( Springer-Verlag 1996
Appl Microbiol Biotechnol (1996) 46:261—267
OR I G I N A L P AP E R
H. E. Valentin · A. Schönebaum · A. Steinbüchel
Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid
and 4-hydroxyoctanoic acid as new constituents
of bacterial polyhydroxyalkanoic acids
Received: 22 April/Received revision: 23 May 1996/Accepted: 2 June 1996
Abstract A recombinant strain of Pseudomonas putida
GPp104 (pHP1014 : : E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of ¹hiocapsa
pfennigii exhibiting an unusual substrate specificity at
a high level was incubated in two-stage batch or fedbatch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second
cultivation phase, copolyesters of 3-hydroxybutyric
acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic
acid (3HHx) plus 5HHx were accumulated as revealed
by gas-chromatographic and 13C-NMR spectroscopic
analysis. When the recombinant P. putida GPp104 was
incubated with 4-hydroxyheptanoic acid (4HHp) as
carbon source in the second cultivation phase,
a copolyester consisting of 3HB, 3-hydroxyvaleric acid
and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the
second cultivation phase led to the accumulation of
a polyester that contained 1—2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3hydroxyvaleric acid and 3HB. In addition to PHA
containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic
acid. Eleven strains from five genera have been also
analysed for their ability to utilize different carbon
sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with
unusual constituents. Although most of the carbon
sources were utilized by some strains for colony
H. E. Valentin
Department of Biology, James Madison University, Harrisonburg,
VA 22807, USA
A. Schönebaum
Institut für Organische Chemie der Georg-August-Universität
Göttingen, Tammannstraße 2, D-37077 Göttingen, Germany
A. Steinbüchel ( )
Institut für Mikrobiologie, Westfälische Wilhelms-Universität
Münster, Corrensstraße 3, D-48149 Münster, Germany
growth, accumulation experiments gave no evidence
for the accumulation of new PHA by these wild-type
strains.
Introduction
Polyhydroxyalkanoic acid (PHA) synthases, which represent the key enzyme of PHA biosynthesis in bacteria,
have been investigated in much detail at a molecular
level in recent years (Steinbüchel et al. 1992, 1995, and
references cited therein; Steinbüchel 1995; Gerngross et
al. 1993). Most bacteria possess a PHA
synthase,
SCL
which preferably incorporates short-chain-length
(SCL) 3-hydroxyalkanoic acids comprising three to five
carbon atoms (Anderson and Dawes 1990; Steinbüchel
1991) but also 4- and 5-hydroxyalkanoic acids
(Kunioka et al. 1988; Valentin et al. 1992; Doi et al.
1987; Steinbüchel and Valentin 1995). Pseudomonads
belonging to the rRNA homology group I possess
PHA
synthases that preferably incorporate 3-hyMCL
droxyalkanoic acids of medium chain length with six or
more carbon atoms (Anderson and Dawes 1990; Steinbüchel 1991).
Recently the PHA synthase structural gene of the
purple-sulphur bacteria ¹hiocapsa pfennigii was cloned
(Liebergesell et al. 1993), and we obtained evidence that
the substrate specificity of this synthase was distinguishable from others. This conclusion was drawn
from the analysis of PHA isolated from cells of a recombinant strain of a PHA-negative mutant of
Pseudomonas putida, which expressed the PHA
synthase of ¹. pfennigii at a high level (Valentin and
Steinbüchel 1994). From octanoic acid these cells accumulated a copolyester consisting of almost equimolar
amounts of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHx) plus small amounts of
3-hydroxyoctanoic acid (3HO) (Liebergesell et al.
1993); from 4-hydroxyhexanoic acid (4HO) these cells
accumulated a terpolyester containing 4HO at a high
262
molar fraction in addition to 3HB acid and 3HHx
(Valentin et al. 1994). These studies prompted us to
explore the capability of this unusual unspecific PHA
synthase for the production of other new PHA.
Materials and methods
Bacterial strains, cultivation of bacteria, and media
All strains investigated in this study are listed in Table 1. In addition, plasmid pHP1014: : E156, which harbours the PHA-biosynthesis genes of ¹. pfennigii (Liebergesell et al. 1993), was used. The
recombinant strain of the PHA-negative mutant of P. putida harbouring this plasmid was referred to as P. putida GPp104-E156. All
bacteria were cultivated at 30°C in nutrient broth (0.8%, w/v), or the
basic mineral salts medium (MSM) described by Schlegel et al.
(1961). Filter-sterilized carbon sources were added as indicated in
the text. Solidified media contained 1.5% (w/v) agar.
The ability to accumulate PHA was investigated in one-stage
fed-batch or in two-stage batch cultivation experiments as described
recently (Valentin et al. 1994) except that 5HHx, 4-hydroxyheptanoic acid (4HHp) or 4HO was used as carbon source instead of
4HHx.
Isolation and gas-chromatographic analysis of PHA
The isolation of PHA from lyophilized cells and precipitation with
ethanol as well as its analysis by gas chromatography was done as
described recently (Valentin et al. 1994 and references cited therein).
Nuclear magnetic resonance (NMR) spectroscopic analysis
NMR spectroscopic analysis of PHA was done as described in
a previous publication (Valentin et al. 1994).
Preparation of carbon sources
5HHx was obtained by reduction of 4-acetylbutyric acid with sodium borohydride under alkaline conditions. Therefore, 0.2 mol
4-acetylbutyric acid was added to 25 ml H O and the pH was
2
adjusted to 10. Sodium borohydride (0.11 mol) was dissolved in 5 ml
slightly alkaline H O (pH 10) and slowly mixed with the ice-chilled
2
aqueous solution of 4-acetylbutyric acid. The mixture was stirred on
ice for 2 h. To purify the resulting 5HHx, 1 M HCl was added to the
reaction mixture until the pH was 2.0. The release of gas during this
process indicated an excess of sodium borohydride. Subsequently,
5HHx was extracted with diethyl ether. To hydrolyse d-hexanolactone, which may have formed during the purification procedures, the
purification product was subjected to alkaline saponification (Valentin et al. 1994).
4HHp acid and 4HO were obtained by alkaline saponification of
the corresponding lactone as described recently for preparation of
4-hydroxyvaleric acid (4HV) (Valentin et al. 1992).
Synthesis of 5-acetoxyhexanoic acid methyl ester
To synthesize 5-acetoxyhexanoic acid methyl ester (5AHM) 2 g
5 HHx (sodium salt) was dissolved in 10 ml methanol/H O (1 : 1 v/v).
2
The pH was adjusted to 4.0 by adding 0.01 M HCl. Diazomethane
(approx. 0.4 M in diethyl ether) was added to the ice-chilled solution,
until it remained yellow. The solvents were evaporated under reduced pressure, and 5 HHx methyl ester was purified by gel
chromatography on Silica Gel 60 (Machery & Nagel, Düren, Germany) with cyclohexane/acetic acid ester (3 : 1, v/v).
5HHx methyl ester (1.5 g), 10 ml dry methylene chloride, 1.1 ml
acetic acid anhydride and 1 ml pyridine were mixed and refluxed for
4 h. The solvents were evaporated under reduced pressure, the
remaining solution was added to 25 ml diethyl ether, and extracted
three times with saturated potassium dihydrogenphosphate. Subsequently, 5AHM was purified by gel chromatography on Silica Gel
60 (Machery & Nagel, Düren, Germany) with cyclohexane/acetic
acid ester (5 : 1, v/v).
Chemicals
4-Acetylbutyric acid was obtained from Aldrich (Steinheim, Germany). c-Heptanolactone and c-octanolactone were obtained from
Haarmann & Reimer GmbH (Holzminden, Germany). Most other
chemicals were from Merck (Darmstadt, Germany).
Results
Growth on various putative precursors for new 4- and
5-hydroxy fatty acid constituents in PHA
Several strains of Alcaligenes eutrophus, Pseudomonas,
and Chromobacterium violaceum were cultivated on
various carbon sources, in order to analyse the utilization of putative precursor substrates for new monomers in PHA and to find suitable substrates for the
synthesis of unusual PHA. Whereas c-octanolactone
seems to be toxic to the strains investigated in this
study, most other lactones were utilized for growth to
a certain extent in solidified mineral salts media
(Table 1). c-Hexanolactone and d-hexanolactone were
utilized for colony growth by all strains of A. eutrophus.
These strains also exhibited colony growth on all 4- or
5-hydroxy- or ketoacids investigated in this study. The
sodium salts of laevulinic acid 4HHx, 5-HV and 5HHx
were also suitable carbon sources for colony growth of
some strains of Pseudomonas species (Table 1). However, application of these carbon sources to wild-type
strains for PHA accumulation did not result in the
incorporation of new constituents into PHA. When
these strains were cultivated under conditions in which
the availability of ammonium limited growth, only
3-hydroxyalkanoic acids, such as 3HB, were detected in
the accumulated polyesters.
PHA accumulation of Pseudomonas putida GPp104
(pHP1014: : E156) from various carbon sources
Since recent studies revealed an unusual substrate specificity of the ¹. pfennigii PHA synthase complex
(Liebergesell et al. 1993; Valentin and Steinbüchel
1994), in this study other precursor substrates were
provided to the recombinant strain of P. putida
GPp104 harbouring the PHA biosynthesis genes of
##
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DSM 291
Worsey and Williams 1975
Huismann et al. 1991
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DSM 1045
ATCC 29347
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### !
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DSM 1707
### ##
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DSM 30191
!
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DSM 428
DSM 541
Srivastava et al. 1982
Don and Pemberton 1981
Alcaligenes eutrophus
H16
PHB-4
HF39
JMP222
Chromobacterium violaceum
Type strain
Pseudomonas aeruginosa
PAO1
P. oleovorans
Type strain
TF4-1L
P. putida
Type strain
KT 2440
GPp104
5HL
5HHx
5VL
5HV
4OL
4HO
4HL
4HHx
LA
4BL
Source or reference
Strain
Table 1 Utilization of different 4- and 5-hydroxy fatty acids and corresponding lactones or keto acids for growth. Colony growth was monitored on mineral salts medium/agar plates after
3 days incubation at 30°C. ### good growth, ## medium growth, # poor growth, $ very poor growth, !, no growth. 4ACB 4-acetylbutyric acid, 4BL c-butyrolactone, 4HL
c-hexanolactone, 4HHx 4-hydroxyhexanoic acid, 5HHx 5-hydroxyhexanoic acid, 5HL d-hexanolactone, 4HO 4-hydroxyoctanoic acid, 3HPN 3-hydroxypropionic acid nitrile, 5HV
5-hydroxyvaleric acid, ¸A laevulinic acid, 4OL c-octanolactone, 5VL d-valerolactone
263
¹. pfennigii in two-stage batch or two-stage fed-batch
accumulation experiments in order to obtain new polyesters. When laevulinic acid was provided as the carbon
source, poly(3-hydroxybutyric acid-co-3-hydroxyvalericacid-co-4-hydroxyvaleric acid) [poly(3HB-co-3HV-co4HV)] accumulated (Table 2). The molar fraction of
4HV was 19.1 mol%, which was four times higher than
the molar fraction of 4HV in poly(3HB-co-3HV-co4HV) accumulated from 4HV by wild-type strains (Valentin et al. 1992), and almost as high as the molar
fraction of 4HV obtained from genetically manipulated
strains of A. eutrophus after incubation with 4HV as
carbon source (Valentin and Steinbüchel 1995).
Providing 5HHx as carbon source resulted in
the accumulation of poly(3HB-co-3HHx-co-5HHx)
(Table 2) or poly(3HB-co-5HHx) (Figs. 1, 2) depending
on the culture conditions. To obtain sufficient amounts
of PHA containing 5HHx for NMR spectroscopic
analysis, P. putida GPp104-E156 was grown in a twostep accumulation experiment with 5HHx as carbon
and energy source in the second stage. The cells were
grown in four 1-l conical flasks, each containing 250 ml
nutrient broth for 16 h. Cells were harvested, washed
with nitrogen-free mineral salts medium (MSM) and
resuspended in the same volume of nitrogen-free MSM.
During the second step of the cultivation cells were fed
with portions of 0.25% (v/v) 5HHx at the inoculation
and after 24 h, 48 h as well as 72 h after inoculation.
After 96 h cultivation, 380 mg cellular dry mass was
obtained. Chloroform extraction of these cells revealed
20 mg poly(3HB-co-5HHx) with a molar fraction of
40.6 mol% 5HHx, as revealed by 1H-NMR spectroscopic analyses (Fig. 1).
When heptanoic acid was provided as carbon source,
a copolyester of 3HHp and 3HV accumulated. In addition, small amounts of 3HB, and traces of 3HHx as well
as of 3HO were found in the polyester (Table 2).
When P. putida GPp104-E156 was grown in twostage batch cultivation experiments with 4HO as sole
carbon and energy source in the second stage, a polyester accumulated that contained 3HB and 3HHx as
major constituents. In addition small amounts of 3HV,
3HO and 1—2 mol% 4HO were detected in lyophilized
cells as well as in the purified polyesters by gaschromatographic analysis (Table 2).
When P. putida GPp104-E156 was grown in twostage batch cultivatioin experiments with 4HHp as sole
carbon and energy source in the second stage, a polyester accumulated, that contained, in addition to the
major constituent 3HV, also small amounts of 4HHp
(4.9 mol%), 3HHp (4.3 mol%) and 3HB (7.8 mol%)
as revealed by gas-chromatographic analysis of
lyophilized cells. The composition of this PHA was
confirmed upon analysis of the polyester isolated from
the cells of the recombinant strain.
In addition to the incorporation of new constituents
into PHA it was also found that 4HB was incorporated
into PHA by P. putida GPp104-E156 when 4HB was
264
Table 2 Polyhydroxyalkanoic acid (PHA)-accumulation by P.
putida GPp104-E156 from different carbon sources. PHA accumulation was analysed in two-stage fed-batch accumulation experiments.
Cells were grown in nutrient broth during the first cultivation phase,
harvested and washed once with nitrogen-free mineral salts medium
(MSM) and transferred to MSM containing the carbon source as
indicated. If not indicated separately, the cultivation period was
24 h. The composition of the polyesters was determined by gasCarbon source
2-Hydroxybutyrate (0.5% w/v)
4-Hydroxy-2-butenoate (0.5%. v/v)
4-Hydroxy-2-butenoate (0.1% v/v)
#4-hydroxybutyrate (0.5% w/v)
Laevulinate (2]0.5% v/v 96 h)
4-Hydroxyhexanoate (2]0.5% v/v, 48 h)
5-Hydroxyhexanoate!
6-Hydroxyhexanoate (0.5% v/v)
Hheptanoate (2]0.25% v/v)
6-Hydroxyhexanoate#hexanoate
[2](0.05% v/v#0.2% v/v), 96 h]
4-hydroxyoctanoate (4]0.2% v/v, 96 h)
PHA content
(% CDM)
chromatographic analysis. CDM cellular dry matter, 3HB
3-hydroxybutyric acid 4HB 4-hydroxybutyric acid; 3HV, 3-hydroxyvaleric acid; 4HV, 4-hydroxyvaleric acid; 3HHx, 3-hydroxyhexanoic acid; 4HHx, 4-hydroxyhexanoic acid, 5HHx
5-hydroxyhexanoic acid, 3HHp 3-hydroxyheptanoic acid, 3HO 3hydroxyoctanoic acid, 4HO 4-hydroxyoctanoic acid, ND not determined, !not detected
Composition of PHA (mol%)
3HB
4HB
3HV
4HV
3HHx 4HHx 5HHx 3HHp 3HO
4HO
3.6
1.0
7.9
92.6
100.0
19.1
—
—
77.4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
7.4
—
3.4
—
—
—
68.3
31.0
ND
1.2
61.6
24.0
1.6
30.5
71.4
100.0
3.4
45.3
—
—
—
—
—
—
79.3
—
—
—
85.7
1.2
19.1
—
—
—
—
—
—
30.9
3.8
—
0.6
52.9
—
27.9
—
—
—
—
—
—
24.8
—
—
—
—
—
—
—
9.8
—
—
—
—
—
0.5
0.7
—
—
—
—
—
—
15.8
81.4
—
1.3
—
13.9
—
—
—
2.1
1.5
For cultivation on 5HHx, cells were incubated with 0.25% (v/v) 5HHx. Additional substrate was provided after 24 h cultivation (0.25%
5HHx, v/v) and after 48 h of cultivation (0.5% 5HHx, v/v). The composition of 5HHx-containing polyesters was analysed by proton NMR
spectroscopy
present in the culture medium (Table 2). However, it
was possible to detect neither the incorporation of 2- or
6-hydroxy acids nor the incorporation of hydroxy acids
such as 4-hydroxy-2-butenoate, which would place
double bonds into the backbone of the polyester.
Gas-chromatographic analyses of PHA containing
4- or 5-hydroxy acids
The presence of 4- or 5-hydroxy acids in PHA resulted
in two additional signals in the gas chromatogram of
the samples after methanolysis. To analyse this phenomenon in more detail, the retention times of the
lactones were compared to the retention times of methanolysis products of the lactones. In all cases the retention time of the lactone was similar to the retention
time of the second signal of the reaction products of
methanolysis (Table 3), indicating that the second signal originated from the lactone whereas the first signal
presumably originated from the methyl esters. The retention time of the putative 5HHx methyl ester was
identical to that of the methyl ester of 3HB. Therefore,
the composition of 5HHx-containing samples was not
determined quantitatively by gas-chromatographic
analysis but by 1H-NMR spectroscopy.
NMR-spectroscopic analysis of 5HHx-containing
PHA
Since the quantitative analysis of 5HHx-containing
polyesters by gas chromatography was difficult, the
quantification was done by 1H-NMR spectroscopic
analysis. Fig. 2 shows the 500-MHz 1H spectrum obtained from poly(3HB-co-5HHx) with a molar composition of 59.4 mol% 3HB and 40.6 mol% 5HHx.
In13C-NMR spectra all signals split into several peaks
indicating a random distribution of the constituents in
the copolyester, as reported for several other co- and
terpolyesters (Doi et al. 1986; Bluhm et al. 1986; Valentin et al. 1992, 1994). The split of the signals is demonstrated clearly in the stretch of the carbonyl carbon
signals (168.9—169.72 ppm and 172—173.2 ppm).
The 1H- and the 13C-NMR resonances of the 3HB
monomers (signals 1—4) were assigned by data comparison (Doi et al. 1986; Bluhm et al. 1986). The methyl
groups (C-4 in 3HB and C-6 in 5HHx) of both monomers in poly(3HB-co-5HHx) occurred in similar chemical environments (Fig. 1). Therefore, it was expected
that these signals might exhibit a similar chemical shift
in the 13C-NMR spectrum. The 13C-NMR signals of
both methyl groups occurred at the same position,
which is indicated by the height of the 3HB monomer
methyl signal, which was almost twice as high as the
signals of the CH and CH carbons (Table 4). There2
fore, and since it was difficult to assign the signals 6,
7and 8 in the 1H- and the 13C-NMR spectra of
poly(3HB-co-5HHx), a model compound, 5AHM (Fig. 1)
was synthesized and NMR spectroscopically analysed.
The 13C chemical shifts of poly(3HB-co-5HHx) and
5AHM are compared in Table 4.
Except for signals 3 and 4, all signals of 5AHM were
assigned by correlated spectroscopy (CH-COSY) (data
not shown). Signals 3 and 4 of 5AHM were assigned by
recording a COLOC spectrum (data not shown), which
265
reveals 1H-13C resonances extending over more than
one bond. The most important signals of the COLOC
spectrum exhibited the following resonances: the carbon nucleus with the resonance at d"20.76 ppm exhibited resonances with the protons of the 2-methylene
group (d"2.3 ppm) and with the protons attached to
the carbon that exhibited a 13C-resonance at
d"35.22 ppm. The latter exhibited spin-spin interactions with the protons of the 2-methylene group
(d"2.3 ppm) and the 6-methyl group (d"1.2 ppm).
The protons of the 6-methyl group (d"1.2 ppm) also
exhibited spin-spin interactions with C-5 (d"
70.33 ppm). In addition, C-6 exhibited interaction with
the protons correlated to the carbon nucleus that exhibited a 13C resonance at d"35.22 ppm. The latter
correlations determine the assignment of the C-3 carbon signal to d"20.76 ppm and the C-4 carbon signal
to d"35.22 ppm, since no mutual effects to C-3 were
detected.
Discussion
The present study provides further evidence for an
unusual substrate specificity of the ¹. pfennigii PHA
synthase complex (Liebergesell et al. 1993; Valentin et
al. 1994). 4-HHx (Valentin et al. 1994), 5HHx, 4HHp
and 4HO have been found as new constituents of PHA
accumulated by P. putida GPp104-E156, when suitable
precursor substrates were provided as carbon sources.
None of these 4- and 5-hydroxy acids was found in
PHA accumulated by the wild-type strains investigated
in this study, even when incubated in the presence of
precursor substrates for PHA accumulation. The only
exception was 4HHx, which was also found in PHA
accumulated by Rhodococcus ruber when 4HHx was
supplied as carbon source (Valentin et al. 1994).
In addition to the accumulation of PHA containing
new constituents, poly(3HB-co-3HV-co-4HV) containing 19.1 mol% 4HV was accumulated from laevulinic
acid by the recombinant strain. This polyester
Table 3 Gas-chromatographic retention times of c- and d-lactones
and of their methanolysis products
Fig. 1 A–F Structural formulas of a model compound and new
poly(hydroxyalkanoates) investigated in this study. Letters indicate
the structural formulas of 5-acetoxyhexanoic acid methyl ester (A),
poly(3HB-co-5HHx) (B), poly(3HB-co-3HHx-co-5HHx) (C),
poly(3HB-co-3HHx-co-5HHx-co-3HO) (D), poly(3HB-co-3HV-co5HHp-co-4HHp) (E) and poly(3HB-co-3HHx-co-5HHx-co-4HO)
(F). 3HB 3-hydroxybutyric acid, 3HHx 3-hydroxyhexanoic acid,
5HHx 5-hydroxyhexanoic acid, 3HHp 3-hydroxyheptanoic acid,
3HO 3-hydroxyoctanoic acid, 4HO 4-hydroxyoctanoic acid
Compound
Treatment
c-Butyrolactone
Methanolysis
None
Methanolysis
None
Methanolysis
None
Methanolysis
None
Methanolysis
None
methanolysis
None
c-Valerolactone
c-Hexanolactone
c-Octanolactone
d-Valerolactone
d-Hexanolactone
Retention time (min)
5.08
5.44
6.81
10.10
6.94
7.48
10.50
10.45
10.07
10.08
11.87
11.79
16.02
15.92
13.78
13.69
13.42
13.43
266
Fig. 2 1H-NMR spectrum of
poly(3HB-co-5HHx). The
spectrum was recorded from
polyester isolated from cells of
P. putida GPp104-E156
cultivated in a two-stage fedbatch cultivation experiment.
After cells were transferred
into nitrogen-free mineral
salts medium and, after 24 h,
48 h and 72 h incubation at
30°C, portions of each 0.25%
(v/v) 5HHx were added as
carbon source. The numbers
refer to those shown in Fig. 1
Table 4 Chemical-shift data
from the 13C-NMR spectra of
poly(3HB-co-5HHx) and
5-acetoxyhexanoic acid methyl
ester (AHM). The numbers in
lane three (Carbon nucleus) refer
to the numbers in Fig. 1
Environment
Monomer
Poly(3HB-co-5HHx
3HB
5AHM
Carbon nucleus Chemical shift (ppm)
1
2
3
4
169.08
40.89
67.20
19.63
—
—
—
—
169.66
41.20
67.78
19.90
5HHx
5
6
7
8
9
10
172.26
34.00
20.74
35.16
70.39
19.63
—
—
—
—
—
—
172.74
34.23
20.86
35.31
70.90
19.90
5HHx
1
2
3
4
5
6
173.59
33.66
20.76
35.22
70.33
19.78
methoxy group
1@
51.35
Acetate
1A
2A
170.51
20.76
amounted to 60 % of the cellular dry matter. In previous studies it was shown that poly(3HB-co-3HV-co4HV) was accumulated from 4HV; the molar fraction
of 4HV in PHA accumulated by wild-type strains
amounted to 8.8 mol% (Valentin et al. 1992); only after
Tn5 mutagenesis and when the copy number of PHAsynthesis genes had been increased, was PHA comprising more than 30 mol% 4HV obtained. However, the
total amount of PHA accumulated by these strains
decreased to less than 30% of the cellular dry matter
(Valentin and Steinbüchel 1995). Since laevulinic acid is
available in large amounts and is much cheaper than
4HV, and since 4HV has to be prepared from c-valerolactone, laevulinic acid is much more suitable for the
production of poly(3HB-co-3HV-co-4HV) on a larger
scale; this will allow the production of larger amounts
of this polyester and the analysis of its physical properties in more detail. This might be of special interest,
since poly(3HB-co-4HB) is still the only PHA containing a 4-hydroxy acid that has been characterized in
detail for its biological and physical properties (Doi et
al. 1989; Mukai et al. 1993). Further investigations may
show whether poly(3HB-co-3HV-co-4HV) exhibits
a higher degradability than other PHA or a tensile
strength comparable to that of poly(3HB-co-4HB) because of the presence of a 4-hydroxy acid.
The accumulation of unusual PHA by P. putida
GPp104-E156 might also be the result of an unusual
pool of metabolites or/and the overexpression of the
¹. pfennigii PHA synthase, which has been reported
267
recently (Valentin and Steinbüchel 1994). Further investigations, in which the PHA synthase activity is
directly analysed by the spectrometric PHA synthase
assay (Valentin and Steinbüchel 1994) or which compare the substrate specificities by overexpressing different PHA synthases in the same host, might provide an
answer to these questions. Since the PHA synthase of
¹. pfennigii exhibits a similar molecular structure to
that of the PHA synthase of Chromatium vinosum
(Liebergesell and Steinbüchel 1992) it might also be
interesting to compare the substrate specificities of
these enzymes and to recombine the open-reading
frames of both gene clusters to analyse which component of the synthase complex determines the substrate
specificity.
Acknowledgements The authors thank B. Witholt (Zürich) and M.
Liebergesell for providing mutant GPp104 and plasmid
pHP1014: : E156 respectively, and Dipl.-Chem. R. Machinek for
recording the NMR spectra. Provision of lactones by Haarmann
& Reimer GmbH (Holzminden, Germany) is gratefully acknowledged. Parts of this study were supported by the Buck Werke
GmbH (Bad Reichenhall, Germany) and by the Fonds der Chemischen Industrie (Frankfurt, Germany).
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