Polymer Degradation and Stability 59 (1998) 177-l 82

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ELSEVIER

Biosynthesis of polyesters in bacteria and

recombinant organisms
Alexander Steinbiichel, Bernd Fiichtenbusch, Volker Gorenflo, Silke Hein, Ralf Jossek,
Stefan Langenbach & Bernd H. A. Rehm
Institut fiIr Mikrobiologie der Westftilischen Wilhelms-Universittit Mtinster, Corrensstrasse 3, D-48149 Mtinster, Germany

(Accepted 18 June 1997)

Many bacteria are able to synthesize polyesters of hydroxyalkanoic acids (PHA),

which occur as insoluble cytoplasmic inclusions in the cell and can contribute
significantly to the cellular dry matter. More than 100 different hydroxyalkanoic
acids have been detected as constituents in PHA. This article summarizes strate-
gies and possibilities to obtain these biodegradable and thermoplastic/elastomeric
polymers by fermentation of wild-type bacteria as well as of mutants and
recombinant strains from renewable resources, waste substrates or special pre-
cursor substrates. In addition, in vitro biosynthesis of PHA employing the puri-
fied polymerizing enzyme (PHA synthase) is described. Throughout analysis and
cloning of the bacteria1 PHA biosynthesis genes enabled scientists to also establish
this pathway in non-PHA producing eukaryotic organisms such as yeast, plants
and animals. This will allow new processes for cheap and economic production of
PHA. 0 1998 Elsevier Science Limited. All rights reserved

1 INTRODUCTION biosynthetic PHA, except polymalic acid, are insol-

Many bacteria are able to synthesize aliphatic
polyesters of hydroxyalkanoic acids and to deposit
large amounts of these polyhydroxyalkanoic acids
(PHA) as insoluble inclusions in the cytoplasm.
Whereas poly(3-hydroxybutyric acid), poly(3HB),
was already detected 70years ago, during the last
20 years more than 100 other hydroxyalkanoic acids
have been detected as constituents of biosynthetic
PHA.2 Poly(3HB) is a representative of PHASCL,
which are polyesters consisting of short chain-
length hydroxyalkanoic acids (3-5 carbon atoms)
and which are synthesized by the majority of PHA-
accumulating bacteria. PHAMcL are polyesters
consisting of medium chain-length hydroxyalkanoic
acids (6-16 carbon atoms) and are synthesized by
pseudomonades belonging to the rRNA homology
group I and only very few other bacteria.
These polyesters reveal several properties that
lend them to various technical, medical, pharma-
ceutical and other applications; therefore, PHA have
attracted the interest of the chemical industry.13 All
*To whom correspondence should be addressed. Fax: +49
(251) 83-38388; e-mail: steinbu@uni-muenster.de
177
uble in water. They exhibit thermoplastic and
elastomeric properties, and some of them can be
processed to useful materials by apparatus used for
the processing of conventional plastics. However,
in contrast to most representatives of the latter,
PHA are readily biodegradable.4 Other interesting
features are the very high molecular weight, which
can be in the range of millions g/mol, and the
uniform stereochemistry. All constituents of bio-
synthetic PHA investigated so far possess the
R-configuration. This makes biosynthetic PHA
interesting candidates for the starting materials for
the synthesis of enantiomeric pure chemicals5
It is surprising that the molecular genetics of the
biosynthesis pathways for these abundant and
widespread polyesters have only been studied for
approximately 10 years. After the cloning and
molecular analysis of the genes encoding the bio-
synthesis pathway from acetyl CoA to poly(3HB)
of Alcaligenes eutrophus, 6,7 the biosynthesis genes
for poly(3HB) and other PHA have been cloned
from approximately 30 different bacteria and many
have been analysed at a molecular leve1.8q9Of par-
ticular interest was the PHA synthase, which
represents the key enzyme of PHA biosynthesis
178 A. Steinb&chelet al.
and which catalyses the polymerization of hydro-
xyalkanoic acids from the corresponding coenzyme
A thioesters. Although PHA synthases exhibit very
low substrate specificities, it is possible to distinguish
between PHAscr and PHAMcL synthases, and
only very few PHA synthases utilize the coenzyme
A thioesters of HAscr as well as of HAMCL as
substrates. In addition to the biosynthesis genes,
genes encoding for structural proteins attached to
the surface of the PHA inclusions and constituting
the major component of the surface layer sur-
rounding the hydrophobic core of the inclusions
were also cloned and analysed.8*9
This article will summarize recent approaches to
identifying new constituents and new polyesters in
bacteria by screening for new wild types, employ-
ing metabolic mutants and employing genetically
engineered bacteria capable of this. In addition, the
impacts of the current knowledge on the molecular
genetics with regard to establishing in vitro PHA
biosynthesis systems and PHA biosynthesis path-
ways in higher organisms will be addressed.
2 PRODUCTION OF MICROBIAL
POLYESTERS BY IN VZIO PHA
BIOSYNTHESIS EMPLOYING WILD-TYPE
STRAINS
Even after more than 90 different hydroxyalkanoic
acids had been detected as constituents of biosyn-
thetic PHA, mainly in the laboratories of R. Lenz,
R. C. Fuller, Y. Doi, B. Witholt, G. Eggink and A.
Steinbtichel, or were detected in environmental
samples by Wallen and Rohwedder (for a recent
review, see Ref. 21), further interesting constituents
were detected in PHA of wild-type microorganisms
that were cultivated in the presence of precursor
substrates. Several examples were recently obtained
in our laboratory (Table 1). We demonstrated that
3-hydroxyalkanoic acids, which are substituted
with methyl groups at the C&carbon atom, i.e. at
that carbon atom that contributes to the backbone
of the polyester chain, can be incorporated into
PHA. These constituents were 2-methyl-3-hydro-
xybutyric acid and 2,2-dimethyl-3-hydro-
xypropionic acid, which were obtained if, for
example, Burkholderia cepacia or Rhodococcus
ruber were cultivated on tiglic acidlo or 3-hydro-
xypivalinic acid (unpublished data), respectively.
We also detected incorporation of 3-hydroxy-4-
pentenoic acid into PHA, if Burkholderia sp. was
cultivated on e.g. gluconate.* Cells of R. ruber
incorporated 4-hydroxyhexanoic acids into PHA if
the cells were cultivated with 4-hydroxyhexanoic
acid as the sole carbon source.12 We recently
developed a process that allowed the production of
kilogram quantities of poly(3-hydroxyvaleric acid)
homopolyester from valeric acid as the carbon
source with Chromobacterium violaceum.3 It was
shown that the homopolyester could be processed
to test bars by injection molding or by press pro-
cessing and to fibers by melt spinning.13
After finding in the literature a report on the
production of poly(/3-L-malic acid), PMA, by a
non-available and taxonomically not further char-
acterized strain of Aureobasidium sp.,14 we applied
a successful screening for PMA-producing micro-
organisms, and we identified several strains of
Aureobasidium pullulans, which were able to
produce large quantities of this water-soluble
polyester as extracellular polymer. Under opti-
mized cultivation conditions up to log PMAjl
occurred in the medium,15 and various effecters
were identified which stimulated or inhibited PMA
production. 6
3 APPLICATIONS OF INHIBITORS AND
EFFECTORS TO DIRECT THE METABOLIC
FLOW TOWARDS PHA
Some strains of Rhodococcus and Nocardia synthe-
sized from unrelated, simple carbon sources such
as, for example, glucose, a copolyester of 3-hydro-
xybutyric acid and 3-hydroxyvaleric acid (3HV).i7
In addition, they accumulated almost equal
amounts of triacylglycerols in their cells;* some
bacteria of this genera accumulated exclusively
triacylglycerols and other lipids or waxes in the
cells.19 In bacteria that accumulated more than one
lipid storage compound, the relative amounts of
these lipids and the molar composition of the
copolyester were affected by various inhibitors and
other components. For example in Rhodococcus
ruber, the PHA content produced from valeric acid
as the carbon source was increased twofold, and
the polyester consisted exclusively of 3HV if the
cells were cultivated in the presence of acrylic acid,
an inhibitor of fatty acid #?-oxidation. The addition
of PEG-200 caused a decrease in their PHA con-
tent but stimulated the incorporation of 3HV units
into PHA in this bacterium. This study demon-
strated that the relative amounts of different
storage lipids and their composition can be affected
by various compounds.
 Biosynthesis of polyesters in bacteria and recombinant organisms
Table 1. Novel biosynthetic PHA or simpler processes to obtain known PHA
Poly(2HP)
Poly(3HV)
Poly(4HB)
Poly(3HB-co-4HV)
Poly(3HB-co-3HV-co-4HV)
Poly(3HB-co-3HPA4)
Poly(3HB-co-3HHx-co-JHHx)
Poly(3HB-co-3HHx-co-4HHx)
4 IN VITRO PHA BIOSYNTHESIS
In addition to fermentative production of PHA by
in vivo synthesis, polyesters can also be synthesized
in vitro with various isolated enzymes. Various
polyesters were synthesized from lactones or
hydroxyfatty acid alkylesters as substrates and
with lipases or even proteases as catalysts by uti-
lizing non-physiological reactions of these enzymes
in the absence of water in organic solvents or in
the liquid lactones. This aspect of polyester bio-
synthesis will be the subject of the contribution of
Kobayashi and coworkers.20
Another possibility for in vitro PHA biosynthesis
is to employ isolated PHA synthases as catalysts
and coenzyme A thioesters of hydroxyfatty acids as
substrates and utilizing the physiological reactions
of these enzymes. Various methods for the synth-
esis of suitable substrates were recently compiled.21
The availability of PHA synthase protein from
recombinant strains of E. coli, which overpress this
enzyme, contributed greatly to the establishment of
such systems. In vitro poly(3HB) biosynthesis has
previously been shown with the purified PHA syn-
thase from A. eutrophus. 22 In our laboratory, we
employed the PHA synthase complex from the
anoxygenic phototrophic bacterium Chromatium
vinosum, which can be easily prepared in large
quantities from recombinant cells of E. coli.23
When this PHA synthase was incubated with R(-)-
3-hydroxybutyryl coenzyme A, up to approximately
95% of the substrate was converted into poly(3HB)
within 15 min under optimized conditions. The
polyester occurred in granules with diameters of up
to 2 pm and exhibited molecular weights (Mw) of
up to 2 x lo6 g/mol with polydispersity indexes (Mw/
M,) ranging from 1.4 to 2.8.24
In vitro PHA biosynthesis systems will allow
kinetic studies on the PHA synthase and mechan-
179
In vitro biosynthesis
Wild-type
Mutants + recombinant bacteria
Wild-type + mutants
Wild-type + mutants
Wild-type
Recombinant bacteria
Wild-type + recombinant bacteria
Recombinant bacteria
Recombinant bacteria
Recombinant bacteria
Wild-type
Wild-type
istic studies on PHA granule formation. It will also
reveal the components and factors that affect the
reaction rate and the real substrate range of these
generally rather unspecified enzymes. However, the
availability of in vitro PHA biosynthesis systems
will also have other impacts, since it might be
possible to obtain new polyesters. For example,
co-monomers might be incorporated into PHA, the
coenzyme A thioesters of which are not synthesized
in cells. This will extend the range of substrates
that can be used by PHA synthases. In our labora-
tory, we recently demonstrated that lactyl-CoA is
utilized by several PHA synthases to some extent as
a substrate.21 It might also become possible to pro-
duce novel copolyesters such as block-copolyesters.
Biosynthetic block-copolyesters so far have not
been obtained, and by in vivo PHA biosynthesis
they are unlikely to be obtained due to the slug-
gishness of bacterial metabolism.
One disadvantage of the in vitro PHA bio-
synthesis systems that have been established so far
is the dependence on coenzyme A thioesters of
hydroxyalkanoic acids. If no additional enzymes
are involved in this system, whibh regenerate the
released coenzyme A, the availability of in vitro
synthesized PHA is limited by the cost of the sub-
strate, and polyesters produced by this approach
will remain an academic oddity.
5 HETEROLOGOUS EXPRESSION OF PHA
SYNTHASE GENES
The availability of the A. eutrophus PHA bio-
synthesis genes, which were analysed in most detail
at a molecular level, allowed the establishment of
poly(3HB) biosynthesis in other organisms. The
genes were not only expressed in a functionally
active form in bacteria (see below) such as in
180 A. Steinbtichel et al.
Escherichia coli,6 Pseudomonas oleovorans25 or in
cyanobacteria, 26 but also in eukaryotes. Poly(3HB)
biosynthesis and accumulation occurred in trans-
genie plants such as Arabidopsis thaliana27 and
Gossypium hirsutum,28 in transgenic cells of the
insect Spodoptera frugiperda29 and in the yeast
Saccharomyces cerevisiae.30
By heterologous expression of functionally active
A. eutrophus PHA synthase in a dehydrase-defi-
cient fatty acid synthase mutant of S. frugiperda, a
novel pathway to link poly(3HB) biosynthesis
directly to fatty acid de novo synthesis was
designed,29 which may be of general importance.
The establishment of the poly(3HB) biosynthesis
pathway in plants opens up a promising perspec-
tive for cheap production of large quantities of
poly(3HB) by agriculture.3T32 Production of
poly(3HB) with recombinant yeast cells from
sucrose may also be an interesting biotechnological
process, since sucrose is a cheap and abundant
carbon source and since mass cultivation of yeasts
is established in industry.30
6 IN VIVO BIOSYNTHESIS
AND
PRODUCTION OF PHA EMPLOYING
RECOMBINANT E. COLI
Wild-type strains of E. coli and all non-recombi-
nant strains of E. coli, which are widely used in
laboratories, are unable to synthesize this or any
other PHA as a carbon storage compound.
Poly(3HB) biosynthesis and accumulation of this
polyester is, however, easily achieved in recombi-
nant strains of E. coli. It requires the expression of
a functionally active poly(3HB) synthase, a B-
ketothiolase and an NADPH-dependent aceto-
acetyl-CoA reductase as well as the provision of
an excess of a suitable carbon source such as glu-
cose, for example. 7 The cells then convert acetyl-
CoA into poly(3HB) and deposit this polyester in
large quantities as insoluble cytoplasmic inclusions.
After this knowledge was obtained during the
cloning of the PHA biosynthesis genes from A.
eutrophus,7 corresponding sets of such genes
from other microorganisms were also expressed in
E. coli.
Based on the knowledge of PHA-biosynthesis in
A. eutrophus, plasmids were constructed and intro-
duced into suitable strains of E. coli in the labora-
tory of S. Y. Lee, which allowed the production of
large quantities of poly(3HB) in high cell density
fermentations after the cultivation conditions had
been optimized. Cell densities exceeding 15Og
cell dry weight per liter and with more than 60%
of poly(3HB) in the biomass make fermentative
production of this polyester with E. coli very
promising.33y34
Beside the production of poly(3HB) other
polyesters have also been produced in the cells of
recombinant strains of E. coli. So far, not all of
these PHA were obtained from glucose or other
carbohydrates, but from precursor substrates.
Dennis and coworkers constructed an E. coli strain
that produced poly(3HB-co-3HV). They expressed
the A. eutrophus phaCAB operon in a fadR atoC
(Con) mutant of E. coli. When this strain was cul-
tivated on glucose and propionic acid, the cells
accumulated the copolyester. The ratio of the
co-monomers depended on the concentrations of
propionic acid and glucose in the medium.3s
In our laboratory, we constructed E. coli strains
that produced PHAMCL. For this, we expressed the
P. aeruginosa phaC1 and phaC2 genes, encoding
for PHA syntheses 1 and 2, respectively, in a fadB
mutant of E. coli.36 When these strains were culti-
vated on fatty acids, the cells accumulated copo-
lyesters consisting of various 3HAMcL (Table 2).
Synthesis of these copolyesters was strictly depen-
dent on the provision of fatty acids as carbon
sources: from glucose no PHA was accumulated.
The composition of the accumulated PHA depen-
ded on the carbon chain-length of the fatty acids.
For example, strains, which expressed phaC1 syn-
thesized a copolyester from decanoic acid with 3-
hydroxydecanoic acid as the main constituent,
whereas 3-hydroxyoctanoic acid was the main
constituent from octanoic acid (Table 2). The
accumulated PHAMCL contributed up to 21% of
the dry weight of the cells of the fadB mutant.
Other mutants affected in fatty acid degradation or
fatty acid biosynthesis as well as strains with the
corresponding wild-type allels accumulated much
less (0.4-2.8% of cellular dry weight) PHA.36 This
indicated that an accumulation of intermediates of
the oxidation cycle, which most likely occurs in the
fadB mutant, promotes PHA biosynthesis.
We also constructed E. coli strains that produced
poly(4HB). For this, we co-expressed the A. eutro-
phus phaC gene and the Clostridium kluyveri orfZ
in various strains of E. coli.37 OrfZ putatively
encodes a 4-hydroxybutyric acid-coenzyme A
transferase and is involved in the anaerobic cata-
bolism of succinic acid to butyric acid in C. kluyveri.
When these strains were cultivated on 4-hydro-
xybutyric acid plus glucose as carbon sources they
 Biosynthesis of polyesters in bacteria and recombinant organisms 181

Table 2. PHA content and composition in cells of E. cofifiB ases the physiological background of the respective
mutant LS1298 expressing phaCj from P. ueruginosa organism could be utilized for the synthesis of
Carbon PHA content Composition of PHA (mol %) PHA. These mutants turned out to be valuable
source (% of CDW) tools not only for genetic complementation studies
3HB 3HHx 3H0 3HD 3HDD but also to obtain various new polyesters. Table 1
Octanoic acid 4.3 co.1 15.3 55.3 27.1 2.1 includes the copolyesters that were obtained with
Decanoic acid 21.1 < 0.1 2.5 20.0 72.5 5.0 recombinant strains of these mutants. Emphasis
Abbreviations: PHA, polyhydroxyalkanoic acids; CDW, should be made to 4-hydroxyhexanoic acid,12 and
cellular dry weight; 3HB, 3-hydroxybutyric acid; 3HHx, to 4-hydroxyheptanoic acid, 4-hydroxyoctanoic
3-hydroxyhexanoic acid; 3H0, 3-hydroxyoctanoic acid; 3HD, acid and 5hydroxyhexanoic acid,40 which were
3-hydroxydecanoic acid; 3HDD, 3-hydroxydodecanoic acid.
For experimental details refer to Ref. 36. obtained as new constituents from such strains
after the cells were cultivated on the corresponding
hydroxyalkanoic acids or lactones as carbon
accumulated the homopolyester; from glucose source. A copolyester consisting of 3HB, 3HV and
alone no poly(4HB) or any other PHA occurred in 4HV as its main constituents was not only
the cells (Table 3). Under optimized cultivation obtained from 4-hydroxyvaleric acid but also from
conditions poly(4HB) contributed up to 80% of levulinic acid. It could be produced in large quan-
the cellular dry weight. 4HB was also incorporated tities, and processing of the material by a melt
by these cells into PHA if y-butyrolaclone was used spinning process was successfully demonstrated.41
instead of 4-hydroxybutyric acid (Table 3). The use of levulinic acid instead of 4-hydroxy-
In summary, these examples demonstrate that it valeric acid as a carbon source is advantageous
will be possible to obtain poly(3HB) and also other since levulinic acid is much cheaper and can be
PHA in reasonable amounts from cells of recom- obtained from renewable resources by chemical
binant E. coli strains if suitable pathways are reactions. Application of mutants allowed us to
designed by metabolic and genetic engineering. increase the molar fraction of 4HV in copoly-
ester.42 Recombinant strains of mutants of A.
eutrophus affected in PHA biosynthesis were also
7 iN VIVO BIOSYNTHESIS AND utilized to obtain poly(4-hydroxybutyric acid)
PRODUCTION OF PHA EMPLOYING homopolyester.43
RECOMBINANT BACTERIA OTHER THAN
E. COLI

In addition to establishing PHA biosynthesis ACKNOWLEDGEMENTS

pathways in recombinant strains of E. coli, several
novel polyesters were obtained by expressing PHA Chemical analysis of new PHA constituents by
biosynthesis genes heterologously in PHA-negative Dirk Fabritius and technical assistance by Marc
mutants of A. eutrophus3* and P. putida. These Waltermann is highly appreciated. During the last
mutants are defective in inherent PHA syntheses, 10 years the studies in the laboratories of the
and if they are substituted by foreign PHA synth- corresponding author on various aspects of PHA
metabolism were supported by grants provided by
the Bundesministerium fur Bildung und For-
Table 3. PHA content of cells of E. cdi grown on various schung, Bundesministerium fur Landwirtschaft
carbon sources expressing pbaC from P. ueruginosa and orfZ und Forsten, European Commission, Deutsche
from C. kluyveri
Forschungsgemeinschaft, ZENECA Seeds, Mon-
Carbon source Poly(4HB) content santo, Boehringer Ingelheim and Buck-Werke.
(% of CDW) This support, which allows intensive research, is
Glucose (0.5%) co.1 gratefully acknowledged.
Glucose (0.5%) 58.5
+ 4-hydroxybutyric acid (0.4%)
Glucose (0.5%) 16.1
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