FEMS Microbiology Reviews 26 (2002) 403^417

www.fems-microbiology.org

Biochemistry, genetics and physiology of

microbial styrene degradation
Niall D. OLeary
a

a;

, Kevin E. OConnor b , Alan D.W. Dobson

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.
5 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.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aerobic styrene degradation . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Side-chain oxidation . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Direct ring cleavage . . . . . . . . . . . . . . . . . . . . . . . . .
3. Anaerobic styrene metabolism . . . . . . . . . . . . . . . . . . . . . . .
4. Genetic organisation of the styrene upper pathway . . . . . . . .
5. The upper pathway regulatory apparatus . . . . . . . . . . . . . . .
5.1.
StyS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
StyR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Pathway-specic regulation of the styrene catabolic operon .
7. Physiological factors aecting styrene degradation . . . . . . . .
7.1.
Catabolite repression . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
Nutrient limitation in continuous culture . . . . . . . . . .
8. Application of styrene-degrading microorganisms in biolters
9. Biotechnological applications of pathway enzymes . . . . . . . .
9.1.
Styrene oxide production . . . . . . . . . . . . . . . . . . . . . .
9.2.
Indigo production . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.
Other potential biotransformations . . . . . . . . . . . . . .
10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

415

* Corresponding author. Tel. : +353 (21) 490 2952 ; Fax: +353 (21) 490 3101.

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E-mail address: ndolearyucc@hotmail.com (N.D. OLeary).

0168-6445 / 02 / $22.00 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 4 5 ( 0 2 ) 0 0 1 2 6 - 2

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1. Introduction
The extensive use of aromatic hydrocarbons in industrial 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 is
employed both as a starting material for synthetic polymers and as a solvent in the polymer-processing industry,
leading to its release in a variety of industrial euents.
Styrene is known to be genotoxic while styrene oxide,
the major in vivo metabolite of styrene, is classied as a
probable carcinogen in humans [2]. Recent reports indicate that styrene may also have an immuno-modulatory
eect on workers exposed to gaseous emissions in an industrial setting [3]. As a result of concerns arising from
these ndings, the last decade has seen intense investigation into various aspects of the microbial degradation of
this compound.
One feature of styrene which has undoubtedly been inuential 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 reected in the diverse range of styrene-degrading
microbes that have been isolated from various soil locations around the globe since the late 1970s. While a dependence on mixed cultures has been reported, pure culture isolates capable of styrene degradation have included
species of Pseudomonas, Rhodococcus, Nocardia, Xanthobacter and Enterobacter as well as the black yeast Exo-

phiala jeanselmei (for a review see Hartmans [6]). However, as the aim of this review is to present the current
understanding of microbial styrene catabolism, the primary focus will repeatedly centre upon the genus Pseudomonas, from which signicant insights have come in recent
years.

2. Aerobic styrene degradation

Two main pathways for the aerobic degradation of styrene have been described, one involving an initial oxidation 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 dinucleotidedependent, two-subunit monooxygenase followed by isomerisation of the epoxystyrene formed to phenylacetaldehyde (PAAL). This compound is subsequently oxidised to
phenylacetic acid (PAA) through the action of either an
NAD - or phenazine methosulfate-dependent dehydrogenase [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 degradative routes yet to be formally demonstrated.

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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 metabolism 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 hybridisation 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] identied 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 coworkers reported in P. uorescens ST the accumulation of
2-hydroxyphenylacetic acid (2-OHPAA) in the culture media of cells grown on styrene and PAA [20]. The authors
proposed that 2-OHPAA could be metabolised via homogentisate to acetoacetate based on the detection of homogentisate-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 reect the role of a
PACoA-catabolon in this strain also [20].
It should be noted that in the styrene-degrading Corynebacterium strains AC-5 and ST-10, there appears to be a
modied 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 monooxygenase 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-

405

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 chemical
dump isolate capable of growth on a diverse range of
aromatic hydrocarbons, degrades styrene via direct oxidation of the aromatic nucleus. Thin layer chromatography
and nuclear magnetic resonance studies revealed that styrene metabolism in this organism proceeded via 3-vinylcatechol [8] (Fig. 1). An NAD -dependent cis-glycol
dehydrogenase activity was detected in cells grown on
nutrient broth and styrene. Cells grown under these conditions 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 specicity.
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 styrene 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 medium [24]. Ring attack has also been proposed in Xanthobacter 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 on
1-phenylethanol. Cripps et al. [25] proposed that the
1-phenylethanol degradation in this Nocardia strain involved initial oxidation of the aromatic nucleus by a dioxygenase.

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 styrene
degradation under these conditions was proposed to involve conversion to PAA via 2-phenylethanol and PAAL.
This pathway bears similarity to the aerobic transformations 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 available water. Divergence of the aerobic and anaerobic pathways 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 encoded by the TOL plasmid, pWW0 [26].

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ferent paa genes from various species capable of PAA

degradation [18], suggesting that independent evolution
of the styrene upper and lower pathways may have occurred.

4. Genetic organisation of the styrene upper pathway

Marconi and co-workers were the rst to report the
cloning and identication of genes involved in styrene catabolism [12]. They cloned two genes from the P. uorescens ST chromosome, encoding enzymes with styrene
monooxygenase and epoxystyrene isomerase activities.
Further genetic characterisation of the locus by this group
identied an upper pathway catabolic operon containing
all enzymes necessary for the conversion of styrene to
PAA [23]. The genes involved were styA and styB, encoding 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 Pseudomonas
strains, namely Pseudomonas sp. strain Y2 [15], Pseudomonas 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 common 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 diverse organisation and sequence variation observed in dif-

5. The upper pathway regulatory apparatus

The initial observation in P. uorescens ST that the sty
genes were clustered on the chromosome raised the possibility that a pathway-specic 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 catabolic operon in E. coli W was signicantly reduced in the
absence of two genes, styS and styR, located upstream
from the styrene catabolic operon. Expression was restored when these genes were provided in trans. Several
studies 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 transcriptionally 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

styS

55.5

226 (frag)

styR

52.2

207

styA

57.9

416

PaaK (437)
PhaE (439)
PaaK (447)
PaaK (440)
PhaE (445)
StyS (982)
StyS (983)
TutC (979)
TodS (978)
StyR (207)
StdR (207)
StyR (207)
TutB (218)
TodT (227)
NodW (227)
FixJ (211)
StdA (415)
StyA (415)
StyA (415)
StyA (415)

96
85
68
67
49
99
88
49
41
98
96
90
55
49
51
40
98
98
95
96

Pseudomonas sp. strain Y2

Pseudomonas putida U
Escherichia coli W
Azoarcus evansii KB740
Bacillus halodurans
Pseudomonas sp. strain Y2
Pseudomonas uorescens ST
Thauera aromatica
Pseudomonas putida F1
Pseudomonas sp. strain Y2
Pseudomonas sp. strain VLB120
Pseudomonas uorescens ST
Thauera aromatica
Pseudomonas putida F1
Bradyrhizobium japonicum
Azorhizobium caulinodans
Pseudomonas sp. strain VLB120
Pseudomonas putida S12
Pseudomonas sp. strain Y2
Pseudomonas uorescens ST

AJ000330
AF029714
X97452
AF176259
AP001507
AJ000330
AF024619
U57900
AF180147
AJ000330
AF031161
AF024619
U57900
AF180147
AF322013
X56658
AF031161
Y13349
AJ000330
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].

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407

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

5.1. StyS
The StyS proteins exhibit amino acid sequence similarity
to a number of sensor kinase proteins (Table 1), particularly the TodS and TutC sensor kinases that regulate toluene 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 act
as 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 characteristic 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 dier slightly and have been further
classied 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 regulators, and belongs to the RA2 receiver subfamily [27]. Adjacent to the receiver is an input region (Input 2), with
signicant similarity to the putative oxygen-sensing domain 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 with
many response regulators of other two-component systems. 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 consensus sequence of putative DNA-binding domains in the
family 3 response regulators is also present in the carboxyl-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 hydrocarbon catabolism [37]. Interestingly, the StyR proteins
also display amino acid similarity with the response regulators NodW and FixJ, involved in nodulation and nitrogen xation, respectively [38,39] and may, as has been
proposed by Diaz and co-workers, represent another example of domain shuing which could have occurred during the parallel evolution of family 3 DNA-binding domains with cluster 1 receiver modules [15,36,40].

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Fig. 3. A: Conserved domains of the StyS proteins from a number of styrene-degrading strains, together with known sensor kinases from toluene degradation pathways. The dierently 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 receiver domains display the typical DDSK of response regulators and these are highlighted in bold face. The aspartate residue which may undergo phosphorylation is underlined. Homologies to PAS and PAC consensus sequences are also highlighted in bold face.

6. Pathway-specic 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 fusion when styrene was present in the medium [34]. Furthermore, reverse-transcription analysis of total RNA
from P. putida CA-3 grown on various carbon sources
also indicated that there were no detectable mRNA transcripts from the sty operon in the absence of styrene [19].
Complementation studies performed in E. coli with elements of the sty operon from both Pseudomonas sp. strain

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409

Fig. 4. A: Conserved domains of the StyR protein from a number of styrene-degrading strains and similar response regulators. The dierently 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 similarity 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.

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 identied a number of key features that support
the proposed role of StyR as the transcriptional activator
of the styrene upper pathway genes. A potential DNAbinding site with the palindromic sequence ATAAACCATGGTTTAT, 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 repeat 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 extended 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 proteins, is presented in Fig. 5.
While very strong structural and functional similarities
exist between the sty operons of the Pseudomonas strains
thus far investigated, a number of observations have been
made which suggest that they may be dierentially regulated. It has been reported that in batch-grown P. uorescens 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 elements 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,

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Fig. 5. Schematic depiction of a possible mode of signal transduction in the regulation of the styrene catabolic genes. (1) Styrene enters the cell membrane 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 domain of the response regulator StyR. (6) The phosphorylated response regulator binds to the 8-bp inverted repeat shown centred at position 341 upstream 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.

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 absence of inducer [19]. The authors also reported that in
CA-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 catabolic 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 degradation. 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 transcription of the CA-3 upper pathway structural genes when
PAA was present [19]. Thus, PAA or its metabolic intermediates may induce a non-styD-encoded acetaldehyde dehydrogenase, resulting in false-positive upper pathway activity under potentially repressive growth conditions.

7. Physiological factors aecting styrene degradation

7.1. Catabolite repression
Catabolite repression has been reported in a number of
Pseudomonas strains for a variety of aromatic carbon assimilation routes including toluene [42], phenol [43], benzylamine [44], chloroaromatics [45] and ethylbenzene [46].
Similarly, it has been reported that a number of non-aromatic carbon sources such as organic acids and/or carbohydrates repress styrene degradation in both P. putida
CA-3 and P. uorescens ST [9,19,34]. Interestingly, the
catabolite repression substrate proles of the latter two
organisms, while sharing some common carbon sources,
are aected quite dierently by others. In CA-3, glucose,
succinate and acetate have no repressing eects [9], while
the same carbon sources strongly repress induction of the
styA promoter in P. uorescens ST [34]. It is unclear as yet
how catabolite repression is specically imposed on sty
gene expression in these strains. Santos and co-workers
postulate that since styS and styR appear to be constitutively expressed in strain ST, catabolite repression may
involve some factor(s) aecting 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 effect 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

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411

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

regulated independently of stySR, is also subject to catabolite repression, with eects being mediated at the transcriptional 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 involvement 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-transcriptional regulation of the pathways positive regulator,
BkdR [50]. Hester et al. have postulated that Crc might
act as a structure-specic ribonuclease degrading bkdR
mRNA or, alternatively, hindering the eciency of bkdR
mRNA translation [50]. The former hypothesis supported
observations made in P. putida CA-3, where the addition
of 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-decient Pseudomonas strains harbouring key genetic elements of the alkane degradation pathway, the authors demonstrated that
mRNA transcript stability during catabolite repression
was not signicantly dierent 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. putida
CA-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 independent mechanisms. In fact, what is currently emerging
from various studies on catabolite repression in other microbial 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-diphosphate (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.

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 physiology. OConnor and co-workers reported that growth of
P. putida CA-3 on limiting concentrations of styrene resulted in far higher upper pathway enzyme activities than
those recorded in batch systems [54]. Subsequent investigation revealed that this eect 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-specic signal induction during carbon starvation [55]. This nding was almost identical to that reported in P. putida mt-2, where limiting concentrations
of the TOL pathway inducer, m-xylene, resulted in increased transcription of the necessary degradative genes
[56]. According to Harder and Dijkhuizen, microorganisms may overcome low concentrations of carbon by either
(a) increased uptake and intracellular accumulation of the
substrate, or (b) enhanced initial metabolism of intracellular 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 nutrient
limitations such as phosphate, sulfur and nitrogen, which
are common environmental conditions, have strikingly
similar repressive eects 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 catabolite repression [54^56]. It is thought that a variety of
global regulatory factors are involved in these co-ordinated 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 has
serious implications with respect to the development
of environmentally-based bioremediation/biodegradation
strategies.

7.2. Nutrient limitation in continuous culture

Despite these challenges, several groups have already

begun to investigate the potential of biolters for the removal of styrene from contaminated waste gases. Cox and
co-workers, using perlite-packed lters to enrich styrene-

Investigations of styrene catabolism under conditions of

organic and inorganic nutrient limitation have thus far

8. Application of styrene-degrading microorganisms in

biolters

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degrading fungi, reported styrene elimination capacities of

70 g m33 lter bed h31 when exposed to inuent gas containing 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 a
long-term adapted mixed microbial culture [60]. The potential of low cost natural lters has also been assessed.
Reittu and co-workers, using peat as lter material, reported maximal styrene elimination capacities of 30 g
m33 lter material h31 . The group identied several bacterial isolates capable of styrene degradation from the genera Tsukamurella, Pseudomonas, Sphingomonas, Xanthomonas and an unidentied 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 microora in the lterpackaging material or upon inoculation with individual
and/or mixed cultures capable of styrene degradation (followed by careful control of the nutrient, pH, and temperature conditions). However, the continued elucidation of
mechanisms regulating the bacterial degradation of styrene
may permit the construction of enhanced styrene-degrading 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 become 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, styrene
monooxygenase 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 reected in
the number of studies undertaken to develop styrene-degrading strains, or bacterial hosts heterologously expressing 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 production of optically pure styrene oxide has been the focus of
attention for a number of groups [65]. Bestetti and coworkers have recently developed a procedure for the production of enantiomerically pure epoxides from a variety
of substituted styrenes with a recombinant E. coli strain
expressing the P. uorescens ST styrene monooxygenase
gene [63,66].
Nothe 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-styrene 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
93 X 3% [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 optically pure styrene oxide have since been carried out by
Panke and co-workers, who investigated the biocatalytic
potential of cloned styrene monooxygenase from Pseudomonas sp. strain VLB120. E. coli JM101 (pT7ST-C) expressing 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 reported 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 monooxygenase 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 aromatic hydrocarbons are capable of producing indigo

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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 dioxygenases was hindered by an even greater problem ^ the
appearance of undesired side products. Indirubin, a heterodimer of 2- and 3-hydroxyindole, is produced as a minor
red by-product of indole biotransformation by organisms
expressing naphthalene dioxygenase [70]. The appearance
of indirubin during indole biotransformation by naphthalene dioxygenase can be explained by the formation of cisindole 2,3-dihydrodiol. Chemical dehydration of the dihydrodiol 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 endproduct purity thus hindered the success of this biotransformation. However, it was possible that an enzyme expressing a monooxygenase rather than a dioxygenase activity could produce the desired 3-oxindole product and
alleviate the problem of impurities. The rst report of
indigo formation by an organism expressing styrene-degrading genes was in an E. coli strain expressing the styrene monooxygenase gene from P. uorescens ST [23].
In further work where the formation of indigo by a range
of aromatic hydrocarbon-degrading organisms was performed, 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 biotransformation assays [71,72]. Thin layer chromatography analysis
showed that the indigo produced by both Pseudomonas
strains contained no detectable levels of the contaminating
pigment 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 styrene through initial attack of the side-chain produce indigo from indole at rates higher than styrene-degrading microorganisms 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-derived 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,

413

and that this conversion is essential for the rapid production of indigo from indole. Since there is no contaminating
indirubin produced, the product of styrene oxide isomerase catalysis must be 3-oxindole. To support this hypothesis, 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-oxindole (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 catalysis [71]. The use of whole cells for the regiospecic
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. Accumulation of such a compound would result in chemical hydrolysis to produce a dihydrodiol. Chemical dehydration
could 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 to
allow 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 precursor of cis-1S,2R-aminoindanol, an intermediate in the
synthesis of the anti-HIV-1 drug Crixavin. The chemical
synthesis of indene oxide is dicult, as reected in the
report by Zhang and co-workers [73]. Thus the bioconversion of indene to indene oxide may overcome this chemical
bottleneck. However, the indene oxide formed by whole
cells of P. putida S12 was through-converted to indanone
by styrene oxide isomerase (Fig. 6) and subsequent manipulation of this strain either through mutation of the isomerase 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 biotechnological potential of styrene monooxygenase and styrene 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-phenylethanol from PAAL, has an extremely broad substrate specicity [22]. The enzyme shows potential as an interesting

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Fig. 6. Representative biotransformations performed by enzymes involved in styrene degradation. SMO, styrene monooxygenase; SOI, styrene oxide isomerase; PDH, phenacetaldehyde dehydrogenase; PAR, phenylacetaldehyde reductase. Adapted from [71].

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 pharmaceuticals [75]. Therefore, this enzyme, like styrene monooxygenase and styrene oxide isomerase, may also have
signicant future biotechnological applications.

10. Conclusions
While there have been signicant 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 transcriptional regulation of the catabolic pathways, it is at
present unclear whether a specic transport system exists
for this compound which could be manipulated to enhance
an organisms ability to accumulate the compound intracellularly. 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 specic phosphorylation cascade governing signal transduction between these regulatory proteins.
This information may be of use in the potential development of stySR-based reporter systems for the detection of
styrene. Such reporter systems have already been constructed for the detection of BTEX compounds, phenols
and polychlorinated biphenyls [76^78]. Finally, there remains the complex task of identifying how global regulatory molecules exert control over the styrene degradation

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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
inuences as these currently represent the greatest obstacles to establishing feasible, environmentally based, styrene biodegradation strategies, as well as inuencing potential methods of whole-cell biocatalysis.

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