the left.

FIG. 6

Analysis of the region between styR and styA. (A) Schematic representation of the region as it is present in pSTFull, which contains the complete 163 bp between the SacI site and the ATG codon of styA, including the vector-derived lacZp. Vector- and insert-derived sequences are indicated. Sequence length numbers refer to the two segments for which mRNA secondary structures were predicted. A putative LacZα fusion peptide is terminated by an insert-derived in-frame stop codon. Known (+1, for lacZa-mRNA) or assumed (lightly shaded arrow, for styA-mRNA) mRNA synthesis starts are indicated. The inverted repeats of the tod box are indicated by repetitive carets, while the larger repeats present in the sty system are indicated by open arrows. (B) Predicted mRNA secondary structures when either the region between the SacI site upstream of the 163-bp sequence and the HincII site downstream of the ATG of styA or the region between the NcoI and the HincII site is transcribed. Free energies for the entire structures are given. Relevant sequences are boxed. The tod box sequence is printed in boldface type, and the AUG codon of styA mRNA is, additionally, italicized. (C) Amino acid sequence comparison of the putative protein regions involved directly in DNA binding for StyR and TodT, with a postulated consensus sequence for the equivalent region of class 3 response regulators derived from the alignment of 19 proteins (see text). Boxed letters indicate identical residues between StyR and TodT; italicized letters indicate identical residues in all three sequences.