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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-


uble in water. They exhibit thermoplastic and
Many bacteria are able to synthesize aliphatic elastomeric properties, and some of them can be
polyesters of hydroxyalkanoic acids and to deposit processed to useful materials by apparatus used for
large amounts of these polyhydroxyalkanoic acids the processing of conventional plastics. However,
(PHA) as insoluble inclusions in the cytoplasm. in contrast to most representatives of the latter,
Whereas poly(3-hydroxybutyric acid), poly(3HB), PHA are readily biodegradable.4 Other interesting
was already detected 70years ago, during the last features are the very high molecular weight, which
20 years more than 100 other hydroxyalkanoic acids can be in the range of millions g/mol, and the
have been detected as constituents of biosynthetic uniform stereochemistry. All constituents of bio-
PHA.2 Poly(3HB) is a representative of PHASCL, synthetic PHA investigated so far possess the
which are polyesters consisting of short chain- R-configuration. This makes biosynthetic PHA
length hydroxyalkanoic acids (3-5 carbon atoms) interesting candidates for the starting materials for
and which are synthesized by the majority of PHA- the synthesis of enantiomeric pure chemicals5
accumulating bacteria. PHAMcL are polyesters It is surprising that the molecular genetics of the
consisting of medium chain-length hydroxyalkanoic biosynthesis pathways for these abundant and
acids (6-16 carbon atoms) and are synthesized by widespread polyesters have only been studied for
pseudomonades belonging to the rRNA homology approximately 10 years. After the cloning and
group I and only very few other bacteria. molecular analysis of the genes encoding the bio-
These polyesters reveal several properties that synthesis pathway from acetyl CoA to poly(3HB)
lend them to various technical, medical, pharma- of Alcaligenes eutrophus, 6,7 the biosynthesis genes
ceutical and other applications; therefore, PHA have for poly(3HB) and other PHA have been cloned
attracted the interest of the chemical industry.13 All from approximately 30 different bacteria and many
have been analysed at a molecular leve1.8q9Of par-
*To whom correspondence should be addressed. Fax: +49 ticular interest was the PHA synthase, which
(251) 83-38388; e-mail: steinbu@uni-muenster.de represents the key enzyme of PHA biosynthesis
177
178 A. Steinb&chelet al.

and which catalyses the polymerization of hydro- incorporated 4-hydroxyhexanoic acids into PHA if
xyalkanoic acids from the corresponding coenzyme the cells were cultivated with 4-hydroxyhexanoic
A thioesters. Although PHA synthases exhibit very acid as the sole carbon source.12 We recently
low substrate specificities, it is possible to distinguish developed a process that allowed the production of
between PHAscr and PHAMcL synthases, and kilogram quantities of poly(3-hydroxyvaleric acid)
only very few PHA synthases utilize the coenzyme homopolyester from valeric acid as the carbon
A thioesters of HAscr as well as of HAMCL as source with Chromobacterium violaceum.3 It was
substrates. In addition to the biosynthesis genes, shown that the homopolyester could be processed
genes encoding for structural proteins attached to to test bars by injection molding or by press pro-
the surface of the PHA inclusions and constituting cessing and to fibers by melt spinning.13
the major component of the surface layer sur- After finding in the literature a report on the
rounding the hydrophobic core of the inclusions production of poly(/3-L-malic acid), PMA, by a
were also cloned and analysed.8*9 non-available and taxonomically not further char-
This article will summarize recent approaches to acterized strain of Aureobasidium sp.,14 we applied
identifying new constituents and new polyesters in a successful screening for PMA-producing micro-
bacteria by screening for new wild types, employ- organisms, and we identified several strains of
ing metabolic mutants and employing genetically Aureobasidium pullulans, which were able to
engineered bacteria capable of this. In addition, the produce large quantities of this water-soluble
impacts of the current knowledge on the molecular polyester as extracellular polymer. Under opti-
genetics with regard to establishing in vitro PHA mized cultivation conditions up to log PMAjl
biosynthesis systems and PHA biosynthesis path- occurred in the medium,15 and various effecters
ways in higher organisms will be addressed. were identified which stimulated or inhibited PMA
production. 6

2 PRODUCTION OF MICROBIAL
POLYESTERS BY IN VZIO PHA 3 APPLICATIONS OF INHIBITORS AND
BIOSYNTHESIS EMPLOYING WILD-TYPE EFFECTORS TO DIRECT THE METABOLIC
STRAINS FLOW TOWARDS PHA

Even after more than 90 different hydroxyalkanoic Some strains of Rhodococcus and Nocardia synthe-
acids had been detected as constituents of biosyn- sized from unrelated, simple carbon sources such
thetic PHA, mainly in the laboratories of R. Lenz, as, for example, glucose, a copolyester of 3-hydro-
R. C. Fuller, Y. Doi, B. Witholt, G. Eggink and A. xybutyric acid and 3-hydroxyvaleric acid (3HV).i7
Steinbtichel, or were detected in environmental In addition, they accumulated almost equal
samples by Wallen and Rohwedder (for a recent amounts of triacylglycerols in their cells;* some
review, see Ref. 21), further interesting constituents bacteria of this genera accumulated exclusively
were detected in PHA of wild-type microorganisms triacylglycerols and other lipids or waxes in the
that were cultivated in the presence of precursor cells.19 In bacteria that accumulated more than one
substrates. Several examples were recently obtained lipid storage compound, the relative amounts of
in our laboratory (Table 1). We demonstrated that these lipids and the molar composition of the
3-hydroxyalkanoic acids, which are substituted copolyester were affected by various inhibitors and
with methyl groups at the C&carbon atom, i.e. at other components. For example in Rhodococcus
that carbon atom that contributes to the backbone ruber, the PHA content produced from valeric acid
of the polyester chain, can be incorporated into as the carbon source was increased twofold, and
PHA. These constituents were 2-methyl-3-hydro- the polyester consisted exclusively of 3HV if the
xybutyric acid and 2,2-dimethyl-3-hydro- cells were cultivated in the presence of acrylic acid,
xypropionic acid, which were obtained if, for an inhibitor of fatty acid #?-oxidation. The addition
example, Burkholderia cepacia or Rhodococcus of PEG-200 caused a decrease in their PHA con-
ruber were cultivated on tiglic acidlo or 3-hydro- tent but stimulated the incorporation of 3HV units
xypivalinic acid (unpublished data), respectively. into PHA in this bacterium. This study demon-
We also detected incorporation of 3-hydroxy-4- strated that the relative amounts of different
pentenoic acid into PHA, if Burkholderia sp. was storage lipids and their composition can be affected
cultivated on e.g. gluconate.* Cells of R. ruber by various compounds.
Biosynthesis of polyesters in bacteria and recombinant organisms 179

Table 1. Novel biosynthetic PHA or simpler processes to obtain known PHA

Poly(2HP) In vitro biosynthesis


Poly(3HV) Wild-type
Poly(4HB) Mutants + recombinant bacteria
Poly(3HB-co-4HV) Wild-type + mutants
Poly(3HB-co-3HV-co-4HV) Wild-type + mutants
Poly(3HB-co-3HPA4) Wild-type
Poly(3HB-co-3HHx-co-JHHx) Recombinant bacteria
Poly(3HB-co-3HHx-co-4HHx) Wild-type + recombinant bacteria
Recombinant bacteria
Recombinant bacteria
Recombinant bacteria
Wild-type
Wild-type

4 IN VITRO PHA BIOSYNTHESIS istic studies on PHA granule formation. It will also
reveal the components and factors that affect the
In addition to fermentative production of PHA by reaction rate and the real substrate range of these
in vivo synthesis, polyesters can also be synthesized generally rather unspecified enzymes. However, the
in vitro with various isolated enzymes. Various availability of in vitro PHA biosynthesis systems
polyesters were synthesized from lactones or will also have other impacts, since it might be
hydroxyfatty acid alkylesters as substrates and possible to obtain new polyesters. For example,
with lipases or even proteases as catalysts by uti- co-monomers might be incorporated into PHA, the
lizing non-physiological reactions of these enzymes coenzyme A thioesters of which are not synthesized
in the absence of water in organic solvents or in in cells. This will extend the range of substrates
the liquid lactones. This aspect of polyester bio- that can be used by PHA synthases. In our labora-
synthesis will be the subject of the contribution of tory, we recently demonstrated that lactyl-CoA is
Kobayashi and coworkers.20 utilized by several PHA synthases to some extent as
Another possibility for in vitro PHA biosynthesis a substrate.21 It might also become possible to pro-
is to employ isolated PHA synthases as catalysts duce novel copolyesters such as block-copolyesters.
and coenzyme A thioesters of hydroxyfatty acids as Biosynthetic block-copolyesters so far have not
substrates and utilizing the physiological reactions been obtained, and by in vivo PHA biosynthesis
of these enzymes. Various methods for the synth- they are unlikely to be obtained due to the slug-
esis of suitable substrates were recently compiled.21 gishness of bacterial metabolism.
The availability of PHA synthase protein from One disadvantage of the in vitro PHA bio-
recombinant strains of E. coli, which overpress this synthesis systems that have been established so far
enzyme, contributed greatly to the establishment of is the dependence on coenzyme A thioesters of
such systems. In vitro poly(3HB) biosynthesis has hydroxyalkanoic acids. If no additional enzymes
previously been shown with the purified PHA syn- are involved in this system, whibh regenerate the
thase from A. eutrophus. 22 In our laboratory, we released coenzyme A, the availability of in vitro
employed the PHA synthase complex from the synthesized PHA is limited by the cost of the sub-
anoxygenic phototrophic bacterium Chromatium strate, and polyesters produced by this approach
vinosum, which can be easily prepared in large will remain an academic oddity.
quantities from recombinant cells of E. coli.23
When this PHA synthase was incubated with R(-)-
3-hydroxybutyryl coenzyme A, up to approximately 5 HETEROLOGOUS EXPRESSION OF PHA
95% of the substrate was converted into poly(3HB) SYNTHASE GENES
within 15 min under optimized conditions. The
polyester occurred in granules with diameters of up The availability of the A. eutrophus PHA bio-
to 2 pm and exhibited molecular weights (Mw) of synthesis genes, which were analysed in most detail
up to 2 x lo6 g/mol with polydispersity indexes (Mw/ at a molecular level, allowed the establishment of
M,) ranging from 1.4 to 2.8.24 poly(3HB) biosynthesis in other organisms. The
In vitro PHA biosynthesis systems will allow genes were not only expressed in a functionally
kinetic studies on the PHA synthase and mechan- active form in bacteria (see below) such as in
180 A. Steinbtichel et al.

Escherichia coli,6 Pseudomonas oleovorans25 or in been optimized. Cell densities exceeding 15Og
cyanobacteria, 26 but also in eukaryotes. Poly(3HB) cell dry weight per liter and with more than 60%
biosynthesis and accumulation occurred in trans- of poly(3HB) in the biomass make fermentative
genie plants such as Arabidopsis thaliana27 and production of this polyester with E. coli very
Gossypium hirsutum,28 in transgenic cells of the promising.33y34
insect Spodoptera frugiperda29 and in the yeast Beside the production of poly(3HB) other
Saccharomyces cerevisiae.30 polyesters have also been produced in the cells of
By heterologous expression of functionally active recombinant strains of E. coli. So far, not all of
A. eutrophus PHA synthase in a dehydrase-defi- these PHA were obtained from glucose or other
cient fatty acid synthase mutant of S. frugiperda, a carbohydrates, but from precursor substrates.
novel pathway to link poly(3HB) biosynthesis Dennis and coworkers constructed an E. coli strain
directly to fatty acid de novo synthesis was that produced poly(3HB-co-3HV). They expressed
designed,29 which may be of general importance. the A. eutrophus phaCAB operon in a fadR atoC
The establishment of the poly(3HB) biosynthesis (Con) mutant of E. coli. When this strain was cul-
pathway in plants opens up a promising perspec- tivated on glucose and propionic acid, the cells
tive for cheap production of large quantities of accumulated the copolyester. The ratio of the
poly(3HB) by agriculture.3T32 Production of co-monomers depended on the concentrations of
poly(3HB) with recombinant yeast cells from propionic acid and glucose in the medium.3s
sucrose may also be an interesting biotechnological In our laboratory, we constructed E. coli strains
process, since sucrose is a cheap and abundant that produced PHAMCL. For this, we expressed the
carbon source and since mass cultivation of yeasts P. aeruginosa phaC1 and phaC2 genes, encoding
is established in industry.30 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-
6 IN VIVO BIOSYNTHESIS
AND lyesters consisting of various 3HAMcL (Table 2).
PRODUCTION OF PHA EMPLOYING Synthesis of these copolyesters was strictly depen-
RECOMBINANT E. COLI dent on the provision of fatty acids as carbon
sources: from glucose no PHA was accumulated.
Wild-type strains of E. coli and all non-recombi- The composition of the accumulated PHA depen-
nant strains of E. coli, which are widely used in ded on the carbon chain-length of the fatty acids.
laboratories, are unable to synthesize this or any For example, strains, which expressed phaC1 syn-
other PHA as a carbon storage compound. thesized a copolyester from decanoic acid with 3-
Poly(3HB) biosynthesis and accumulation of this hydroxydecanoic acid as the main constituent,
polyester is, however, easily achieved in recombi- whereas 3-hydroxyoctanoic acid was the main
nant strains of E. coli. It requires the expression of constituent from octanoic acid (Table 2). The
a functionally active poly(3HB) synthase, a B- accumulated PHAMCL contributed up to 21% of
ketothiolase and an NADPH-dependent aceto- the dry weight of the cells of the fadB mutant.
acetyl-CoA reductase as well as the provision of Other mutants affected in fatty acid degradation or
an excess of a suitable carbon source such as glu- fatty acid biosynthesis as well as strains with the
cose, for example. 7 The cells then convert acetyl- corresponding wild-type allels accumulated much
CoA into poly(3HB) and deposit this polyester in less (0.4-2.8% of cellular dry weight) PHA.36 This
large quantities as insoluble cytoplasmic inclusions. indicated that an accumulation of intermediates of
After this knowledge was obtained during the the oxidation cycle, which most likely occurs in the
cloning of the PHA biosynthesis genes from A. fadB mutant, promotes PHA biosynthesis.
eutrophus,7 corresponding sets of such genes We also constructed E. coli strains that produced
from other microorganisms were also expressed in poly(4HB). For this, we co-expressed the A. eutro-
E. coli. phus phaC gene and the Clostridium kluyveri orfZ
Based on the knowledge of PHA-biosynthesis in in various strains of E. coli.37 OrfZ putatively
A. eutrophus, plasmids were constructed and intro- encodes a 4-hydroxybutyric acid-coenzyme A
duced into suitable strains of E. coli in the labora- transferase and is involved in the anaerobic cata-
tory of S. Y. Lee, which allowed the production of bolism of succinic acid to butyric acid in C. kluyveri.
large quantities of poly(3HB) in high cell density When these strains were cultivated on 4-hydro-
fermentations after the cultivation conditions had 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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