Bacterial Polyesters

8/6/2019 Bacterial Polyesters

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Reviews

Bacterial Polyesters: Biosynthesis, Biodegradable Plastics andBiotechnology

Robert W. Lenz*, and Robert H. Marchessault

Polymer Science & Engineering Department, University of Massachusetts,Amherst, Massachusetts 01003-4530, and Department of Chemistry, McGill University,

3420 University St., Montreal, QC, H3A 2A7 Canada

Received May 21, 2004; Revised Manuscript Received September 23, 2004

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate),

PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked

the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized

as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development

effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering

and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple

PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different

aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and

substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural

polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and

polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to

personalities and locations involved with the events covering a discovery timespan of 75 years.

In 1982, Imperial Chemical Industries Ltd. (ICI) in

England announced a product development program on a

new type of thermoplastic polyester which was totally

biodegradable and could be melt processed into a wide

variety of consumer products including plastics, films, and

fibers.1 The polymer was to be manufactured by a large-

scale fermentation process not unlike the brewing of beerbut which, in this case, involved the production of the

polymer inside the cells of bacteria grown in high densities

and containing as much as 90% of their dry weight as

polymer. The bacterium capable of performing this feat was

Alcaligenes eutrophus, since renamed Ralstonia eutropha

(more recently changed again to Wautersia eutropha) and

the commercial polyester product, tradenamed Biopol,

was a copolyester containing randomly arranged units of

[R]-3-hydroxybutyrate, HB, and [R]-3-hydroxyvalerate,

HV:2

Discovery of Bacterial Polyesters

That bacteria could produce polyesters was unknown to

polymer chemists before 1960 and even to most biochemists

and microbiologists before 1958, although their presence in

bacterial cells in isolable amounts, their chemical composi-

tion, and even the fact that they were polymers, were reported

* To whom correspondence should be addressed. Tel.: 1-413-545-3060.Fax: 1-413-545-0082. E-mail: [email protected].

University of Massachusetts. McGill University.

Copyright 2005 by the American Chemical Society

January/February 2005 Published by the American Chemical Society Volume 6, Number 1

10.1021/bm049700c CCC: $30.25 2005 American Chemical SocietyPublished on Web 11/23/2004


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in the literature as early as 1926. These natural polyesters

remained unknown to a wider scientific community for so

long because their discoverer, Maurice Lemoigne, published

his results in little-read French journals at a time whenmicrobiologists had no interest in lipids, as they were referred

to by Lemoigne, that were not ether soluble, and many

organic chemists refused to believe that there were such

things as polymers. Lemoigne (Figure 1) was a bacteriologist

with training in analytical chemistry, and his series of

papers published over the five-year period from 1923 to

1927 are remarkable for their breadth of research and

prescience.3-8

The polyester that Lemoigne isolated and characterized

was poly-3-hydroxybutyrate, PHB, shown below. PHB is the

reserve polymer found in many types of bacteria, which can

grow in a wide variety of natural environments and who have

the ability to produce and polymerize the monomer, [R]-3-

hydroxybutyric acid:

As indicated in this structure, the repeating unit of PHB

has a chiral center, and Lemoigne reported that the polymer

is optically active.4 In fact, PHB is only the parent member

of a family of natural polyesters having the same three-carbon

backbone structure but differing in the type of alkyl group

at the or 3 position. These polymers are referred to in

general as polyhydroxyalkanoates, PHAs, and all such natural

polyesters have the same configuration for the chiral center

Figure 1. Photograph of Maurice Lemoigne, Head of Services de Fermentation at Institut Pasteur, Paris 1949. Courtesy of Institut PasteurArchives, with permission.

2 Biomacromolecules, Vol. 6, No. 1, 2005 Lenz and Marchessault


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at the 3 position, which is very important both for their

physical properties and for the activities of the enzymes

involved in their biosynthesis and biodegradation.1,9

At the time of his discovery of PHB in bacteria, Lemoigne

was the Director of the Fermentation Laboratory of the

Pasteur Institute in Lille, France. He became involved with

PHB in an attempt to determine the cause of the acidification

of aqueous suspensions of the bacterium Bacillus megaterium

when it was kept under an oxygen-free atmosphere. In 1923,Lemoigne reported that the acid produced by the bacteria

was 3-hydroxybutyric acid,3 and in 1927, he described the

isolation of a solid material obtained from the cell which he

characterized as a polymer of 3-hydroxybutyric acid.8 He

came to that conclusion by carefully hydrolyzing the solid

into a series of water-soluble oligomers of 3-hydroxybutyric

acid, which he characterized for molecular weight and

melting point. He named the source of the acid lipide--

hydroxybutyrique, and, remarkably for his time, he even

suggested that the polymer was produced within the cell by

a dehydration polymerization.7

Lemoigne published these observations and interpretations

at the time when Herman Staudinger at the University ofFreiburg, Germany was being ridiculed by his colleagues in

organic chemistry in Europe for proposing the existence of

high molecular weight molecules or polymers, which he

termed macromolecules. Fortunately, Lemoigne was free

of such prejudices, and he was probably familiar with the

work of Emil Fischer, who demonstrated as early as 1906

that proteins are large molecules of enchained amino acid

units or polypeptides, a term he originated.10 Eventually

Staudingers concepts about synthetic polymers won out, but

not until the 1930s and the publication of the definitive

research of Wallace Carothers at duPont Experimental

Station, Wilmington, Delaware, on the synthesis and char-

acterization of aliphatic polyesters and polyamides. In 1953,

Staudinger was awarded the Nobel Prize in Chemistry for

his work on polymer synthesis and for his staunch defense

of the concept of macromolecules.11 There is no indication

that either Staudinger or Carothers were ever aware of

Lemoignes discovery of natures polyesters, which remained

hidden from organic and polymer chemists for over 30 years

even though PHB was described in biochemistry textbooks,

where, however, it was referred to as a lipid not a polyester.

Lemoigne and co-workers reported on their PHB studies

in 27 publications from 1923 until 1951, and in their later

work they found that the cells ofB. megaterium could contain

as much as 44% of their dry weight of PHB depending ongrowth conditions.12,63 Lemoigne was the first to describe

an analytical method for quantifying PHB, and he showed

that PHB could be cast into a transparent film like the then

well-known cellulose nitrate material, collodion.7 In follow-

up studies, he and co-workers also reported that a variety of

bacteria could produce PHB, but apparently he never became

involved in determining the function of such polyesters in

cell metabolism even though he labeled it as a reserve

material. It was not until microbial physiologists recognized,

in the late 1950s, the important role that PHB played in the

overall metabolism of bacterial cells that the significance of

Lemoignes earlier discoveries was realized.

Rediscovery of PHB

The rediscovery of PHB occurred simultaneously and was

published independently in 1957 and 1958 by microbiologists

in Great Britain and the United States. At the University of

Edinburgh in Scotland, Wilkinson and co-workers became

interested in the relationship between the presence of the

intracellular lipid granules in bacteria, which had been known

since 1901, and the large amounts of PHB found in some

species as reported by Lemoigne and co-workers, and thefunction of PHB in the cells.13 During the same time period

at the University of California in Berkeley, Stanier,

Doudoroff and co-workers found that PHB was the primary

product of the oxidative and photosynthetic assimilation of

organic compounds by phototropic bacteria, and they at-

tempted to detail the biosynthesis and breakdown mechanism

of PHB in the cells.14

Well before these studies, Weibull in 1953 had isolated

the granules ofB. megaterium by dissolution of the cell wall

with a lysozyme, and he confirmed the claim made by

Lemoigne in 1944 that PHB was the major constituent of

the granules.15 A typical example of such granules inside a

cell is shown in Figure 2 for the bacterium A. Chrococcum.In 1958, Wilkinson and co-workers obtained morphologically

intact granules from Bacillus cereus by disrupting the cells

with alkaline hypochlorite solution and determined the

amount of PHB in the granules, but this reagent, the well-

known eau de javelle laundry bleach, was later shown by

Lundgren and co-workers at Syracuse University to degrade

these polyesters and yield, only low molecular weight

polymers.16,17 Only cells that were chloroform extracted

yielded high molecular weight PHB when reliable methods

were used later, starting in 1965.17a

In 1961, Doudoroff and Merrick isolated what they

described as native PHB granules of two chemohetero-

tropic bacteria, Rhodospirillum rubrum and B. megaterium.

Native granules are intact granules which are carefully

isolated from the cell and purified to retain the active

synthase.18 R. rubrum native granules also retain the

depolymerase enzyme that can degrade PHB to the monomer,

but the B. megaterium native granules have only the

associated synthase. Doudoroff and co-workers also studied

the enzyme-catalyzed hydrolysis of PHB extracted from the

cell and free of all proteins by the extracellular depolymerases

which are excreted by a variety of bacteria that can use the

polymer as a carbon source as discussed below.19

Biosynthesis of PHB

Stanier and Wilkinson and their co-workers determined

that the PHB granules in bacteria serve as an intracellular

food and energy reserve and that the polymer is produced

by the cell in response to a nutrient limitation in the

environment in order to prevent starvation if an essential

element becomes unavailable. The nutrient limitation acti-

vates a metabolic pathway, which shunts acetyl units from

the tricarboxylic cycle into the production of PHB. The latter

is ideal as a carbon-storage polymer because it is water

insoluble, chemically and osmotically inert, and can be

readily reconverted to acetic acid by a series of enzymatic

reactions inside the cell.20

Historical Review of Bacterial Polyesters Biomacromolecules, Vol. 6, No. 1, 2005 3


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The reactions involved in the metabolic pathway respon-

sible for the biosynthesis of PHB from acetic acid were first

identified by Stanier and co-workers in 1959 in their studies

on PHB formation in R. rubrum. However, the specific

enzymes which catalyzed the reactions for the synthesis of

3-hydroxybutyric acid, the monomer for PHB, were not

identified until 1973, when Schlegel at the University of

Gottingen, Germany and Dawes at the University of Hull,

England, working independently, were able to isolate and

characterize those enzymes.21,22 Schlegel and co-workers

carried out their investigations on the metabolic cycle for

PHB production in Alcaligenes eutrophus while Dawes and

co-workers studied the cycle for PHB production in Azoto-

bacter beijerinckii.

Schlegel first began his research on A. eutrophus in the

late 1950s as part of a study of the oxidation of molecular

hydrogen by Hydrogenomonas bacteria. In 1961, they

observed that A. eutrophus, a member of this group, could

accumulate very large amounts of PHB during growth in

nitrogen-limited media.23 Coincidentally, Dawes became

interested in PHB while preparing a review on microbial

metabolism in 1962 before joining the University of Hull.He began his research program there with a study of the

accumulation of PHB in A. beijerinckii, which he found

capable of accumulating as much as 70% of its dry weight

of polymer.24 In the same 1973 issue ofBiochemical Journal,

Schlegel and Dawes published simultaneously their discover-

ies on the identification of the two enzymes involved in the

reactions for converting acetic acid to 3-hydroxybutyric acid

in the two different bacteria.21,22 For both bacteria, the

enzymes were a ketothiolase (1), which catalyzes the

dimerization of the Coenzyme A derivative of acetic acid,

acetyl-CoA, to acetoacetyl-CoA, and a reductase (2), which

catalyzes the hydrogenation of the latter to [R]-3-hydroxy-

butyryl-CoA, the monomer that is polymerized to PHBby a synthase (3), as shown in the following reaction

scheme:

As discussed above, this cycle becomes activated when

acetyl-CoA is restricted from entering the tricarboxylic acid

cycle because of a deficiency in nutrients (generally eitherphosphorus, nitrogen or oxygen) needed by the cell to further

metabolize acetyl-CoA for cell growth.

Although the first two enzymes, the ketothiolase (1) and

the reductase (2) which are responsible for monomer

synthesis, were not fully identified and characterized until

the investigations of Schlegel and Dawes in 1973, the enzyme

responsible for the polymerization process, the synthase or

polymerase (3), was initially recognized by Doudoroff,

Merrick and co-workers as early as 1964, and it was

characterized by Merrick and co-workers in 1968 in their

studies on the production of PHB in both R. rubrum and B.

megaterium.25,26 They made their initial recognition of the

existence and role of the synthase in their work on the

isolation and characterization of the active native PHB

granules from these bacteria. These granules could be used

in an aqueous suspension for the in vitro polymerization

of [R]-3-hydroxybutyryl-CoA to PHB. With their native

PHB granules, Merrick and co-workers were also able to

carry out kinetic studies on the polymerization reaction to

determine the Michaelis-Menten constants for the reaction.

They even proposed in their 1968 studies that the active site

of the synthase contains a cysteine unit which provides a

thiol group that covalently bonds to the growing polymer

chain as a thioester.26

A detailed mechanism for the polymerization reaction,

which was based on Merricks suggestion, was proposed by

Ballard and co-workers at ICI in 1987 and further elaborated

by Doi and co-workers at the RIKEN Institute in Japan in

1992.27,28 They proposed a mechanism in which two thiol

groups are involved in the active site for both the initiation

and propagation reactions of the polymerization. For initia-

tion, the two thiol groups form thioesters with two molecules

of monomer, which then undergo a thioester-oxyester

interchange reaction at the active site to form a dimer andrelease one of the thiol groups for the propagation reaction,

as follows:

Propagation ensues by bonding another monomer to the

free thiol group of the active site followed by another

thioester-oxyester exchange reaction to form the trimer, and

so on. These reactions are thermodynamically favorable

because of the higher bond strength of the oxyester compared

to the thioester. The synthase, therefore, both initiates and

catalyzes the polymerization process, which proceeds by a

continuous series of insertion reactions in much the same

manner as in the stereoregular polymerization of olefins by

Ziegler-Natta catalysts. In this case, the enzyme is specific

for monomers with the [R] configuration and will not

polymerize identical compounds having the [S] configuration

as initially reported by Dawes and co-workers in 1989,29

soas a result, all natural PHAs are completely isotactic.

Biotechnology

As mentioned at the start of this review, bacterial

polyesters became an article of commerce when ICI began

their production of Biopol in 1982, but Biopol was not

PHB. PHB has a high melting point (180 C) and forms

highly crystalline solids which crystallize slowly and form

large spherulitic structures that impart poor mechanical

properties in molded plastics and films, although, addition

of nucleating agents and suitable posttreatment after extrusion



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or casting can lead to much improved properties.30 Because

of its high melting point, PHB is also susceptible to thermal

degradation during melt processing by ester pyrolysis of the

aliphatic secondary esters of the repeating units. These

deficiencies were partly eliminated when it was found that,

when A. eutrophus is grown on a mixture of glucose and

propionic acid, the storage polyester formed is a random

copolyester of HB and HV units which has a lower melting

point.30 As a result, the copolymers have better processing

characteristics and considerably improved mechanical prop-

erties for use as plastics as shown in Figure 3. Nevertheless,

like PHB, the copolymer is fully biodegradable in a wide

variety of natural environments as well as in waste disposal

facilities, especially in municipal compost sites.

The ability of bacteria to produce storage polyesters with

compositions other than PHB was not realized until 1974

when Wallen and Rohwedder at the USDA Northern

Regional Research Laboratory reported that a polyester

isolated from activated sludge contained both HB and HV

units, but they were not able to identify the microbial species

in sludge which produced the polyester.31 In 1983, White

and co-workers at Florida State University demonstrated thatthe hydroxyalkanoic acid units present in polyesters extracted

from bacteria in marine sediments included even more than

HB and HV units.32 They analyzed the ethyl esters of the

units, which were obtained by ethanolysis of the polyester,

by gas chromatography and showed the presence of at least

11 types of repeating units, including both linear and

branched 3-hydroxyalkanoic acids with compositions varying

from four to eight carbon atoms. White and co-workers also

showed that Lemoignes original bacterium B. megaterium

could produce polyesters containing at least six different

types of units, although HB units still comprised ap-

proximately 95% of the contents. In 1983, Witholt and co-

workers at the University of Groningen, The Netherlands,found that Pseudomonas oleoVorans grown on alkanes

produced a large number of granules containing polyesters

with units of 6-10 carbon atoms.33 These bacterial polyesters

have low glass transition temperatures and much lower

crystallinities than PHB, and as a result, they display

elastomeric properties. In 1988, Doi and co-workers obtained

PHAs with 4-hydroxybutyric acid repeat units from bacteria

grown on carbon substrates having these structures. 34

ICI became involved in the commercial development of

bacterial polyesters after terminating a program on the large-

scale production of single cell proteins, SCP, by bacteria for

use as fodder.

35

After evaluating a variety of methods forthat purpose, they had concentrated on the use of methylo-

tropic bacteria to produce SCPs from methanol, but the

project was terminated in 1976 because of consumer

resistance. The company then turned instead to the possible

large-scale production of PHB by the same bacteria for their

entree into industrial biotechnology and bioprocessing.36 In

this case, they were partially motivated by the major

petroleum crisis of the 1970s, which made the production

of plastics from renewable resources economically attrac-

tive.35

During the 1950s, Schlegel had also studied the production

of SCPs by bacteria, eventually selecting the Hydrogenomas

bacterium A. eutrophus for that purpose. In those investiga-

tions, he and co-workers observed that this bacterium was

capable of producing very large amounts of PHB under

selected growth conditions, and in 1979, Schlegel provided

samples of several strains of A. eutrophus to ICI for their

PHB process development program.23 ICI selected one of

these strains for intensive study after Holmes and co-workers

in their research laboratory found that the bacterium could

produce up to 80% of its dry weight of the HB/HV

copolymer when grown on a mixture of glucose and

propionic acid as mentioned above.35 Furthermore, the

composition of the copolymer could be varied over a wide

range by varying the composition of the feed mixture.

ICI was not the first company to consider the commercial

development of bacterial polyesters for consumer products.

Their efforts were preceded by a much earlier attempt at the

W. R. Grace Company in Maryland, where Baptist and

Werber initiated a similar program in 1960.37 Baptist had

joined the Research Division of Grace in 1959 after a

postdoctoral in biochemistry at the University of Michigan,

where he had learned about bacterial production of PHB.

Baptist and Werber recognized that PHB was a stereoregularpolymer with a melting point close to that of polypropylene,

which suggested to them that PHB might be able to compete

with polyolefins as a thermoplastic but with the added

advantage of being biodegradable. Baptist used a sample of

Rhizobium obtained from Hayward and co-workers at the

Colonial Microbiological Research Institute in Trinidad, who

reported in 1958 that their strain of that bacterium could

produce PHB to 58% of its dry cell weight.38 With this

bacterium Baptist was able to produce large quantities of

PHB for evaluation, initially for molded plastics and later

for absorbable sutures. The latter subject was of interest

because PHB, as a natural polyester, was assumed to be a

biocompatible polymer in humans, which it has been found

to be in more recent studies, but it is very slowly resorbable.

They improved the mechanical properties of PHB plastics

when they found that PHB was compatible with a variety of

plasticizers, which greatly improved its processing and

solid-state properties. Nevertheless, the project was termi-

nated in 1962 because of the poor thermal stability of PHB,

and their work on the synthesis and properties of PHB was

reported in 1964 in the Transactions of the Society of

Plastics Engineers.37 However, Chemistry and Engineering

News in the March 18, 1963, issue published an extensive

research report titled: Bacteria Produce Polyester Thermo-

plastic, which was apparently the first time that most

polymer chemists became aware of this thermoplastic

biopolyester, which occurred as inclusions in bacterial cells

(Figure 2).

Coincidentally, in 1962, Marchessault and co-workers

began a program at the State University of New York in

Syracuse on characterization of the structure and properties

of PHB both in the solid state and in solution.12,39 Samples

of the polymer were provided to them by Lundgren, who

had been studying the presence of PHB in bacteria for several

years in the Microbiology Department of Syracuse Univer-

sity. Issues such as obtaining high molecular weight poly-

mers, optical rotation, and X-ray crystal structure were settled



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in definitive experiments. Also, at about the same time,

Merrick joined Lundgrens Department and continued his

studies on the composition and activity of the native PHB

granules of B. megaterium.26,40 They observed that the

carefully purified, or native, granules (cf. Figure 2) were

surrounded by a protein membrane. They also continued and

expanded the earlier studies of Merrick and Doudoroff on

the depolymerization of PHB by soluble depolymerases,

which they obtained from a variety of bacteria that produce

and release these enzymes and are capable of utilizing PHB

as a sole carbon source.19,41,42 The protein membranes

covering the PHA granules of P. oleVorans were studied in

detail by Fuller and co-workers at the University of Mas-

sachusetts, Amherst. In 1995, they reported that the granules

are enclosed in two separate protein membranes, and the

synthase is associated with the inner membrane.

42

Biodegradable Polymers

Because PHB is stored by bacteria for eventual breakdown

and utilization as a carbon source when extracellular carbon

is no longer available, there must be an effective and rapid

mechanism within the cell for the biodegradation of this high

molecular weight polyester into simple organic compounds.

As discussed above, Lemoigne had been led into his study

of PHB by finding that [R]-3-hydroxybutyric acid is released

by B. megaterium in an aqueous environment. In 1958,

Wilkinson and co-workers also observed the release of both

acetoacetic acid and acetic acid during the utilization of PHBreserves by that bacterium.43 Subsequently, in 1962, Merrick

and co-workers demonstrated that native granules from B.

rubrum were self-hydrolyzing, and they isolated the enzyme

responsible for this reaction which they referred to as a

depolymerase or hydrolase (1).44 In 1967, Williamson and

co-workers identified a specific dehydrogenase (2) that

converted [R]-3-hydroxybutyric acid to acetoacetic acid,45

and in 1973, Dawes and co-workers identified an enzyme

for the conversion of acetoacetic acid to acetic acid (3), 22

so the entire intracellular pathway for the reconversion of

PHB to acetic acid was established to include the following

steps:

PHB can also be rapidly hydrolyzed to the monomer by

extracellular depolymerase enzymes secreted by a wide

variety of bacteria and fungi that can utilize this compound

after it is liberated by the death and lyses of bacteria in which

it is stored. Initial observations made by Schlegel and

Chowdhury in 1963 with strains of Pseudomonas obtained

from soil and compost samples established this concept,46

and in 1965 Delafield, Doudoroff and co-workers isolated

and characterized a number of pseudomonads capable of

utilizing extracellular PHB as their sole source of carbon

and energy.47

It is now known that microorganisms exist in all natural

environments that are capable of degrading PHB andmetabolizing [R]-3-hydroxybutyric acid by enzyme-catalyzed

reactions, so by definition, PHB is a biodegradable polymer.

In more recent studies, depolymerases have also been found

for the PHAs with long alkyl chains.48 As mentioned above,

these polyesters have much different physical and mechanical

properties, and they can also be utilized as biodegradable

polymers in applications such as elastomers and adhesives.

Many different types of intracellular and extracellular

polyester depolymerases have now been isolated and char-

acterized. As reported by Doi and co-workers, all of these

enzymes consist of a single polypeptide chain in the

molecular weight range of approximately 40 000-60 000.49

The structural genes of a large number of extracellular

depolymerases of different microorganisms have been iso-

lated and analyzed, and they appear to have three charac-

teristics in common along the polypeptide chain, including

(1) a catalytic domain (termed a lipid box), (2) a substrate-

binding domain, and (3) a linking region connecting these

two domains. In that manner, they have the same features

as the depolymerizing enzymes for insoluble polysaccharides

such as cellulose and chitin.

Genetic Engineering

In 1988, Dennis and co-workers at James Madison

University cloned the entire set of genes in R. eutropha forthe three enzymes involved in the synthesis of PHB from

acetyl CoA as described above.50 The three genes are

clustered in one operon, and Dennis and co-workers were

able to introduce this operon into E. coli. The genetically

engineered E. coli containing the operon can express all three

enzymes and can synthesize PHB in large quantities from a

wide range of organic compounds. Some recombinant strains

of E. coli can also produce the HB/HV copolymer,51 or

alternatively as reported by Sinskey and co-workers at the

Massachusetts Institute of Technology in 1994, strains

containing only the synthase gene can express this protein

in sufficiently large quantities for isolation and purification.52

Figure 2. Transmission electron micrograph of ultrathin section ofAzotobacter chroococcum cell treated with phenylacetic acid. FromNuti et al., ref 17a, herein, with permission.



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The purified enzyme is stable in aqueous solution and has

been used for in vitro polymerization reactions of a wide

variety of 3- and 4-hydroxyalkanoate-CoA monomers.53 Lenz

and co-workers at the University of Massachusetts reported

in 2000 that these in vitro polymerization reactions can form

living polymers, which means that the polymerization

process has no polymer chain termination reaction, so the

propagating end group remains active indefinitely and very

high molecular weight polymers can be prepared in vitro. 54

In another application of genetic engineering for bacterial

polyester synthesis, Somerville and co-workers at Michigan

State University reported in 1992 that the reductase and

synthase genes of A. eutrophus can be inserted into a plant,

Arabidopsis thaliana, which can also produce acetoacetyl-

CoA, and the transgenic plant can then accumulate PHBgranules, Figure 4, to approximately 14% of its dry weight. 55

PHB and the Macromolecule Controversy

Reserve polymers also played a role in the controversy

between Staudinger and his colleagues in organic chemistry

in Germany during the 1920s over the very existence of

macromolecules. In his book on the history of polymer

science, Morawetz discusses how organic chemists at that

time considered starch, which is a reserve polymer for plants,

and cellulose to be colloidal aggregates of glucose molecules

rather than long chain polymers.56 A leading German organic

chemist at that time, Karrer, reasoned that, because starch is

utilized by plants as a food and energy reserve, one has to

be surprised that the view of hundreds or thousands of

glucose molecules joined together by glucosidic bonds into

long chains could have remained unchallenged because it

is improbable that a plant in converting sugar to a reserve

substance from which it might soon have to be recovered

would perform such complex work as would be required in

the build-up of a polyglucoside. 56 Had he known about

PHB, Karrer would undoubtedly have made the same

argument against Lemoignes contention of the existence of

high molecular weight reserve polyesters in bacteria. So

much for conventional wisdom.

Outlook

Despite the 75 years, on and off, of research on PHAs

and 20 years of intense industrial interest, PHAs still appear

to be far removed from large scale production. At this

writing, two development programs on these biopolymers

are receiving attention, namely (1) a joint program by the

Proctor & Gamble Co. and Kaneka Corp. on a family of

short and medium chain copolymers, especially on poly(3-

hydroxybutyrate-co-3-hydroxyhexanoate), and (2) a programat Metabolix Inc. on PHAs for medical applications. The

lack of commercialization of the initially promising bacterial

poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers

has been generally attributed to the high investment for the

fermentation and product recovery processes on a large scale

and to the cost of the substrates. To reduce the latter

limitation, alternative substrates are receiving much attention,

including starch and vegetable oils, but no major break-

throughs in this area have been announced. Nevertheless, in

the long run, it is possible that advances in our understanding

and control of the genetic pathways involved in the biosyn-

thesis of PHAs in microorganisms and plants could make

the industrial scale production of these biopolymers competi-

tive with oil-based synthetic polymers.

As for the agricultural production of PHAs, the feasibility

of this route has been demonstrated in small plants such as

Arabidopsis thaliana,57 but the transfer of this technology

into crops such as canola with acceptable production levels

is still in the research stage. On the other hand, the chemical

modification of medium chain PHAs produced by bacteria

is a promising approach to the commercialization of high-

value polymers for specialty applications.58-60 Indeed, by

either direct bacterial synthesis or by the chemical modifica-

tion of bacterially produced PHAs, polyesters with more than

one hundred different types of repeating units have beenidentified and characterized.61 Very recently, it was even

found possible to produce a thioester analogue of the PHAs

with bacteria,62 so it is apparent that there is still much more

to be discovered about the synthesis of bacterial polyesters.

Acknowledgment. We wish to recognize the important

contributions by Professors Schlegel, Dawes, Merrick, and

Fuller for the factual details herein. In addition, Dr. Bernard

Hautecoeur and Archives of the Institut Pasteur provided

historical background concerning Professor Maurice Lem-

oigne as surveyed by Rene Dujarric de la Riviere.63 Dr.

Francis Werber kindly informed us on the development of

Figure 3. Moulded PHB objects for various applications. In soil burialor composting experiments, such objects biodegrade in about threemonths.

Figure 4. PHB granules in the choloroplast of Arabidopsis thaliana.With permission from Yves Poirier.



8/8

PHB at W.R. Grace Company, where he was V.P. research.

Finally, we are grateful to Professor Alexander Steinbuchel

who provided a critical review of our manuscript.

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