FEMS Microbiology Letters 253 (2005) 111–118

www.fems-microbiology.org

Polyhydroxyalkanoate accumulating diversity of Pseudomonas

species utilising aromatic hydrocarbons

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Karen M. Tobin, Kevin E. O
Connor *

Department of Industrial Microbiology, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and
Biomedical Research, National University of Ireland, University College Dublin, Belfield, Dublin 4, Republic of Ireland

Received 1 July 2005; received in revised form 14 September 2005; accepted 17 September 2005

First published online 5 October 2005

Edited by A. Steinbuchel

Abstract

A number of Pseudomonas strains accumulated polyhdroxyalkanoate (PHA) from a variety of aromatic hydrocarbons. In many
strains the level of PHA accumulation was dependent on the side chain length of the phenylalkanoic acid provided for growth. 4 of
the 8 strains accumulated increased levels of PHA as the side chain length of the phenylalkanoic acid substrate increased. PHA
accumulated from styrene and phenylacetic acid was composed of aliphatic monomers only. The PHA accumulated from any
one of the phenylalkanoic acids with 5 carbons or more in their side chain (n P 5) was almost identical for all strains with PHA
composed of both aromatic and aliphatic monomers. The predominant monomers accumulated were 3-hydroxyphenylvaleric acid
and 3-hydroxyphenylhexanoic acid. The addition of the metabolic pathway inhibitors acrylic acid and 2-bromoctanoic acid resulted
in decreased levels of PHA from phenylacetic acid, suggesting a role for both b-oxidation and fatty acid synthesis in PHA accumu-
lation from phenylacetic acid.
Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Polyhdroxyalkanoate; Aromatic hydrocarbons; Pseudomonas species; Metabolic diversity

1. Introduction A number of reports on accumulation of aliphatic

Polyhydroxyalkanoates (PHAs) are a group of biode-
gradable polymers accumulated by bacteria as an intra-
cellular carbon storage material generally in response to
inorganic nutrient limitation in the presence of excess
carbon [1]. PHAs are a versatile set of biodegradable
plastics whose uses depend on their monomeric compo-
sition [2]. The presence of aromatic monomers in PHA
can dramatically change its physical properties, leading
to new biomedical and biotechnological applications
[3–7].
*
Corresponding author. Tel.: +353 1 716 1307; fax: +353 1 716
1183.
E-mail address: kevin.oconnor@ucd.ie (K.E. O
Connor).
and aromatic PHA from aromatic hydrocarbons have
been published [3–18]. However, the majority of these
studies have been limited to a single substrate and a sin-
gle Pseudomonas species. PHA accumulation from aro-
matic hydrocarbons has been shown in only three
Pseudomonas species, i.e., Pseudomonas putida [3–
7,13,14,16–18], Pseudomonas oleovorans [9–13,15] and
P. citronellolis [5,8,16]. Consequently there is a lack of
knowledge on the diversity of PHA accumulating abili-
ties within and across Pseudomonas species from a range
of structurally related aromatic hydrocarbons.
Aliphatic PHA accumulation from the aromatic sub-
strates styrene and phenylacetic acid has only been
shown in P. putida CA-3 [18]. The ability of various
Pseudomonas species to accumulate PHA from these

0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.09.025
112 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118
substrates is examined to increase knowledge on meta-
bolic diversity of PHA accumulation from aromatic car-
bon sources. Furthermore styrene is an environmental
pollutant and the conversion of such a compound to
PHA could have a doubly beneficial effect on the envi-
ronment by preventing pollution and concomitantly
producing an environmentally friendly polymer. Phenyl-
acetic acid is an intermediate of styrene degradation [19–
21] and other aromatic compounds [22–24], thus the
ability of a variety of strains to accumulate PHA from
phenylacetic acid is metabolically interesting.
This study investigates the effect of the aromatic sub-
strate supplied on the ability of various Pseudomonas
species to accumulate PHA as well as the monomer
composition of the PHA accumulated. In addition the
ability of Pseudomonas fluorescens and Pseudomonas
jessenii to accumulate PHA from aromatic substrates
is reported for the first time. This study provides a com-
prehensive look at the diversity in PHA accumulation
and composition amongst Pseudomonas species from a
range of aromatic hydrocarbons.
2. Materials and methods
2.1. Bacterial strains
P. putida S12 is a soil bacterium, isolated by the abil-
ity to grow on styrene [19]. P. putida CA-1 is a styrene
bioreactor isolate. P. putida F6 [25] and Pseudomonas
strain B2 are soil isolates with the ability to utilise
para-hydroxyphenylacetic acid for growth. Pseudomo-
nas strains H4, B3, C8 and D5 are soil isolates with
the ability to utilise phenylacetic acid for growth. Pseu-
domonas strains B2, H4, B3, C8 and D5 were identified
by 16S rDNA sequencing. Sequences were compared
with those in the Genbank database [26] using the batch
BLAST [27] feature of Bioedit [28]. Based on sequence
similarities, the strains were identified as P. fluorescens
B2, P. putida H4, P. fluorescens B3, P. jessenii C8 and
P. putida D5.
2.2. Chemicals
Phenylvaleric acid, phenylhexanoic acid, phenylhep-
tanoic acid and phenyloctanoic acid were supplied by
Lancaster Synthesis (UK). All other chemicals were sup-
plied by Sigma–Aldrich (Dublin, Ireland).
2.3. Media and growth conditions
Bacterial strains were grown as previously described
[18]. Cells were grown on styrene as previously des-
cribed [18], supplying 300 ll of styrene. Optical density
(OD) was determined at 540 nm using a UV–visible
spectrometer.
2.4. PHA accumulation
Nitrogen-limited E2 media [29] was used for PHA
production experiments by supplying 0.63 g/L NaNH4-
HPO4 Æ 4H2O. Bacterial strains were grown in batch
culture as previously described [17].
2.5. Alternative method for PHA accumulation
Where certain strains failed to accumulate PHA from
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phenylacetic acid when grown as described above, these
strains accumulated PHA using an alternative method
as follows: bacterial cells were inoculated (1%) into
50 ml E2 medium with additions of growth substrate
(5 mM), vitamins, trace elements (TE) and MgSO4 Æ
7H2O at the time of inoculation and incubated for 24 h.
The cell suspension was centrifuged at 10,000 r.p.m. for
15 min. The cells were resuspended aseptically in E2 med-
ium with 0.08 g/L NaNH4HPO4 Æ 4H2O. Growth sub-
strate (5 mM), vitamins, TE and MgSO4 Æ 7H2O and
were added to the growth flasks and the cells incubated
for a further 24 h.
2.6. Inhibition experiments
Cells were grown, washed and resuspended as de-
scribed in Section 2.5. Growth substrate (5 mM), vita-
mins, TE, MgSO4 Æ 7H2O and inhibitor were added to
the growth flasks and the cells incubated for 24 h.
2.7. PHA monomer determination
PHA monomer composition was determined using
the method previously described [30]. Samples were ana-
lysed on a Fisons GC-8000 series gas chromatograph.
PHA standards from P. oleovorans (aliphatic) and P.
putida U and P. putida CA-3 (aromatic) were used for
peak identification.
3. Results and discussion
3.1. PHA accumulation from styrene and phenylacetic
acid
To date only one microorganism, P. putida CA-3, has
been reported to accumulate PHA from styrene and
phenylacetic acid [18]. Since styrene is an environmental
pollutant and phenylacetic acid an intermediate of sty-
rene metabolism [19–21] and other aromatic hydrocar-
bons [22–24], the ability of other Pseudomonas species
to accumulate PHA from these carbon sources is both
environmentally and metabolically interesting. Only
two strains (P. putida S12 and CA-1) grew with styrene
as a sole source of carbon and energy. Both strains accu-
mulate mcl-PHA to 14% and 8% of the bacterial cell dry
 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118 113

weight (CDW), respectively (Table 1). The monomer
composition of the PHA accumulated comprises
3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and
3-hydroxydecanoic acid in a general ratio of 2:26:72
(Table 1). The level of PHA accumulated by P. putida
S12 and CA-1 from styrene is 1.5- and 2.6-fold lower
than that reported for P. putida CA-3 [18]. P. putida
S12 and P. putida CA-3 are reported to have similar
pathways for styrene degradation [19,20] with P. putida
S12 known to consume styrene at almost twice the rate
ase enzyme (phaG) which has been referred to as (R)-3-
hydroxydeaconyl-ACP-CoA transferase [33].
3.2. PHA accumulation from phenylalkanoic acids n P 4
When the Pseudomonas strains are supplied with
phenylbutyric acid as growth substrate little or no
growth is observed in 5 of the 8 strains after incubation
periods of up to 264 h (OD540 < 0.2). Consequently this
substrate was not tested for PHA accumulation in these

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observed for P. putida CA-3 [31]. Thus the pathway for strains. P. putida CA-1, P. putida F6, and P. putida S12
styrene metabolism to PHA in P. putida S12 may have a grew after 8 days of incubation with phenylbutyric acid.
bottleneck lower down the metabolic pathway that is However, none of these strains accumulate PHA from
absent in P. putida CA-3. phenylbutyric acid.
All the bacterial strains tested exhibit good growth on Only 2 of the 8 strains tested grow well (POD540 of
phenylacetic acid and accumulate PHA from this sub- 1.1) when supplied with phenylvaleric acid (10 mM) as
strate at moderate to low levels (Table 1). P. putida the sole source of carbon and energy. P. putida F6 accu-
S12 accumulated approximately the same level of PHA mulates moderate levels of PHA (26% CDW) with P.
from styrene and phenylacetic acid. However P. putida putida H4 accumulating low levels of PHA (5% CDW)
CA-1 accumulated half the level of PHA from phenyla- (Table 2). There is a notable change in the ability of
cetic acid compared to styrene. Further investigation of the strains in this study to accumulate PHA from phe-
the biochemical fate of styrene in P. putida CA-1 is re- nylalkanoic acids once the acyl carbon side chain length
quired before the latter observation can be explained. is increased from 5 to 6 carbons. All 8 strains tested are
Despite differences in the levels of PHA accumulated capable of accumulating PHA from phenylhexanoic
from phenylacetic acid by various Pseudomonas strains, acid (Table 2). P. fluorescens B2 and P. jessenii C8 accu-
the ratio of the monomer composition is similar with 3- mulate the highest level of PHA from phenylhexanoic
hydroxydecanoic acid as the predominant monomer acid and phenylheptanoic acid, respectively, while P.
(Table 1). The predominance of (R)-3-hydroxydecanoic jessenii C8 and P. fluorescens B3 accumulate the highest
acid monomers from PHA unrelated aliphatic substrates level of PHA from phenyloctanoic acid (Table 2). There
(substrates whose chemical structure does not resemble appears to be a broad diversity amongst Pseudomonas
the monomers of PHA) has been reported previously strains with respect to the relationship between the level
[18,32,33]. This has been attributed to the substrate of PHA accumulation and the number of carbons on the
selectivity of the (R)-3-hydroxyacyl-ACP-CoA transfer- side chain of phenylalkanoic acids. P. putida S12, P. put-
ida H4 and P. fluorescens B3 exhibit an increase in PHA
levels with an increase in side chain length of the growth
substrate (Table 2). This has been previously observed in
Table 1 P. putida U [3]. P. jessenii C8 and P. putida D5 show a
PHA accumulation from unrelated carbon sources styrene and dramatic increase in the level of PHA accumulation
phenylacetic acid when the number of carbons in the side chain is
Strain PHA accumulation (% CDW)a when
grown on:
Styrene Phenylacetic acid Table 2
P. putida S12 14 {2:26:72}b
12 {2:25:73} PHA accumulation from phenylalkanoic acids
P. putida CA-1 8 {1:22:77} 4 {0:19:81} Strains PHA accumulation (% CDW) when grown on:
P. fluorescens B2 – 1.5 {0:17:83}
P. putida H4 – 3c {0:16:84} PhVal PhHex PhHept PhOct
P. putida F6 – 1.4c {0:8:92} P. putida S12 0 8 10 21
P. fluorescens B3 – 12 {3:26:71} P. putida CA-1 0 12 14 11
P. jessenii C8 – 1.7 {6:20:74} P. fluorescens B2 0 25 14 10
P. putida D5 – 1.8 {3:26:71} P. putida H4 5 16 31 36
– = No growth. P. putida F6 26 21 36 22
a
All data are the average of at least three independent determina- P. fluorescens B3 0 19 31 42
tions. Standard error is less than 10%. P. jessenii C8 0 22 41 42
b
{A:B:C} = wt% monomer composition: {(R)-3-hydroxyhexanoic P. putida D5 0 9 34 32
acid: (R)-3-hydroxyoctanoic acid: (R)-3-hydroxydecanoic acid}. All data are the average of at least three independent determinations.
c
PHA accumulation using alternative PHA accumulating method as Standard error is less than 5%.
PHA accumulation from phenylacetic acid was not observed under PhVal, phenylvaleric acid; PhHex, phenylhexanoic acid; PhHept, phe-
normal PHA-accumulating conditions (see Section 2). nylheptanoic acid; PhOct, phenyloctanoic acid.
114 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118

increased from 6 to 7 (Table 2). However both strains

accumulate the same levels of PHA from phenyloctanoic
acid as phenylheptanoic acid (Table 2). In contrast P.
fluorescens B2 accumulates a higher level of PHA from
substrates with shorter side chains i.e., it accumulates al-
most 1.8- and 2.5-fold higher levels of PHA from phe-
nylhexanoic acid compared to cells grown with
phenylheptanoic acid and phenyloctanoic acid, respec-
tively. P. putida F6 accumulates higher levels of PHA
from phenylalkanoic acids with an uneven number of

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carbons in the side chain. P. putida CA-1 accumulates
PHA to approximately the same (low) level from phe-
nylhexanoic acid, phenylheptanoic acid and phenylocta-
noic acid (Table 2). P. putida H4 and F6 are the only
two strains capable of PHA accumulation with all four
phenylalkanoic acids tested in this study. Thus a diver-
sity of abilities and levels of PHA accumulation exists
within the Pseudomonas species tested in this study.
Interestingly strains from the same species did not exhi-
bit species-specific trends with similar PHA accumulat-
ing abilities and increased PHA levels being observed
across species rather than within (Table 2).
A number of enzymes could be responsible for the
variation in PHA accumulation from phenylalkanoic
acids, e.g., phenylacetyl-CoA ligase responsible for acti-
vating phenylalkanoic acids [34,35] may act on different
substrates at different rates, the enzymes responsible for
transfer of b-oxidation intermediates towards PHA
polymerase may exhibit a broad or narrow specificity,
or indeed PHA polymerase itself may exhibit a prefer-
ence for substrates with a particular chain length [1].
An examination of the monomer composition of PHA
accumulated from these substrates will generate infor-
mation on the substrate specificity of the PHA accumu-
lating enzymes within the Pseudomonas strains tested in Fig. 1. The monomeric composition of PHA accumulated from
this study. phenylalkanoic acids as sole carbon and energy substrates by a variety
of Pseudomonas species. The monomer composition is expressed as a
weight percentage (wt%). All data are the average of at least three
3.3. Monomer composition of PHA accumulated from
independent determinations. (a) Monomer composition of PHA accu-
phenylalkanoic acids mulated from phenylhexanoic acid. (R)-3-hydroxyoctanoic acid;
(R)-3-hydroxydecanoic acid; (R)-3-hydroxyphenylbutyric acid; (R)-
Despite significant differences in the level of PHA 3-hydroxyphenylhexanoic acid. (b) Monomer composition of PHA
accumulated from phenylalkanoic acids (Table 2) the accumulated from phenylheptanoic acid. (R)-3-hydroxyoctanoic acid;
(R)-3-hydroxydecanoic acid; (R)-3-hydroxyphenylvaleric acid;
trend in monomer composition of PHA accumulated
(R)-3-hydroxyphenylheptanoic acid. (c) Monomer composition of PHA
from individual substrates was surprisingly similar in accumulated from phenyloctanoic acid. (R)-3-hydroxyoctanoic acid;
all strains (Fig. 1(a)–(c)). All the bacteria accumulated (R)-3-hydroxydecanoic acid; (R)-3-hydroxyphenylbutyric acid;
PHA containing predominantly aromatic monomers (R)-3-hydroxyphenylhexanoic acid; (R)-3-hydroxyphenyloctanoic
with traces of aliphatic monomers (Fig. 1(a)–(c)). While acid.
Pseudomonas strains have been shown to accumulate
PHA from phenylalkanoic acids [3–17], up until now phenylheptanoic acid the predominant monomer in
the occurrence of a heteropolymer of aromatic and ali- all cases is either 3-hydroxyphenylhexanoic acid
phatic monomers has been limited [5,7,13,16,17]. (Fig. 1(a)) or 3-hydroxyphenyvaleric acid (Fig. 1(b)),
The monomer composition of PHA accumulated by respectively. 3-hydroxyphenylheptanoic acid monomers
both P. putida H4 and F6 from phenylvaleric acid is al- were also observed in PHA accumulated from
most identical (3-hydroxyphenylvaleric acid monomer phenylheptanoic acid in all strains tested (Fig. 1(b)).
(95%) and traces of 3-hydroxydecanoic acid (5%)). Interestingly, strains incapable of PHA accumulation
When strains are supplied with phenylhexanoic acid or from phenylvaleric acid accumulated PHA with
 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118
3-hydroxyphenylvaleric acid monomers (Fig. 1(b)). Fur-
thermore, despite the inability of strains to either grow
or accumulate PHA from phenylbutyric acid all strains
accumulated 3-hydroxyphenylbutyric acid as a minor
monomer from phenylhexanoic acid (Fig. 1(a)).
The monomer composition of PHA accumulated
from phenyloctanoic acid was very similar in 7 of the
8 strains with 3-hydroxyphenylhexanoic acid as the pre-
dominant monomer, 3-hydroxyphenyloctanoic acid as
the second most predominant aromatic monomer, and
3-hydroxyphenylbutyric acid as the minor aromatic
monomer (Fig. 1(c)). The exceptional strain (P. fluores-
cens B2) accumulated PHA with a ratio of 3-hydroxy-
phenylhexanoic acid to 3-hydroxyphenyloctanoic acid
of almost 1:1 (Fig. 1(c)). This suggests that with the
exception of a single strain on a single substrate the
PHA accumulating enzymes from all strains for all of
the substrates exhibit a very similar substrate range.
Thus while the monomer composition of PHA does
not significantly vary (Fig. 1), the ability to accumulate
PHA varies dramatically (Table 1). PHA monomer
composition can be dictated by PHA polymerase or an
earlier enzyme in the PHA accumulation pathway
[32,33], e.g., the monomer composition of PHA accumu-
lated from unrelated substrates is determined by PhaG
[32,33]. PhaG exhibits a preference for (R)-3-hydroxy-
decanoyl-ACP intermediates and thus transfers more
10 carbon units to PHA polymerase resulting in PHA
composed predominantly of 10 carbon units [32,33]. Fu-
ture biochemical experiments will determine which en-
zyme activities are affecting PHA accumulation and
monomer composition in these strains.
Increased diversity in the ratio of aromatic monomers
allows for greater diversity in polymer properties and
thus increased opportunities for PHA polymer applica-
tions [5,7,36]. The similarity in monomer composition
across a range of strains suggests that the approach to
increasing the range and diversity of aromatic PHA
14
12
10
% CDW PHA
0
S12
Fig. 2. PHA accumulation from phenylacetic acid in the presence of 2-bromooctanoic acid. Cells are grown with phenylacetic acid (5 mM) for 24 h,
centrifuged and resuspended in fresh medium containing 2-bromooctanoic acid (10 mM) for a further 24 h. All data are the average of at least three
independent determinations. control – phenylacetic acid; phenylacetic acid and 2- bromooctanoic acid.
115
(i.e., tailored biosynthesis of PHA) is possibly best
achieved through altered substrate feeding strategies
(i.e., substrate co-feeding) rather than screening wild
type strains for PHA polymerases or other PHA accu-
mulating enzymes with differing substrate specificity
[5,7,10,11,13,16].
3.4. The effect of 2-bromooctanoic acid on PHA
accumulation from phenylacetic acid and phenyloctanoic
acid
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The fatty acid synthesis pathway is known to rebuild
acetyl-CoA molecules to produce aliphatic (R)-3-hydrox-
yacyl-ACP molecules, which are transformed by (R)-3-
hydroxyacyl-ACP-CoA transferase (encoded by phaG)
to (R)-3-hydroxyacyl-CoA which acts as a substrate for
PHA polymerase [32,33]. 2-Bromooctanoic acid is a
known inhibitor of (R)-3-hydroxyacyl-ACP-CoA
transferase (phaG) [37].
Growth of the Pseudomonas species in this study with
phenylacetic acid as the sole source of carbon and energy
in the presence of 2-bromooctanoic acid (10 mM) re-
sulted in a significant decrease in the level of PHA accu-
mulation in the majority of strains (Fig. 2). However,
PHA accumulation was only completely inhibited in a
single strain (P. putida F6) and only moderately affected
in P. jessenii C8 (Fig. 2). The monomer composition of
the PHA accumulated in the presence of 2-bromoocta-
noic acid was unchanged from that accumulated in its
absence (data not shown). These observations suggest
that for the majority of strains (R)-3-hydroxyacyl-
ACP-CoA transferase plays an important role in PHA
accumulation from phenylacetic acid.
PHA accumulated by Pseudomonas strains from
phenyloctanoic acid in this study contained aromatic
and aliphatic monomers suggesting a role for b-oxida-
tion and fatty acid synthesis, respectively. Thus the
addition of 2-bromooctanoic acid, to cell suspensions
CA-1 B2 H4 F6 B3 C8 D5
Strains
116 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118
30
25
% CDW PHA
20
15
10
0
S12
Fig. 3. PHA accumulation from phenyloctanoic acid in the presence of acrylic acid. Cells are grown with phenyloctanoic acid (5 mM) for 24 h,
centrifuged and resuspended in fresh medium containing acrylic acid (5 mM) for a further 24 h. All data are the average of at least three independent
determinations. control – phenyloctanoic acid; phenyloctanoic acid and acrylic acid.
should allow aromatic monomer accumulation but in-
hibit the accumulation of aliphatic monomers from
aromatic substrates [37]. However, cells incubated with
2-bromooctanoic acid and phenyloctanoic acid showed
little or no decrease in the levels of both aromatic and
aliphatic PHA monomers (data not shown). The failure
to inhibit aliphatic monomer accumulation is in keep-
ing with observations made for P. putida CA-3 where
2-bromooctanoic acid was shown to be a leaky inhibi-
tor incapable of inhibition of aliphatic PHA accumula-
tion from phenylalkanoic acids [17].
3.5. Effect of acrylic acid on PHA accumulation from
phenylacetic acid and phenyloctanoic acid
Acrylic acid inhibits the activity of 3-ketoacyl-CoA
thiolase, the enzyme that catalyses the final step of b-oxi-
dation releasing acetyl-CoA from 3-ketoacyl-CoA [37–
39]. As an unrelated substrate whose metabolic route
to PHA should involve fatty acid synthesis only, acrylic
acid should not affect PHA accumulation from phenyla-
cetic acid. Surprisingly, when cells are supplied with
phenylacetic acid as the sole carbon source in the pres-
ence of acrylic acid, a notable decrease in the final OD
of cultures and PHA synthesis is observed (data not
shown). It is known that microbial metabolism of phen-
ylacetic acid proceeds via phenylacetyl-CoA ligase,
through the action of an acyl-CoA synthase (fadD) and
that the product of the reaction proceeds through a b-
oxidation like pathway [21,22]. The b-oxidation like
pathway contains a ketothiolase enzyme (phaD) that is
essential for phenylacetic acid metabolism [22]. Acrylic
acid could potentially inhibit one or both of these en-
zymes [37–39] and consequently inhibit growth and
PHA accumulation. Thus for all strains tested in this
study it is likely that b-oxidation and fatty acid synthesis
play a role in PHA accumulation from phenylacetic acid.
Where PHA accumulation from phenylacetic acid is ob-
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CA-1 B2 H4 F6 B3 C8 D5
Strains
served the predominant monomer remains 3-hydroxy-
decanoic acid.
Previously, attempts to increase PHA accumulation
from PHA related substrates (those whose chemical
structure resembles PHA monomers, e.g., alkanoic
and phenylalkanoic acids) have employed the enzyme
inhibitor acrylic acid to cause a build up of PHA inter-
mediates from b-oxidation [38,39]. As acrylic acid
inhibits the growth of the Pseudomonas strains on phe-
nyloctanoic acid, cells were grown as described in
materials and methods Section 2.5. The addition of ac-
rylic acid to cell suspensions grown on phenyloctanoic
acid decreased the level of PHA accumulated by all of
the strains tested in comparison to cell suspensions un-
treated with acrylic acid (Fig. 3). Increasing the con-
centration of acrylic acid to 10 mM decreases the
levels of PHA even further (data not shown). The
monomer composition of the PHA accumulated by
all strains is unaffected by the presence of acrylic acid
(data not shown).
In conclusion there is a broad diversity in the ability
of Pseudomonas species to accumulate PHA from aro-
matic hydrocarbons. With the exception of a single
strain the monomer composition for all the strains
tested in this study is almost identical. Data generated
from the addition of fatty acid synthesis and b-oxida-
tion inhibitors to cells grown with phenylacetic acid
suggest a role for both pathways in PHA accumulation
from this unrelated carbon source. The addition of b-
oxidation inhibitors did not result in an increase in
PHA accumulation from phenyloctanoic acid in any
of the strains tested.
Acknowledgements
We thank Dr. Nora Carroll, University College
Dublin, for assistance with strain identification using
16S rDNA sequencing. Karen Tobin is a recipient of a
 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118
University College Dublin funded postgraduate
scholarship.
References
[1] Madison, L.L. and Huisman, G.W. (1999) Metabolic engineering
of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol.
Mol. Biol. Rev. 63, 21–53.
[2] Witholt, B. and Kessler, B. (1999) Perspectives of medium chain
length poly (hydroxyalkanoates), a versatile set of bacterial
bioplastics. Curr. Opin. Biotechnol. 10, 279–285.
[3] Garcia, B., Olivera, E.R., Miñambres, B., Fernandez-Valverde,
M., Canedo, L.M., Prieto, M.A., Garcia, J.L., Martinez, M. and
Luengo, J.M. (1999) Novel biodegradable aromatic plastics from
a bacterial source. J. Biol. Chem. 274, 29228–29241.
[4] Abraham, G.A., Gallardo, A., San Roman, J., Olivera, E.R.,
Jodra, R., Garcia, B., Miñambres, B., Garcia, J.L. and Luengo,
J.M. (2001) Microbial synthesis of poly(b-hydroxyalkanoates)
bearing phenyl groups from Pseudomonas putida: chemical
structure and characterization. Biomacromol 2, 562–567.
[5] Song, J.J., Choi, M.H., Yoon, S.C. and Huh, N.E. (2001)
Cometabolism of phenylalkanoic acids with butyric acid for the
efficient production of aromatic polyesters in Pseudomonas putida
BM01. J. Microbiol. Biotechnol. 11, 435–442.
[6] Olivera, E.R., Carnicero, D., Garcia, B., Miñambres, B., Moreno,
M.A., Cañedo, L., DiRusso, C.C., Naharro, G. and Luengo, J.L.
(2001) Two different pathways are involved in the b-oxidation of
n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U:
genetic studies and biotechnological applications. Mol. Microbiol.
39, 863–874.
[7] Olivera, E.R., Carnicero, D., Jodra, R., Miñambres, B., Garcia,
B., Abraham, G.A., Gallardo, A., San Roman, J., Garcia, J.L.,
Naharro, G. and Luengo, J.M. (2001) Genetically engineered
Pseudomonas: a factory of new bioplastics with broad applica-
tions. Environ. Microbiol. 3, 612–618.
[8] Choi, M.H. and Yoon, S.C. (1994) Polyester biosynthesis
characteristics of Pseudomonas citronellolis grown on various
carbon sources, including 3-methyl-branched substrates. Appl.
Env. Mirobiol. 60, 3245–3254.
[9] Fritzsche, K., Lenz, R.N. and Fuller, R.C. (1990) An unusual
bacterial polyester with a phenyl pendent group. Macromol.
Chem. 191, 1957–1965.
[10] Kim, Y.B., Lenz, R.W. and Fuller, R.C. (1991) Preparation and
characterisation of poly(b-hydroxyalkanoates) obtained from
Pseudomonas oleovorans grown with mixtures of 5-phenylvaleric
acid and n-alkanoic acids. Macromol 24, 5256–5260.
[11] Curley, J.M., Hazer, B., Lenz, R.W. and Fuller, R.C. (1996)
Production of poly(3-hydroxyalkanoates) containing aromatic
substituents by Pseudomonas oleovorans. Macromol 29, 1762–1766.
[12] Curley, J.M., Lenz, R.W. and Fuller, R.C. (1996) Sequential
production of 2 different polyesters in the inclusion bodies of
Pseudomonas oleovorans. Int. J. Biol. Macromol. 19, 29–34.
[13] Hazer, B., Lenz, R.W. and Fuller, R.C. (1996) Bacterial produc-
tion of poly-3-hydroxyalkanoates containing arylalkyl substituent
groups. Polymer 37, 5951–5957.
[14] Song, J.J. and Yoon, S.C. (1996) Biosynthesis of novel aromatic
copolyesters from insoluble 11-phenoxyundecanoic acid by Pseu-
domonas putida BM01. Appl. Environ. Microbiol. 62, 536–544.
[15] Kim, Y.B., Kim, D.Y. and Rhee, Y.H. (1999) PHAs produced by
Pseduomonas putida and Pseudomonas oleovorans grown with n-
alkanoic acids containing aromatic groups. Macromol 32, 6058–
6064.
[16] Chung, D.M., Choi, M.H., Song, J.J., Yoon, S.C., Kang, I.K. and
Huh, N.E. (2001) Intracellular degradation of two structurally
different polyhydroxyalkanoic acids accumulated in Pseudomonas
117
putida and Pseudomonas citronellolis from mixtures of octanoic acid
and 5-phenylvaleric acid. Int. J. Biol. Macromol. 29, 243–250.
[17] Ward, P.G. and O
Connor, K.E. (2005) Bacterial synthesis of
polyhydroxyalkanoates containing aromatic and aliphatic mono-
mers by Pseudomonas putida CA-3. Int. J. Biol. Macromol. 35,
127–133.
[18] Ward, P.G., de Roo, G. and O
Connor, K.E. (2005) Accumu-
lation of polyhydroxyalkanoate from styrene and phenylacetic
acid by Pseudomonas putida CA-3. Appl. Environ. Microbiol. 71,
2046–2052.
[19] Hartmans, S., Van der Werf, M.J. and de Bont, J.A.M. (1990)
Bacterial degradation of styrene involving a novel flavin adenine
Downloaded from https://academic.oup.com/femsle/article-abstract/253/1/111/516508 by guest on 17 September 2019
dinucleotide-dependent styrene monooxygenase. Appl. Env.
Microbiol. 56, 1347–1351.
[20] O
Connor, K., Buckley, C.M., Hartmans, S. and Dobson,
A.D.W. (1995) Possible regulatory role for nonaromatic carbon
sources in styrene degradation by Pseudomonas putida CA-3.
Appl. Env. Microbiol. 61, 544–548.
[21] O
Leary, N.D., O
Connor, K.E. and Dobson, A.D.W. (2002)
Biochemistry, genetics and physiology of microbial styrene
degradation. FEMS Microbiol. Rev. 26, 403–417.
[22] Olivera, E.R., Miñambres, B., Garcia, B., Muñiz, C., Moreno,
M.A., Ferrández, A., Diaz, E., Garcia, J.L. and Luengo, J.M.
(1998) Molecular characterisation of the phenylacetic acid cata-
bolic pathway in Pseudomonas putida U: the phenylacetyl-CoA
catabolon. Proc. Natl. Acad. Sci. 95, 6419–6424.
[23] Luengo, J.M., Garcia, J.L. and Olivera, E.R. (2001) The
phenylacetyl-CoA catabolon: a complex catabolic unit with broad
biotechnological applications. Mol. Microbiol. 39, 1434–1442.
[24] Prieto, M.A., Galan, B., Torres, B., Ferrández, A., Ferrández, C.,
Miñambres, B., Garcia, J.L. and Diaz, E. (2004) Aromatic
metabolism versus carbon availability: the regulatory network
that controls the catabolism of less-preferred carbon sources in
Escherichia coli. FEMS Microbiol. Rev. 28, 503–518.
[25] O
Connor, K.E., Witholt, B. and Duetz, W. (2001) p-Hydroxy-
phenylacetic acid metabolism in Pseudomonas putida F6. J.
Bacteriol. 183, 928–933.
[26] Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J.,
Rapp, B.A. and Wheeler, D.L. (2002) GenBank. Nucleic Acids
Res. 30, 17–20.
[27] Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang,
Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-
BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25, 3389–3402.
[28] Hall, T.A. (1999) BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows 95/98/NT.
Nucleic Acids Symp. Ser. 41, 95–98.
[29] Vogel, H.J. and Bonner, D.M. (1956) DM Acetylornithinase of
E.coli: partial purification and some properties. J. Biol. Chem.
218, 97–106.
[30] Lageveen, R.G., Huisman, G.W., Preusting, H., Ketelaar, P.,
Eggink, G. and Witholt, B. (1988) Formation of polyesters
by Pseudomonas oleovorans: effect of substrates on formation
and composition of poly-(R)-3-hydroxyalkanoates and poly-
(R)-3-hydroxyalkenoates. Appl. Env. Mirobiol. 54, 2924–
2932.
[31] O
Connor, K.E. (1998) Indigo formation by aromatic hydrocar-
bon-degrading bacteria. Biotechnol. Lett. 20, 219–223.
[32] Rehm, B.H.A., Krüger, N. and Steinbüchel, A. (1998) A new
metabolic link between fatty acid de novo synthesis and
polyhydroxyalkanoic acid synthesis. J. Biol. Chem. 273, 24044–
24051.
[33] Fiedler, S., Steinbüchel, A. and Rehm, B.H.A. (2000) PhaG-
mediated synthesis of poly(3-hydroxyalkanoates) consisting of
medium-chain-length constituents from nonrelated carbon
sources in recombinant Pseduomonas fragi. Appl. Env. Microbiol.
66, 2117–2124.
118 K.M. Tobin, K.E. O’Connor / FEMS Microbiology Letters 253 (2005) 111–118
[34] Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and
Luengo, J.M. (1990) Purification and biochemical characteriza-
tion of phenylacetyl-CoA ligase from Pseudomonas putida. J. Biol.
Chem. 265, 7084–7090.
[35] Schleissner, C., Olivera, E.R., Fernández-Valverde, M. and
Luengo, J.M. (1994) Aerobic catabolism of phenylacetic acid in
Pseudomonas putida U: biochemical characterisation of a specific
phenylacetic acid transport system and formal demonstration that
phenylacetyl-coenzyme A is a catabolic intermediate. J. Bacteriol.
176, 7667–7676.
[36] Steinbüchel, A. and Valentin, H.E. (1995) Diversity of bacterial
polyhydroxyalkanoic acids. FEMS Microbiol. Lett. 128, 219–
228.
[37] Lee, H.J., Choi, M.H., Kim, T.U. and Yoon, S.C. (2001)
Accumulation of polyhydroxyalkanoic acid containing large
amounts of unsaturated monomers in Pseudomonas fluorescens
BM07 utilizing saccharides and its inhibition by 2-bromooctanoic
acid. Appl. Env. Microbiol. 67, 4963–4974.
[38] Huijberts, G.N., de Rijk, T.C., de Waard, P. and Eggink, G.
(1994) 13C nuclear magnetic resonance studies of Pseudomonas
putida fatty acid metabolic routes involved in poly(3-hydroxyalk-
anoate) synthesis. J. Bacteriol. 176, 1661–1666.
[39] Qi, Q., Steinbüchel, A. and Rehm, B.H.A. (1998) Metabolic
routing towards polyhydroxyalkanoic acid synthesis in recombi-
nant Escherichia coli (fadR): inhibition of fatty acid b-oxidation
Downloaded from https://academic.oup.com/femsle/article-abstract/253/1/111/516508 by guest on 17 September 2019
by acrylic acid. FEMS Microbiol. Lett. 167, 89–94.