biochemistry genetics and physiology

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Biochemistry, genetics and physiology of

microbial styrene degradation

Niall D. O’Leary a;, Kevin E. O’Connor b, Alan D.W. Dobson a

a Microbiology Department, National Food Biotechnology Centre, National University of Ireland, Cork, Ireland b Department of Industrial Microbiology, National University of Ireland, Dublin, Ireland

Received 24 January 2002; received in revised form 27 June 2002; accepted 8 August 2002

First published online 11 September 2002

Abstract

The last few decades have seen a steady increase in the global production and utilisation of the alkenylbenzene, styrene. The compound

is of major importance in the petrochemical and polymer-processing industries, which can contribute to the pollution of natural resources

via the release of styrene-contaminated effluents and off-gases. This is a cause for some concern as human over-exposure to styrene, and/

or its early catabolic intermediates, can have a range of destructive health effects. These features have prompted researchers to investigate

routes of styrene degradation in microorganisms, given the potential application of these organisms in bioremediation/biodegradation

strategies. This review aims to examine the recent advances which have been made in elucidating the underlying biochemistry, genetics and

physiology of microbial styrene catabolism, identifying areas of interest for the future and highlighting the potential industrial importance

of individual catabolic pathway enzymes.

2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Phenylacetic acid; Two-component; Regulation; Biodegradation; Biotransformation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404

2. Aerobic styrene degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404

2.1. Side-chain oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404

2.2. Direct ring cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

3. Anaerobic styrene metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

4. Genetic organisation of the styrene upper pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406

5. The upper pathway regulatory apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406

5.1. StyS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

5.2. StyR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

6. Pathway-speci¢c regulation of the styrene catabolic operon . . . . . . . . . . . . . . . . . . . . . . . 408

7. Physiological factors a¡ecting styrene degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

7.1. Catabolite repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

7.2. Nutrient limitation in continuous culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

8. Application of styrene-degrading microorganisms in bio¢lters . . . . . . . . . . . . . . . . . . . . . . 411

9. Biotechnological applications of pathway enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

9.1. Styrene oxide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

9.2. Indigo production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

9.3. Other potential biotransformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415

0168-6445 / 02 / $22.00 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 8 - 6 4 4 5 ( 0 2 ) 0 0 1 2 6 - 2

* Corresponding author. Tel.: +353 (21) 490 2952; Fax: +353 (21) 490 3101. E-mail address: [email protected] (N.D. O’Leary).

FEMS Microbiology Reviews 26 (2002) 403^417

www.fems-microbiology.org

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1. Introduction

The extensive use of aromatic hydrocarbons in industri-

al processes, coupled with inadequate waste management

strategies, has led to the widespread introduction of these

compounds into our environment [1]. Styrene, the simplest

of the alkenylbenzenes, is one such compound which isemployed both as a starting material for synthetic poly-

mers and as a solvent in the polymer-processing industry,

leading to its release in a variety of industrial e¥uents.

Styrene is known to be genotoxic while styrene oxide,

the major in vivo metabolite of styrene, is classi¢ed as a

probable carcinogen in humans [2]. Recent reports indi-

cate that styrene may also have an immuno-modulatory

e¡ect on workers exposed to gaseous emissions in an in-

dustrial setting [3]. As a result of concerns arising from

these ¢ndings, the last decade has seen intense investiga-

tion into various aspects of the microbial degradation of

this compound.One feature of styrene which has undoubtedly been in-

£uential in the evolution of microbial styrene catabolic

routes is the natural occurrence of this compound, e.g.

via fungal decarboxylation of cinnamic acid [4,5]. This is

perhaps re£ected in the diverse range of styrene-degrading

microbes that have been isolated from various soil loca-

tions around the globe since the late 1970s. While a de-

pendence on mixed cultures has been reported, pure cul-

ture isolates capable of styrene degradation have included

species of Pseudomonas, Rhodococcus, Nocardia, Xantho-

bacter and Enterobacter as well as the black yeast Exo-

phiala jeanselmei (for a review see Hartmans [6]). How-

ever, as the aim of this review is to present the current

understanding of microbial styrene catabolism, the pri-

mary focus will repeatedly centre upon the genus Pseudo-

monas, from which signi¢cant insights have come in recent

years.

2. Aerobic styrene degradation

Two main pathways for the aerobic degradation of sty-

rene have been described, one involving an initial oxida-

tion of the vinyl side-chain [7] and the other based on

direct attack on the aromatic nucleus [8] (Fig. 1).

2.1. Side-chain oxidation

The side-chain oxidation pathway involves epoxidation

of the vinyl side-chain by a £avin adenine dinucleotide-dependent, two-subunit monooxygenase followed by iso-

merisation of the epoxystyrene formed to phenylacetalde-

hyde (PAAL). This compound is subsequently oxidised to

phenylacetic acid (PAA) through the action of either an

NADþ- or phenazine methosulfate-dependent dehydroge-

nase [6]. This conversion of styrene to PAA is generally

referred to as the upper pathway of styrene degradation

and appears to operate in many of the bacterial strains

studied to date; examples include Pseudomonas putida

CA-3 [9], Xanthobacter strain 124X [10], Xanthobacter

strain S5 [11], Pseudomonas £uorescens ST [12], Pseudomo-

Fig. 1. A summary of the major pathways of bacterial styrene degradation. The number(s) associated with each pathway identify the organism(s) that

have been shown to perform the particular transformation: 1, P. putida CA-3; 2, Xanthobacter strain 124X; 3, Xanthobacter strain S5; 4, P. £uorescens

ST; 5, Pseudomonas sp. strain Y2; 6, Corynebacterium strain ST-10; 7, Rhodococcus rhodochrous NCIMB 13259. Dotted lines indicate proposed degra-

dative routes yet to be formally demonstrated.

N.D. O’Leary et al. / FEMS Microbiology Reviews 26 (2002) 403^417 404



nas sp. strain VLB120 [13] and Pseudomonas sp. strain Y2

[14,15] (Fig. 1).

The lower pathway is thought to involve the conversion

of PAA to Krebs cycle intermediates but this has not been

formally demonstrated in any styrene-degrading bacterium

thus far. The current understanding of the microbial me-

tabolism of PAA owes itself to studies in P. putida U [16]and Escherichia coli W [17]. In both strains, PAA is ¢rst

activated to phenylacetyl-CoA (PACoA), before passing

through a L-oxidation-like, enzyme-catalysed conversion

process to yield acetyl-CoA moieties. Similarly, it has

been reported for both strains that PACoA, not PAA,

acts as the true inducer of the catabolic core. It alleviates

the negative regulation imposed on the pathway by the

DNA-binding transcriptional repressor PaaX in E. coli

and PaaN in P. putida U [18]. This degradative route,

referred to as the PACoA-catabolon, represents the hy-

bridisation of a typically anaerobic step such as coenzyme

A activation, with an aerobic L-oxidation-like process.Analysis of genome sequences has revealed that genetic

elements of this catabolon are found in various bacterial

genera, suggesting that it may represent a common, central

catabolic route for bacterial PAA metabolism [18]. There

are several, cautious lines of evidence to suggest that this

catabolon may also operate in styrene-degrading strains.

Genetic analysis of Pseudomonas sp. strain Y2 [15] and

P. putida CA-3 [19] identi¢ed a PACoA ligase gene,

paaK , directly upstream from the genes responsible for

the conversion of styrene to PAA, which would facilitate

activation of PAA to PACoA. In addition, Baggi and co-

workers reported in P. £uorescens ST the accumulation of

2-hydroxyphenylacetic acid (2-OHPAA) in the culture me-dia of cells grown on styrene and PAA [20]. The authors

proposed that 2-OHPAA could be metabolised via homo-

gentisate to acetoacetate based on the detection of homo-

gentisate-1,2-dioxygenase activity. However, it has been

demonstrated that disruption of the PACoA-catabolon

paaZ gene, encoding an aldehyde dehydrogenase involved

in ring cleavage of 2-OHPACoA in E. coli W, results in

the hydrolysis of accumulated 2-OHPACoA to 2-OHPAA,

which can be detected in the growth medium [17]. Thus

the presence of 2-OHPAA in styrene- and PAA-grown

cultures of P. £uorescens ST may re£ect the role of a

PACoA-catabolon in this strain also [20].It should be noted that in the styrene-degrading Cory-

nebacterium strains AC-5 and ST-10, there appears to be a

modi¢ed form of the upper pathway with PAAL being

reduced to 2-phenylethanol by a PAAL reductase enzyme

[21,22]. Interestingly, an accumulation of 2-phenylethanol

was reported in E. coli JM109 cells when transformed with

the P. £uorescens ST genes encoding styrene monooxyge-

nase and epoxystyrene isomerase, due to the presence of

endogenous PAAL reductase activity, despite the inability

of the natural host to utilise styrene [12,23]. A relatively

minor pathway involving 1-phenylethanol, acetophenone,

salicylate and 2-OHPAA has also been reported to oc-

cur in Pseudomonas sp. strain Y2 [14], suggesting that

more than one degradative route may operate in a single

strain.

2.2. Direct ring cleavage

Rhodococcus rhodochrous NCIMB 13259, a chemicaldump isolate capable of growth on a diverse range of

aromatic hydrocarbons, degrades styrene via direct oxida-

tion of the aromatic nucleus. Thin layer chromatography

and nuclear magnetic resonance studies revealed that sty-

rene metabolism in this organism proceeded via 3-vinyl-

catechol [8] (Fig. 1). An NADþ-dependent cis-glycol

dehydrogenase activity was detected in cells grown on

nutrient broth and styrene. Cells grown under these con-

ditions were also able to oxidise toluene cis-glycol. The

styrene cis-glycol enzyme activity behaves very similarly

to toluene cis-glycol dehydrogenase and is believed to

be the same enzyme with a broad substrate speci¢city.3-Vinylcatechol is further degraded via meta-cleavage

to acetaldehyde and pyruvate. The role of catechol

2,3-dioxygenase in this process was established when

3-vinylcatechol accumulated in cells incubated with sty-

rene and 3-£uorocatechol, an inhibitor of the dioxygenase.

Evidence of direct ring cleavage has also been reported

in P. putida MST where cells grown on styrene release

1,2-dihydroxy-3-ethenyl-3-cyclohexene into the culture me-

dium [24]. Ring attack has also been proposed in Xantho-

bacter strain 124X [10]. The transient accumulation of

a yellow colour in the media of cells grown on styrene

and 1-phenylethanol, but not styrene oxide, corresponds

with observations made in Nocardia species T5 grown on1-phenylethanol. Cripps et al. [25] proposed that the

1-phenylethanol degradation in this Nocardia strain in-

volved initial oxidation of the aromatic nucleus by a di-

oxygenase.

3. Anaerobic styrene metabolism

Degradation of styrene under anaerobic conditions was

observed in an anaerobic consortium enriched on styrene

and ferulic acid [26]. Although a wide range of aromatic

intermediates was detected, the principal route of styrenedegradation under these conditions was proposed to in-

volve conversion to PAA via 2-phenylethanol and PAAL.

This pathway bears similarity to the aerobic transforma-

tions of styrene. The initial step in aerobic degradation of

styrene utilises molecular oxygen while it is thought that

anaerobic bacteria derive the necessary oxygen from avail-

able water. Divergence of the aerobic and anaerobic path-

ways arises with the respective downstream processing of

PAA. Anaerobic consortia are thought to convert PAA to

benzoic acid via benzyl alcohol and benzaldehyde, much

like the meta-cleavage pathway of toluene degradation en-

coded by the TOL plasmid, pWW0 [26].




4. Genetic organisation of the styrene upper pathway

Marconi and co-workers were the ¢rst to report the

cloning and identi¢cation of genes involved in styrene ca-

tabolism [12]. They cloned two genes from the P. £uores-

cens ST chromosome, encoding enzymes with styrene

monooxygenase and epoxystyrene isomerase activities.Further genetic characterisation of the locus by this group

identi¢ed an upper pathway catabolic operon containing

all enzymes necessary for the conversion of styrene to

PAA [23]. The genes involved were styA and styB , encod-

ing a two-subunit styrene monooxygenase responsible

for the transformation of styrene to epoxystyrene, while

styC was reported to encode an epoxystyrene isomerase

that converted styrene oxide to PAAL. The concluding

step of the upper pathway, the formation of PAA, was

performed by the styD-encoded PAAL dehydrogenase

(PAALDH). This operonic organisation was subsequently

reported in three other styrene-degrading Pseudomonasstrains, namely Pseudomonas sp. strain Y2 [15], Pseudomo-

nas sp. strain VLB120, [13], and P. putida CA-3 [19]. The

high degree of sequence similarity between the styA genes

thus far cloned and sequenced (Table 1) suggests a com-

mon evolutionary origin for this catabolic route. This is

further supported by the conserved operonic structure of

the upper pathway between these Pseudomonas strains,

which corresponds with the order of the catabolic steps

(Fig. 2). This observation contrasts sharply with the di-

verse organisation and sequence variation observed in dif-

ferent paa genes from various species capable of PAA

degradation [18], suggesting that independent evolution

of the styrene upper and lower pathways may have oc-

curred.

5. The upper pathway regulatory apparatus

The initial observation in P. £uorescens ST that the ‘sty’

genes were clustered on the chromosome raised the possi-

bility that a pathway-speci¢c regulatory apparatus could

also be in close proximity. This was indeed established by

Velasco et al. following complementation studies in E. coli

W with elements of the Pseudomonas sp. strain Y2 sty

operon [15]. It was reported that expression of the cata-

bolic operon in E. coli W was signi¢cantly reduced in the

absence of two genes, styS and styR, located upstream

from the styrene catabolic operon. Expression was re-

stored when these genes were provided in trans. Severalstudies have since reinforced the essential function of

stySR in styrene catabolism and their role in pathway

regulation will be dealt with in more detail below. styS

and styR (Table 1 ; Fig. 2) encode two proteins which

display a high level of amino acid similarity with members

of the superfamily of two-component signal transduction

systems found in both prokaryotes and eukaryotes [27,32].

Based on sequence analysis and RT-PCR investigations, it

appears that both genes are expressed in a transcription-

ally coupled fashion [19].

Table 1

Comparison of P. putida CA-3 styrene pathway gene products with other proteins of known function

Gene % G+C Product (aa) Similar polypeptide % ID Organism Accession No.

paaK 62.7 437 PaaK (437) 96 Pseudomonas sp. strain Y2 AJ000330

PhaE (439) 85 Pseudomonas putida U AF029714

PaaK (447) 68 Escherichia coli W X97452

PaaK (440) 67 Azoarcus evansii KB740 AF176259

PhaE (445) 49 Bacillus halodurans AP001507

styS 55.5 226 (frag) StyS (982) 99 Pseudomonas sp. strain Y2 AJ000330

StyS (983) 88 Pseudomonas £uorescens ST AF024619

TutC (979) 49 Thauera aromatica U57900

TodS (978) 41 Pseudomonas putida F1 AF180147

styR 52.2 207 StyR (207) 98 Pseudomonas sp. strain Y2 AJ000330

StdR (207) 96 Pseudomonas sp. strain VLB120 AF031161StyR (207) 90 Pseudomonas £uorescens ST AF024619

TutB (218) 55 Thauera aromatica U57900

TodT (227) 49 Pseudomonas putida F1 AF180147

NodW (227) 51 Bradyrhizobium japonicum AF322013

FixJ (211) 40 Azorhizobium caulinodans X56658

styA 57.9 416 StdA (415) 98 Pseudomonas sp. strain VLB120 AF031161

StyA (415) 98 Pseudomonas putida S12 Y13349

StyA (415) 95 Pseudomonas sp. strain Y2 AJ000330

StyA (415) 96 Pseudomonas £uorescens ST AF292524

PaaK, phenylacetyl-coenzyme A ligase (PACoAL) of the styrene degradative pathway [15] ; PaaK and PhaE, aerobic PACoAL from PAA catabolic

pathways [18] ; StyS and StyR, sensor histidine kinase and response regulator from styrene degradative pathway [15,34,41] ; StdR and StdA, regulator

and styrene monooxygenase (large component) [41] ; TodS and TodT, sensor kinase and response regulator, respectively, involved in aerobic toluene

degradation [28] ; TutC and TutB, sensor kinase and response regulator of toluene degradation [29] ; NodW and FixJ, response regulators involved in

nodulation and nitrogen ¢xation, respectively [38,39] ; StyA, oxygenase component of styrene monooxygenases [12,15,41].




5.1. StyS

The StyS proteins exhibit amino acid sequence similarity

to a number of sensor kinase proteins (Table 1), particu-

larly the TodS and TutC sensor kinases that regulate tolu-

ene catabolism in P. putida F1 and DOT-T1E [28,29].

Interestingly, recent work on toluene degradation by

P. putida F1 and DOT-T1E indicates that styrene can actas an inducer of the tod operon [30,31]. StyS consists of

¢ve distinct domains (Fig. 3). Two of these are histidine

kinase domains, HK1 and HK2, both containing the char-

acteristic kinase amino acid blocks H, N, G1, F and G2

(Fig. 3). The occurrence of two perfectly duplicated kinase

cores was previously reported as being unique to the TodS

sensor kinase [31]. It should be noted that the HK1 and

HK2 domains in StyS di¡er slightly and have been further

classi¢ed into the kinase subfamilies 1a and 4, respectively

[27]. The receiver domain of StyS contains the D, D, S, K

amino acid residues typical of bacterial response regula-

tors, and belongs to the RA2 receiver subfamily [27]. Ad- jacent to the receiver is an input region (Input 2), with

signi¢cant similarity to the putative oxygen-sensing do-

main of TodS [28]. Located within this domain are the

S1 (PAS) and S2 (PAC) sensory boxes, typical of PAS

domains found in a variety of prokaryotic and eukaryotic

sensory mechanisms [33]. Zennaro and co-workers suggest

that the presence of the PAS domain may play a role in

the detection of styrene as a stress factor, perhaps as a

consequence of the redox potential generated in the cell

due to the toxicity of the intermediates styrene oxide or

PAAL [34]. However, the observation that input domains

are usually located at the N-terminus of a sensor kinase

has led to the suggestion that another input domain (Input

1) may also be present in StyS [15]. It is not yet known

precisely how the input domain responds to the presence

of styrene.

5.2. StyR

The StyR proteins display amino acid identity withmany response regulators of other two-component sys-

tems. Given the similarity between StyS and the sensor

kinases of the toluene degradation regulatory apparatus,

TodS and TutB, it is not surprising that StyR also exhibits

high level amino acid identity with the cognate response

regulators, TodT and TutC (Table 1). StyR contains a

receiver domain and a DNA-binding domain joined by a

Q-linker region (Fig. 4). The receiver domain belongs to

the RA4 receiver subfamily and contains the essential,

conserved D, D, T and K amino acid residues found in

a variety of bacterial response regulators [27,35]. A con-

sensus sequence of putative DNA-binding domains in thefamily 3 response regulators is also present in the carbox-

yl-terminal region of the proposed protein [36]. The StyS

and StyR proteins are one of only four two-component

regulatory systems known to be involved in aromatic hy-

drocarbon catabolism [37]. Interestingly, the StyR proteins

also display amino acid similarity with the response regu-

lators NodW and FixJ, involved in nodulation and nitro-

gen ¢xation, respectively [38,39] and may, as has been

proposed by Diaz and co-workers, represent another ex-

ample of domain shu¥ing which could have occurred dur-

ing the parallel evolution of family 3 DNA-binding do-

mains with cluster 1 receiver modules [15,36,40].

Fig. 2. Structural organisation of the chromosomally encoded styrene degradative operons in a number of styrene-degrading strains of the genusPseudomonas. The speci¢c transformations are illustrated above the genetic elements encoding the enzyme(s) required for each step. 1, styrene mono-

oxygenase (SMO) subunits 1 and 2 (styA and styB ); 2, styrene oxide isomerase (SOI) styC ; 3, phenylacetaldehyde dehydrogenase (PAALDH) styD ;

4, phenylacetyl-coenzyme A ligase (PACoAL) paaK . White areas indicate regions for which sequence data are unavailable.




6. Pathway-speci¢c regulation of the styrene

catabolic operon

Styrene-dependent induction of the sty operon has been

demonstrated by several groups. Northern blotting and

transcription start site analysis by primer extension in

Pseudomonas sp. strain Y2 revealed that the presence of

styrene was essential for upper pathway gene transcription

[15]. Zennaro and co-workers have reported that L-galac-

tosidase activity was only detectable in P. £uorescens ST

cells harbouring a plasmid-borne styA promoter: :lacZ fu-

sion when styrene was present in the medium [34]. Fur-

thermore, reverse-transcription analysis of total RNA

from P. putida CA-3 grown on various carbon sources

also indicated that there were no detectable mRNA tran-

scripts from the sty operon in the absence of styrene [19].

Complementation studies performed in E. coli with ele-

ments of the sty operon from both Pseudomonas sp. strain

Fig. 3. A: Conserved domains of the StyS proteins from a number of styrene-degrading strains, together with known sensor kinases from toluene degra-

dation pathways. The di¡erently shaded boxes identify the domains. B: Partial amino acid sequence alignments of domain regions. Both histidine kinase

domains contain the conserved amino acids H, N, G1, F, G2 (underlined), but only the sequence alignment of histidine kinase 1 is shown here. The re-

ceiver domains display the typical DDSK of response regulators and these are highlighted in bold face. The aspartate residue which may undergo phos-phorylation is underlined. Homologies to PAS and PAC consensus sequences are also highlighted in bold face.




Y2 and Pseudomonas sp. strain VLB120, demonstrated

that the StyS and StyR proteins were required for the

styrene-dependent induction of the sty upper pathway

genes [15,41]. It has also been reported in P. putida

CA-3 that transcription of the styrene upper pathway

genes is entirely dependent upon expression of stySR [19].

Analysis of the promoter region upstream from the styA

gene has identi¢ed a number of key features that support

the proposed role of StyR as the transcriptional activator

of the styrene upper pathway genes. A potential DNA-binding site with the palindromic sequence ATAAAC-

CATGGTTTAT, centred at position 341, was located in

the styA promoter region of Pseudomonas sp. strain Y2

[15]. This sequence is almost identical to the inverted re-

peat ATAAACCATCGTTTAT of the TodT-binding site

in the tod operon [28]. Sequence analysis of the potential

styA promoter region in P. £uorescens ST [23] and

P. putida CA-3 [19] revealed that this is also likely to be

the case in these strains. In Pseudomonas sp. strain

VLB120, a ‘sty’ box was located 75 bp upstream from

the styA start codon and it was shown that 5P deletion

of this region prevented styA expression [41]. As these

strains do not possess a putative 335 c70 binding site in

their styA promoter region, it is likely that StyR exerts

control over the upper pathway by binding at this 341

region sty box and attracting RNA polymerase to the ex-

tended 310 box (TGTTAGCTT) present in the promoter.

A proposed model of the styrene-induced phosphorylation

cascade, leading to transcriptional activation of the upper

pathway genes by the two-component StyS and StyR pro-

teins, is presented in Fig. 5.

While very strong structural and functional similaritiesexist between the sty operons of the Pseudomonas strains

thus far investigated, a number of observations have been

made which suggest that they may be di¡erentially regu-

lated. It has been reported that in batch-grown P. £uores-

cens ST, expression of stySR is constitutive, irrespective of

the presence or absence of styrene, but that styrene is

required to activate transcription of the upper pathway

genes [34]. However, primer extension analyses with ele-

ments of the sty operon from species Y2, expressed in

E. coli , failed to identify the stySR transcription start site

and the authors believed this was the result of low level

stySR expression in the absence of styrene [15]. Similarly,

Fig. 4. A: Conserved domains of the StyR protein from a number of styrene-degrading strains and similar response regulators. The di¡erently shaded

boxes indicate the domains. B : Partial amino acid sequence alignments of the response regulators. These residues are highlighted in the alignment in

bold face. The aspartate residue most likely to be phosphorylated is underlined. StyR also possesses a helix-turn-helix domain with considerable similar-

ity to the LuxR DNA-binding motif. The LuxR helix-turn-helix (HTH) consensus sequence is shown in bold face in the lower alignment and identical

residues within the response regulators are correspondingly given in bold face.




in P. putida CA-3, reverse transcription analysis of total

RNA revealed that stySR mRNA transcripts were not

detectable in the absence of styrene, suggesting basal level

transcription and/or poor transcript stability in the ab-

sence of inducer [19]. The authors also reported that inCA-3 the addition of the lower pathway substrate, PAA,

into styrene-growing cultures caused a complete loss of

detectable upper pathway activity and that this was linked

to transcriptional repression of stySR. This would also

lead us to the conclusion that stySR have no role in the

regulation of the lower pathway. This hypothesis is further

supported by reports of the transcriptional repressors

PaaN and PaaX controlling expression of the PACoA cat-

abolic core in P. putida U and E. coli W, respectively

[16,17]. It is unclear as to the mechanism by which PAA

imposes this negative regulation on the upper pathway,

but the phenomenon is not universal in styrene degrada-tion. The presence of PAA does not prevent detection of

styrene oxide isomerase activity in the styrene-degrading

Xanthobacter strain 124X [10]. The authors of the latter

work also report detectable PAAL dehydrogenase activity.

However, it may be worth noting that early work in CA-3

reported similar activity during growth on PAA [9], until it

was later demonstrated that there was clearly no transcrip-

tion of the CA-3 upper pathway structural genes when

PAA was present [19]. Thus, PAA or its metabolic inter-

mediates may induce a non-styD-encoded acetaldehyde de-

hydrogenase, resulting in false-positive upper pathway ac-

tivity under potentially repressive growth conditions.

7. Physiological factors a¡ecting styrene degradation

7.1. Catabolite repression

Catabolite repression has been reported in a number of Pseudomonas strains for a variety of aromatic carbon as-

similation routes including toluene [42], phenol [43], ben-

zylamine [44], chloroaromatics [45] and ethylbenzene [46].

Similarly, it has been reported that a number of non-ar-

omatic carbon sources such as organic acids and/or car-

bohydrates repress styrene degradation in both P. putida

CA-3 and P. £uorescens ST [9,19,34]. Interestingly, the

catabolite repression substrate pro¢les of the latter two

organisms, while sharing some common carbon sources,

are a¡ected quite di¡erently by others. In CA-3, glucose,

succinate and acetate have no repressing e¡ects [9], while

the same carbon sources strongly repress induction of thestyA promoter in P. £uorescens ST [34]. It is unclear as yet

how catabolite repression is speci¢cally imposed on sty

gene expression in these strains. Santos and co-workers

postulate that since styS and styR appear to be constitu-

tively expressed in strain ST, catabolite repression may

involve some factor(s) a¡ecting signal transduction of

the two-component system, either through repression of

the sensor kinase activity or by direct repression of the

styA promoter [34]. However, in CA-3, the repressive ef-

fect of citrate was shown to involve reduced transcription

of stySR and styA for as long as the alternative carbon

source persisted in the medium. It was also reported in

Fig. 5. Schematic depiction of a possible mode of signal transduction in the regulation of the styrene catabolic genes. (1) Styrene enters the cell mem-

brane and (2) interacts with Input 1, a sensory input domain in StyS. (3) Oxygen changes indicating a reduction in cell energy levels may also be sensed

by another input domain, Input 2. (4) Depending on the input domain activated, one of the kinases phosphorylates its associated His residue (bold

face). (5) The phosphoryl group is ultimately transferred to an Asp residue in the receiver domain of StyR, activating the carboxy DNA-binding do-

main of the response regulator StyR. (6) The phosphorylated response regulator binds to the 8-bp inverted repeat shown centred at position 341 up-

stream from the styA start codon. (7) This attracts RNA polymerase (RNA pol) to the promoter region and transcription of the upper pathway genes

(ABCD) takes place.




CA-3 that the lower pathway paaK , which appears to be

regulated independently of stySR, is also subject to catab-

olite repression, with e¡ects being mediated at the tran-

scriptional level [19]. It has been reported that cAMP is

not involved in catabolite repression in P. putida CA-3 [47]

and other Pseudomonas species where cAMP pools do not

£uctuate with carbon source, nor does the addition of cAMP relieve repression of catabolite responsive pathways

[48]. Recent reports have, however, documented the in-

volvement of a catabolite repression control protein

(Crc) in both P. putida and Pseudomonas aeruginosa [49].

Catabolite repression of the P. putida branched-chain keto

acid pathway by Crc was proposed to involve post-tran-

scriptional regulation of the pathways positive regulator,

BkdR [50]. Hester et al. have postulated that Crc might

act as a structure-speci¢c ribonuclease degrading bkdR

mRNA or, alternatively, hindering the e⁄ciency of bkdR

mRNA translation [50]. The former hypothesis supported

observations made in P. putida CA-3, where the additionof citrate caused a dramatic reduction in detectable levels

of stySR mRNA transcripts [19]. However, a recent study

by Yuste and Rojo into catabolite repression of the P. putida

GPo1 alkane degradation pathway provided a further

insight [51]. Using wild-type and Crc-de¢cient Pseudo-

monas strains harbouring key genetic elements of the al-

kane degradation pathway, the authors demonstrated that

mRNA transcript stability during catabolite repression

was not signi¢cantly di¡erent in the presence or absence

of crc. These ¢ndings suggest that Crc does not function

as a ribonuclease and is unlikely to be responsible for the

low levels of stySR mRNA transcripts detected during

catabolite repression of styrene degradation in P. putidaCA-3. In addition, and perhaps more interestingly, Yuste

and Rojo demonstrated that Crc-dependent repression was

only observed in Luria^Bertani rich media and was not

involved in the catabolite repression observed in GPo1

when mineral salts medium containing a single, repressing

organic acid was used. Thus, catabolite repression of the

hydrocarbon degradative pathway in this P. putida GPo1

strain, and potentially in others, involves at least two in-

dependent mechanisms. In fact, what is currently emerging

from various studies on catabolite repression in other mi-

crobial systems is that an interplay of cellular factors may

be involved including integration host factor (IHF), PtsN(IIANtr) protein, the alarmone guanine 5-diphosphate 3-di-

phosphate (ppGpp), c factor stability and interaction with

RNA polymerase and alterations in c factor dependence

[37,52,53]. This will be a complex issue to resolve but its

importance is obvious if one intends to apply the styrene

catabolic potential of an organism to environments where

the carbon composition is varied.

7.2. Nutrient limitation in continuous culture

Investigations of styrene catabolism under conditions of

organic and inorganic nutrient limitation have thus far

only been performed with P. putida CA-3. However, the

¢ndings reported strongly resemble observations made

with the TOL pathway during continuous culture of

P. putida mt-2 harbouring pWW0 and may therefore be

representative of a common feature in the regulation of

catabolic pathways in Pseudomonas species which, through

evolution, have become integrated with overall cell physi-ology. O’Connor and co-workers reported that growth of

P. putida CA-3 on limiting concentrations of styrene re-

sulted in far higher upper pathway enzyme activities than

those recorded in batch systems [54]. Subsequent investi-

gation revealed that this e¡ect coincided with increased

stySRABCD and paaK gene transcript levels in response

to limiting concentrations of pathway substrates. It was

not possible to detect transcription of the upper or lower

pathway genes when succinate was the limiting carbon

source, thereby ruling out the possibility that increased

transcription of the two-component stySR apparatus was

due to non-speci¢c signal induction during carbon starva-tion [55]. This ¢nding was almost identical to that re-

ported in P. putida mt-2, where limiting concentrations

of the TOL pathway inducer, m-xylene, resulted in in-

creased transcription of the necessary degradative genes

[56]. According to Harder and Dijkhuizen, microorgan-

isms may overcome low concentrations of carbon by either

(a) increased uptake and intracellular accumulation of the

substrate, or (b) enhanced initial metabolism of intracel-

lular substrate [57]. Although little is currently known with

respect to active intracellular styrene accumulation, the

observed increase in the levels of sty mRNA transcripts

supports the use of the latter mechanism.

Conversely, it has been reported that inorganic nutrientlimitations such as phosphate, sulfur and nitrogen, which

are common environmental conditions, have strikingly

similar repressive e¡ects on the catabolic activities of the

P. putida CA-3 sty operon and the P. putida mt-2 TOL

pathway, as well as making them highly sensitive to catab-

olite repression [54^56]. It is thought that a variety of

global regulatory factors are involved in these co-ordi-

nated responses but the concentration trends and potential

roles of these factors are poorly understood at present

[37,58]. Nevertheless, the transcriptional repression of

the sty operon in CA-3 and potentially in other styrene

degraders during inorganic nutrient-limiting growth hasserious implications with respect to the development

of environmentally-based bioremediation/biodegradation

strategies.

8. Application of styrene-degrading microorganisms in

bio¢lters

Despite these challenges, several groups have already

begun to investigate the potential of bio¢lters for the re-

moval of styrene from contaminated waste gases. Cox and

co-workers, using perlite-packed ¢lters to enrich styrene-




degrading fungi, reported styrene elimination capacities of

70 g m33 ¢lter bed h31 when exposed to in£uent gas con-

taining styrene amounts ranging from 290 to 675 mg m33

[59]. In a subsequent study, Weigner et al. achieved 85%

elimination of styrene from an organic load of 170 g m33

h31, 18 days after the start-up of four serially aligned,

perlite-packed ¢lters that had been inoculated with along-term adapted mixed microbial culture [60]. The po-

tential of low cost natural ¢lters has also been assessed.

Reittu and co-workers, using peat as ¢lter material, re-

ported maximal styrene elimination capacities of 30 g

m33 ¢lter material h31. The group identi¢ed several bac-

terial isolates capable of styrene degradation from the gen-

era Tsukamurella, Pseudomonas, Sphingomonas, Xantho-

monas and an unidenti¢ed member of the Proteobacteria

[61]. However, an elimination capacity of 63 g m33 h31

was subsequently reported using a ¢lter packed with a

mixture (4:1) of peat and glass beads inoculated with

the styrene degrader R. rhodochrous NCIMB 13259 [62].All of the studies thus far have been based either upon the

functional utilisation of natural micro£ora in the ¢lter-

packaging material or upon inoculation with individual

and/or mixed cultures capable of styrene degradation (fol-

lowed by careful control of the nutrient, pH, and temper-

ature conditions). However, the continued elucidation of

mechanisms regulating the bacterial degradation of styrene

may permit the construction of enhanced styrene-degrad-

ing strains capable of maximising the degradative potential

of these ¢lter systems.

9. Biotechnological applications of pathway enzymes

While most of the early work on elucidation of the

pathways for microbial styrene degradation in a variety

of microorganisms was primarily undertaken with a view

to assessing the potential of these organisms for use in the

bioremediation of contaminated environments, it has be-

come clear that a number of the pathway enzymes are of

potential use in organic synthesis.

9.1. Styrene oxide production

Of the enzymes involved in styrene degradation, styrenemonooxygenase has perhaps received the most attention,

in part due to the product of the reaction it catalyses,

styrene oxide, but also due to its potential as a broad

range catalyst for the production of epoxides. Styrene

monooxygenase is found in a number of Pseudomonas

species (Table 1), and the ability of the enzyme to produce

styrene oxide from a cheap starting material is re£ected in

the number of studies undertaken to develop styrene-de-

grading strains, or bacterial hosts heterologously express-

ing styrene monooxygenase genes, as biocatalysts [63,64].

Optically active compounds such as epoxides are desired

for their use as chiral building blocks (synthons) for the

chemical synthesis of pharmaceutical drugs. The produc-

tion of optically pure styrene oxide has been the focus of

attention for a number of groups [65]. Bestetti and co-

workers have recently developed a procedure for the pro-

duction of enantiomerically pure epoxides from a variety

of substituted styrenes with a recombinant E. coli strain

expressing the P. £uorescens ST styrene monooxygenasegene [63,66].

No« the and Hartmans were the ¢rst to report in detail

the biocatalytic production of styrene oxide from styrene

with a nitrosoguanidine (NTG) mutant of P. putida S12

and a Mycobacterium strain E3 [67]. P. putida S12 mutant

M2 produced S -styrene oxide while E3 produced R-sty-

rene oxide. The rate of consumption of styrene was 40

times higher for the Pseudomonas strain (200 nmoles

min31 (mg dry wt.)31) than for strain E3. Both strains

produced styrene oxide to an enantiomeric excess (e.e.)

of s 98%. Wubbolts and co-workers had earlier reported

that xylene oxygenase from P. putida mt-2 was capable of producing S -(+)-styrene oxide from styrene to an e.e. of

933% [68]. The styrene degraders P. putida S12 and

Mycobacterium E3 were also capable of transforming

4-chlorostyrene to 4-chlorostyrene oxide to an e.e. of

s 98%, while P. putida mt-2 achieved an e.e. of 37% [67].

The most extensive studies on the production of opti-

cally pure styrene oxide have since been carried out by

Panke and co-workers, who investigated the biocatalytic

potential of cloned styrene monooxygenase from Pseudo-

monas sp. strain VLB120. E. coli JM101 (pT7ST-C) ex-

pressing styrene monooxygenase (StyAB) from strain

VLB120 produced styrene oxide to an e.e. s 99% at a

rate of 79 nmoles min31 (mg dry wt.)31. While the rate

of styrene oxide production was less than half that re-

ported for P. putida S12 mutant strain M2, the e.e. value

for the styrene oxide produced was greater [13]. Further

work attempted to produce styrene oxide in a continuous

two-liquid phase system by expressing the styrene mono-

oxygenase on the chromosome of a recombinant P. putida

KT2440 strain [64]. The rate of styrene oxide formation

was 6 nmoles min31 (mg dry wt.)31. This compared to

shake £ask biotransformation rates of 30 nmoles min31

(mg dry wt.)31. While the generation of new biocatalysts

through genetic engineering has produced valuable data

on the genetic stability of heterologously expressed genes,it is the improved stereoselectivity and production rate

that have primarily driven research in this area. Ironically,

despite the progress that has been made in the area of

engineered biocatalysts, the highest rate of styrene oxide

formation achieved to date still remains that observed in

the NTG-derived mutants of P. putida S12.

9.2. Indigo production

It has long been established that many microorganisms

capable of growth on toluene, naphthalene and other ar-

omatic hydrocarbons are capable of producing indigo




from indole [69]. However, the rate of indigo formation by

these microorganisms was reported only in arbitrary units,

due in part to the low transformation rates achieved. The

biotransformation of indole to indigo by aromatic dioxy-

genases was hindered by an even greater problem ^ the

appearance of undesired side products. Indirubin, a hetero-

dimer of 2- and 3-hydroxyindole, is produced as a minorred by-product of indole biotransformation by organisms

expressing naphthalene dioxygenase [70]. The appearance

of indirubin during indole biotransformation by naphtha-

lene dioxygenase can be explained by the formation of cis-

indole 2,3-dihydrodiol. Chemical dehydration of the dihy-

drodiol can result in the formation of the two compounds

3-oxindole (indoxyl) and 2-oxindole, dimerisation of which

results in the formation of indirubin. Indigo is a dimer of

3-oxindole. The problems of transformation rate and end-

product purity thus hindered the success of this biotrans-

formation. However, it was possible that an enzyme ex-

pressing a monooxygenase rather than a dioxygenase ac-tivity could produce the desired 3-oxindole product and

alleviate the problem of impurities. The ¢rst report of

indigo formation by an organism expressing styrene-de-

grading genes was in an E. coli strain expressing the sty-

rene monooxygenase gene from P. £uorescens ST [23].

In further work where the formation of indigo by a range

of aromatic hydrocarbon-degrading organisms was per-

formed, both P. putida CA-3 and P. putida S12 were

shown to be capable of producing stoichiometric amounts

of indigo from indole (1 :2) in whole-cell biotransforma-

tion assays [71,72]. Thin layer chromatography analysis

showed that the indigo produced by both Pseudomonas

strains contained no detectable levels of the contaminatingpigment indirubin. The maximum rate of indigo formation

achieved by P. putida S12 and P. putida CA-3 was 10.9

nmoles indigo min31 (mg dry wt.)31[71]. When the indigo

formation rate of both P. putida strains was compared

with that of a number of aromatic hydrocarbon-degrading

strains, expressing both mono- and dioxygenases for either

toluene or naphthalene degradation, it was shown to be as

much as 83-fold higher [72]. Interestingly, P. putida PpG7

expressing the well characterised naphthalene dioxygenase

gene was capable of producing indigo at a rate between

25% and 50% lower than that observed for P. putida CA-3

and S12 [72]. In addition, microorganisms degrading sty-rene through initial attack of the side-chain produce indi-

go from indole at rates higher than styrene-degrading mi-

croorganisms expressing a ring-hydroxylating dioxygenase

[71].

The ability of styrene-grown cells to produce indigo

from indole stoichiometrically was due only in part to

the presence of styrene monooxygenase, since NTG-de-

rived mutants of P. putida S12 that lacked styrene oxide

isomerase enzyme activity produced indigo following a lag

phase and at a much lower rate [67]. This indicates that

the product of styrene monooxygenase catalysis can be

chemically converted to indigo by styrene oxide isomerase,

and that this conversion is essential for the rapid produc-

tion of indigo from indole. Since there is no contaminating

indirubin produced, the product of styrene oxide isomer-

ase catalysis must be 3-oxindole. To support this hypoth-

esis, experiments with 2-oxindole as a substrate failed to

yield any indigo for either P. putida CA-3 or S12 [71].

Thus, the conversion of indole to indigo is the result of a two-step biotransformation with indole oxide and 3-ox-

indole (indoxyl) as intermediates in the reaction (Fig. 6),

and microorganisms expressing styrene monooxygenase

and styrene oxide isomerase show a distinct advantage

over organisms with other mono- or dioxygenases in

that they produce a pure product at a higher rate of ca-

talysis [71]. The use of whole cells for the regiospeci¢c

production of indigo from indole may be a better option

than cell-free systems. In the latter, the indole epoxide

formed from styrene monooxygenase is unstable. Accumu-

lation of such a compound would result in chemical hy-

drolysis to produce a dihydrodiol. Chemical dehydrationcould result in mixed product formation (indoxyl and

2-oxindole). Dimerisation of the products would result

in the formation of mixed products, creating the same

by-product problem observed for whole-cell biocatalysts

with wild-type dioxygenase activity [70]. In any case, the

styrene-degrading P. putida CA-3 and S12 strains possess

monooxygenase and styrene oxide isomerase activities that

may prove useful in the future biological production of

indigo.

9.3. Other potential biotransformations

Styrene monooxygenase has already been reported toallow conversion of indole to indole oxide [65], but the

ability of styrene monooxygenase to produce epoxides

from substrates structurally related to styrene has not

been well studied. One report cites the ability of styrene

monooxygenase to produce indene oxide from indene in

whole cells of P. putida S12 [71]. Indene oxide is a pre-

cursor of cis-1S ,2R-aminoindanol, an intermediate in the

synthesis of the anti-HIV-1 drug Crixavin. The chemical

synthesis of indene oxide is di⁄cult, as re£ected in the

report by Zhang and co-workers [73]. Thus the bioconver-

sion of indene to indene oxide may overcome this chemical

bottleneck. However, the indene oxide formed by wholecells of P. putida S12 was through-converted to indanone

by styrene oxide isomerase (Fig. 6) and subsequent manip-

ulation of this strain either through mutation of the iso-

merase or the use of cell-free systems would be required in

order to accumulate indene oxide from indene.

While the majority of attention has focused on the bio-

technological potential of styrene monooxygenase and sty-

rene oxide isomerase, Itoh and co-workers have reported

the presence of a PAAL reductase from Corynebacterium

strain ST-10 that, in addition to producing 2-phenyletha-

nol from PAAL, has an extremely broad substrate speci-

¢city [22]. The enzyme shows potential as an interesting




biocatalyst given that it is capable of the production of a

number of chiral alcohols, ketones and 2-alkanones, with

yields varying from 29% to 100% [74]. The stereoisomers

of these alcohols are important starting materials for the

synthesis of agrochemicals, liquid crystals and pharma-ceuticals [75]. Therefore, this enzyme, like styrene mono-

oxygenase and styrene oxide isomerase, may also have

signi¢cant future biotechnological applications.

10. Conclusions

While there have been signi¢cant advances over the last

decade in understanding bacterial styrene degradation at

the molecular level, and in the application of pathway

enzymes to achieve various chemical conversions, there

are numerous important aspects which remain unresolved.

Despite the current understanding of styrene-induced tran-

scriptional regulation of the catabolic pathways, it is at

present unclear whether a speci¢c transport system exists

for this compound which could be manipulated to enhance

an organisms ability to accumulate the compound intra-cellularly. Similarly, while the requirement for StyS and

StyR in the activation of the upper pathway genes has

been clearly demonstrated, further work is required to

shed light on the speci¢c phosphorylation cascade govern-

ing signal transduction between these regulatory proteins.

This information may be of use in the potential develop-

ment of stySR-based reporter systems for the detection of

styrene. Such reporter systems have already been con-

structed for the detection of BTEX compounds, phenols

and polychlorinated biphenyls [76^78]. Finally, there re-

mains the complex task of identifying how global regula-

tory molecules exert control over the styrene degradation

Fig. 6. Representative biotransformations performed by enzymes involved in styrene degradation. SMO, styrene monooxygenase; SOI, styrene oxide iso-

merase; PDH, phenacetaldehyde dehydrogenase; PAR, phenylacetaldehyde reductase. Adapted from [71].




pathway in response to the physiological and metabolic

status of the cell. In this endeavour particular emphasis

should be placed on those that exert negative regulatory

in£uences as these currently represent the greatest ob-

stacles to establishing feasible, environmentally based, sty-

rene biodegradation strategies, as well as in£uencing po-

tential methods of whole-cell biocatalysis.

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