Published by the American Chemical Society
Volume 6, Number 1
© Copyright 2005 by the American Chemical Society
Bacterial Polyesters: Biosynthesis, Biodegradable Plastics and Biotechnology
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 largescale fermentation process not unlike the brewing of beer but 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
* To whom correspondence should be addressed. Tel.: 1-413-545-3060. Fax: 1-413-545-0082. E-mail: firstname.lastname@example.org. † University of Massachusetts. ‡ McGill University.
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 composition, and even the fact that they were polymers, were reported
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Figure 1. 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 β or 3 position. Photograph of Maurice Lemoigne.3-8 The polyester that Lemoigne isolated and characterized was poly-3-hydroxybutyrate. Paris 1949. PHB. No.
in the literature as early as 1926. These natural polyesters remained unknown to a wider scientific community for so long because their discoverer. and many organic chemists refused to believe that there were such things as polymers. Maurice Lemoigne.4 In fact. Vol. These polymers are referred to in general as polyhydroxyalkanoates. the repeating unit of PHB has a chiral center. 1.2
Biomacromolecules. and his series of papers published over the five-year period from 1923 to 1927 are remarkable for their breadth of research and prescience. as they were referred to by Lemoigne. 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. with permission. that were not ether soluble. published his results in little-read French journals at a time when microbiologists had no interest in lipids. and all such natural polyesters have the same configuration for the chiral center
. Head of Services de Fermentation at Institut Pasteur. [R]-3hydroxybutyric acid:
As indicated in this structure. Lemoigne (Figure 1) was a bacteriologist with training in analytical chemistry. PHAs. and Lemoigne reported that the polymer is optically active. Courtesy of Institut Pasteur Archives. 6. shown below.
63 Lemoigne was the first to describe an analytical method for quantifying PHB. and. which is very important both for their physical properties and for the activities of the enzymes involved in their biosynthesis and biodegradation. which remained hidden from organic and polymer chemists for over 30 years even though PHB was described in biochemistry textbooks. Wilmington.17 Only cells that were chloroform extracted yielded high molecular weight PHB when reliable methods were used later. Fortunately. only low molecular weight polymers. the wellknown “eau de javelle” laundry bleach. Stanier. 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. and the large amounts of PHB found in some species as reported by Lemoigne and co-workers. Chrococcum. Wilkinson and co-workers became interested in the relationship between the presence of the intracellular lipid granules in bacteria. and in their later work they found that the cells of B.18 R. which had been known since 1901. 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.8 He came to that conclusion by carefully hydrolyzing the solid into a series of water-soluble oligomers of 3-hydroxybutyric acid. 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. but this reagent. Doudoroff and Merrick isolated what they described as “native” PHB granules of two chemoheterotropic bacteria. He named the source of the acid lipide-βhydroxybutyrique. and he was probably familiar with the work of Emil Fischer.20
. and they attempted to detail the biosynthesis and breakdown mechanism of PHB in the cells. The nutrient limitation activates a metabolic pathway. In 1958. which shunts acetyl units from the tricarboxylic cycle into the production of PHB. In 1923. It was not until microbial physiologists recognized.3 and in 1927. collodion. megaterium by dissolution of the cell wall with a lysozyme. in the late 1950s.10 Eventually Staudinger’s concepts about synthetic polymers won out.13 During the same time period at the University of California in Berkeley. 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 Lemoigne’s discovery of nature’s polyesters. however. but not until the 1930s and the publication of the definitive research of Wallace Carothers at duPont Experimental Station. 2005 3
at the 3 position. he even suggested that the polymer was produced within the cell by a “dehydration polymerization”. where.1. a term he originated.7 Lemoigne published these observations and interpretations at the time when Herman Staudinger at the University of Freiburg. on the synthesis and characterization of aliphatic polyesters and polyamides. “Native” granules are intact granules which are carefully isolated from the cell and purified to retain the active synthase.14 Well before these studies. In 1953. which he characterized for molecular weight and melting point. and he showed that PHB could be cast into a transparent film like the then well-known cellulose nitrate material. starting in 1965. Lemoigne was free of such prejudices. At the University of Edinburgh in Scotland. who demonstrated as early as 1906 that proteins are large molecules of “enchained” amino acid units or “polypeptides”.7 In followup studies.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. Delaware. and the function of PHB in the cells.15 A typical example of such granules inside a cell is shown in Figure 2 for the bacterium A. France. Germany was being ridiculed by his colleagues in organic chemistry in Europe for proposing the existence of high molecular weight molecules or polymers. 6.17a In 1961. 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”. remarkably for his time.Historical Review of Bacterial Polyesters
Biomacromolecules. The latter is ideal as a carbon-storage polymer because it is water insoluble. he and co-workers also reported that a variety of bacteria could produce PHB. he described the isolation of a solid material obtained from the cell which he characterized as a polymer of 3-hydroxybutyric acid. Doudoroff and co-workers found that PHB was the primary product of the oxidative and photosynthetic assimilation of organic compounds by phototropic bacteria. Rhodospirillum rubrum and B. and can be readily reconverted to acetic acid by a series of enzymatic reactions inside the cell. the important role that PHB played in the overall metabolism of bacterial cells that the significance of Lemoigne’s 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. megaterium could contain as much as 44% of their dry weight of PHB depending on growth conditions.12. Lemoigne was the Director of the Fermentation Laboratory of the Pasteur Institute in Lille. Vol. rubrum “native” granules also retain the depolymerase enzyme that can degrade PHB to the monomer. 1.9 At the time of his discovery of PHB in bacteria. was later shown by Lundgren and co-workers at Syracuse University to degrade these polyesters and yield. and he confirmed the claim made by Lemoigne in 1944 that PHB was the major constituent of the granules. Lemoigne and co-workers reported on their PHB studies in 27 publications from 1923 until 1951. but the B. megaterium “native” granules have only the associated synthase. which he termed “macromolecules”. chemically and osmotically inert. Weibull in 1953 had isolated the granules of B. megaterium.16. Lemoigne reported that the acid produced by the bacteria was 3-hydroxybutyric acid. it was referred to as a “lipid” not a polyester. No.
22 For both bacteria. which then undergo a thioester-oxyester interchange reaction at the active site to form a dimer and release one of the thiol groups for the propagation reaction. was initially recognized by Doudoroff. nitrogen or oxygen) needed by the cell to further metabolize acetyl-CoA for cell growth. rubrum and B.26 A detailed mechanism for the polymerization reaction. These reactions are thermodynamically favorable because of the higher bond strength of the oxyester compared to the thioester.4
Biomacromolecules. the specific enzymes which catalyzed the reactions for the synthesis of 3-hydroxybutyric acid. The synthase. With their “native” PHB granules. the monomer for PHB. Schlegel and Dawes published simultaneously their discoveries on the identification of the two enzymes involved in the reactions for converting acetic acid to 3-hydroxybutyric acid in the two different bacteria. Although the first two enzymes. 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. the monomer that is polymerized to PHB by a synthase (3). 2005
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The reactions involved in the metabolic pathway responsible 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. this cycle becomes activated when acetyl-CoA is restricted from entering the tricarboxylic acid cycle because of a deficiency in nutrients (generally either phosphorus.21. were not identified until 1973. both initiates and catalyzes the polymerization process. which catalyzes the hydrogenation of the latter to [R]-3-hydroxybutyryl-CoA.25. which catalyzes the dimerization of the Coenzyme A derivative of acetic acid. 6. the two thiol groups form thioesters with two molecules of monomer. therefore. In this case.24 In the same 1973 issue of Biochemical Journal. For initiation. England. the ketothiolase (1) and the reductase (2) which are responsible for monomer synthesis. working independently. addition of nucleating agents and suitable posttreatment after extrusion
. eutrophus. which was based on Merrick’s suggestion. However.27. although. Biotechnology As mentioned at the start of this review.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. Vol.21. and it was characterized by Merrick and co-workers in 1968 in their studies on the production of PHB in both R. 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. and so on. Germany and Dawes at the University of Hull. the enzymes were a ketothiolase (1). megaterium. These granules could be used in an aqueous suspension for the “in vitro” polymerization of [R]-3-hydroxybutyryl-CoA to PHB. 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. eutrophus in the late 1950s as part of a study of the oxidation of molecular hydrogen by Hydrogenomonas bacteria. 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. Schlegel first began his research on A. the synthase or polymerase (3). could accumulate very large amounts of PHB during growth in nitrogen-limited media. No. bacterial polyesters became an article of commerce when ICI began their production of “Biopol” in 1982. In 1961. they observed that A. acetyl-CoA. when Schlegel at the University of Gottingen. 1. but “Biopol” was not PHB. a member of this group. and a reductase (2).26 They made their initial recognition of the
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. beijerinckii. Merrick and co-workers as early as 1964. the enzyme responsible for the polymerization process. rubrum. were able to isolate and characterize those enzymes.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 Azotobacter beijerinckii. which he found capable of accumulating as much as 70% of its dry weight of polymer. to acetoacetyl-CoA.29 so as a result. Dawes became interested in PHB while preparing a review on microbial metabolism in 1962 before joining the University of Hull. 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. all natural PHAs are completely isotactic. as shown in the following reaction scheme:
existence and role of the synthase in their work on the isolation and characterization of the active “native” PHB granules from these bacteria.23 Coincidentally. He began his research program there with a study of the accumulation of PHB in A. as follows:
As discussed above. were not fully identified and characterized until the investigations of Schlegel and Dawes in 1973. 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.
he and co-workers observed that this bacterium was capable of producing very large amounts of PHB under selected growth conditions. The latter subject was of interest because PHB. they had concentrated on the use of methylotropic bacteria to produce SCPs from methanol. which greatly improved its processing and solid-state properties. They improved the mechanical properties of PHB plastics when they found that PHB was compatible with a variety of plasticizers. the copolymers have better processing characteristics and considerably improved mechanical properties for use as plastics as shown in Figure 3. the storage polyester formed is a random copolyester of HB and HV units which has a lower melting point. 6. Issues such as obtaining high molecular weight polymers. Chemistry and Engineering News in the March 18. 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. The Netherlands.Historical Review of Bacterial Polyesters
Biomacromolecules.37 However. Baptist and Werber recognized that PHB was a stereoregular polymer with a melting point close to that of polypropylene. and their work on the synthesis and properties of PHB was reported in 1964 in the Transactions of the Society of Plastics Engineers. found that Pseudomonas oleoVorans grown on alkanes produced a large number of granules containing polyesters with units of 6-10 carbon atoms. Schlegel had also studied the production of SCPs by bacteria. In 1988.30 As a result. but they were not able to identify the microbial species in sludge which produced the polyester. which were obtained by ethanolysis of the polyester. and as a result. Nevertheless. initially for molded plastics and later for absorbable sutures. Their efforts were preceded by a much earlier attempt at the W.32 They analyzed the ethyl esters of the units.37 Baptist had joined the Research Division of Grace in 1959 after a postdoctoral in biochemistry at the University of Michigan.39 Samples of the polymer were provided to them by Lundgren. 1. SCP.36 In ´ this case. In 1983. These deficiencies were partly eliminated when it was found that. 1963. which was apparently the first time that most polymer chemists became aware of this thermoplastic biopolyester. megaterium could produce polyesters containing at least six different types of units. eutrophus to ICI for their PHB process development program. including both linear and branched 3-hydroxyalkanoic acids with compositions varying from four to eight carbon atoms. Grace Company in Maryland. they were partially motivated by the major petroleum crisis of the 1970s. which occurred as inclusions in bacterial cells (Figure 2). Coincidentally. 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. although HB units still comprised approximately 95% of the contents. like PHB. Schlegel provided samples of several strains of A. who reported in 1958 that their strain of that bacterium could produce PHB to 58% of its dry cell weight. R.38 With this bacterium Baptist was able to produce large quantities of PHB for evaluation. White and co-workers at Florida State University demonstrated that the hydroxyalkanoic acid units present in polyesters extracted from bacteria in marine sediments included even more than HB and HV units. but it is very slowly resorbable.33 These bacterial polyesters have low glass transition temperatures and much lower crystallinities than PHB. who had been studying the presence of PHB in bacteria for several years in the Microbiology Department of Syracuse University. they display elastomeric properties. Vol. In those investigations.35 During the 1950s. eutrophus for that purpose. by gas chromatography and showed the presence of at least 11 types of repeating units. but the project was terminated in 1976 because of consumer resistance. Nevertheless. White and co-workers also showed that Lemoigne’s original bacterium B. and in 1979.31 In 1983. where he had learned about bacterial production of PHB. by bacteria for use as fodder. eutrophus is grown on a mixture of glucose and propionic acid. 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. in 1962. which it has been found to be in more recent studies. was assumed to be a biocompatible polymer in humans.30 Because of its high melting point. the copolymer is fully biodegradable in a wide variety of natural environments as well as in waste disposal facilities. ICI was not the first company to consider the commercial development of bacterial polyesters for consumer products.35 Furthermore. as a natural polyester.35 After evaluating a variety of methods for that purpose. Baptist used a sample of Rhizobium obtained from Hayward and co-workers at the Colonial Microbiological Research Institute in Trinidad.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. 2005 5
or casting can lead to much improved properties. Doi and co-workers obtained PHAs with 4-hydroxybutyric acid repeat units from bacteria grown on carbon substrates having these structures. when A.34 ICI became involved in the commercial development of bacterial polyesters after terminating a program on the largescale production of single cell proteins. eventually selecting the Hydrogenomas
bacterium A. and X-ray crystal structure were settled
. issue published an extensive research report titled: “Bacteria Produce Polyester Thermoplastic”. where Baptist and Werber initiated a similar program in 1960. which suggested to them that PHB might be able to compete with polyolefins as a thermoplastic but with the added advantage of being biodegradable. the project was terminated in 1962 because of the poor thermal stability of PHB. which made the production of plastics from renewable resources economically attractive. optical rotation. especially in municipal compost sites. No. Witholt and coworkers at the University of Groningen. the composition of the copolymer could be varied over a wide range by varying the composition of the feed mixture. PHB is also susceptible to thermal degradation during melt processing by ester pyrolysis of the aliphatic secondary esters of the repeating units.12.
1. so by definition. and (3) a linking region connecting these two domains. or native. Merrick joined Lundgren’s Department and continued his studies on the composition and activity of the “native” PHB granules of B. PHB is a biodegradable polymer.52
.51 or alternatively as reported by Sinskey and co-workers at the Massachusetts Institute of Technology in 1994. Williamson and co-workers identified a specific dehydrogenase (2) that converted [R]-3-hydroxybutyric acid to acetoacetic acid. ref 17a. In 1995.42 Biodegradable Polymers Because PHB is stored by bacteria for eventual breakdown and utilization as a carbon source when extracellular carbon is no longer available. From Nuti et al. Also. Transmission electron micrograph of ultrathin section of Azotobacter chroococcum cell treated with phenylacetic acid.44 In 1967. In more recent studies. coli can also produce the HB/HV copolymer. coli. coli containing the operon can express all three enzymes and can synthesize PHB in large quantities from a wide range of organic compounds. Wilkinson and co-workers also observed the release of both acetoacetic acid and acetic acid during the utilization of PHB reserves by that bacterium.50 The three genes are clustered in one operon. and they can also be utilized as biodegradable polymers in applications such as elastomers and adhesives. 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. eutropha for the three enzymes involved in the synthesis of PHB from acetyl CoA as described above.48 As mentioned above. They also continued and expanded the earlier studies of Merrick and Doudoroff on the depolymerization of PHB by soluble depolymerases. As discussed above. with permission.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.. and Dennis and co-workers were able to introduce this operon into E. Merrick and co-workers demonstrated that “native” granules from B. these polyesters have much different physical and mechanical properties. Doudoroff and co-workers isolated and characterized a number of pseudomonads capable of utilizing extracellular PHB as their sole source of carbon and energy. As reported by Doi and co-workers. Amherst. megaterium. Vol.46 and in 1965 Delafield.42 The protein membranes covering the PHA granules of P. and they isolated the enzyme responsible for this reaction which they referred to as a depolymerase or hydrolase (1). Initial observations made by Schlegel and Chowdhury in 1963 with strains of Pseudomonas obtained from soil and compost samples established this concept. and they appear to have three characteristics in common along the polypeptide chain. herein. The genetically engineered E. Dawes and co-workers identified an enzyme for the conversion of acetoacetic acid to acetic acid (3). In 1958.45 and in 1973.
in definitive experiments. there must be an effective and rapid mechanism within the cell for the biodegradation of this high molecular weight polyester into simple organic compounds. 6. No.47 It is now known that microorganisms exist in all natural environments that are capable of degrading PHB and metabolizing [R]-3-hydroxybutyric acid by enzyme-catalyzed reactions. they have the same features as the depolymerizing enzymes for insoluble polysaccharides such as cellulose and chitin. oleVorans were studied in detail by Fuller and co-workers at the University of Massachusetts. (2) a substratebinding domain. at about the same time. megaterium in an aqueous environment. and the synthase is associated with the inner membrane.26. Figure 2) were surrounded by a protein membrane. granules (cf.6
Biomacromolecules.49 The structural genes of a large number of extracellular depolymerases of different microorganisms have been isolated and analyzed.40 They observed that the carefully purified. in 1962.43 Subsequently.41. In that manner. Many different types of intracellular and extracellular polyester depolymerases have now been isolated and characterized. 2005
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Figure 2. Genetic Engineering In 1988. they reported that the granules are enclosed in two separate protein membranes. Some recombinant strains of E. all of these enzymes consist of a single polypeptide chain in the molecular weight range of approximately 40 000-60 000. rubrum were self-hydrolyzing.19. strains containing only the synthase gene can express this protein in sufficiently large quantities for isolation and purification. including (1) a catalytic domain (termed a “lipid” box). Lemoigne had been led into his study of PHB by finding that [R]-3-hydroxybutyric acid is released by B. depolymerases have also been found for the PHAs with long alkyl chains. Dennis and co-workers at James Madison University cloned the entire set of genes in R.
In addition.58-60 Indeed. on PHAs for medical applications. the chemical modification of medium chain PHAs produced by bacteria is a promising approach to the commercialization of highvalue polymers for specialty applications. 2005 7
utilized by plants as a food and energy reserve. which can also produce acetoacetylCoA. PHB granules in the choloroplast of Arabidopsis thaliana.Historical Review of Bacterial Polyesters
Biomacromolecules. on and off. and cellulose to be colloidal aggregates of glucose molecules rather than long chain polymers. Moulded PHB objects for various applications.63 Dr. Karrer would undoubtedly have made the same argument against Lemoigne’s contention of the existence of high molecular weight reserve polyesters in bacteria. in the long run.62 so it is apparent that there is still much more to be discovered about the synthesis of bacterial polyesters. Merrick. to approximately 14% of its dry weight. 6. We wish to recognize the important contributions by Professors Schlegel. including starch and vegetable oils.61 Very recently. So much for conventional wisdom. because starch is
Despite the 75 years. namely (1) a joint program by the Proctor & Gamble Co. which means that the polymerization process has no polymer chain termination reaction. As for the agricultural production of PHAs. 1. Figure 4. At this writing. Outlook
Figure 3.54 In another application of genetic engineering for bacterial polyester synthesis. reasoned that. alternative substrates are receiving much attention. Somerville and co-workers at Michigan State University reported in 1992 that the reductase and synthase genes of A. such objects biodegrade in about three months. Dawes. which is a reserve polymer for plants.57 but the transfer of this technology into crops such as canola with acceptable production levels is still in the research stage. so the propagating end group remains active indefinitely and very high molecular weight polymers can be prepared in vitro. it was even found possible to produce a thioester analogue of the PHAs with bacteria.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”. and Kaneka Corp. on a family of short and medium chain copolymers. In his book on the history of polymer science. No. the feasibility of this route has been demonstrated in small plants such as Arabidopsis thaliana. two development programs on these biopolymers are receiving attention. by either direct bacterial synthesis or by the chemical modification of bacterially produced PHAs. Dr. ´ ` Francis Werber kindly informed us on the development of
. it is possible that advances in our understanding and control of the genetic pathways involved in the biosynthesis of PHAs in microorganisms and plants could make the industrial scale production of these biopolymers competitive with oil-based synthetic polymers. With permission from Yves Poirier. and Fuller for the factual details herein. of research on PHAs and 20 years of intense industrial interest. but no major breakthroughs in this area have been announced. On the other hand. and (2) a program at Metabolix Inc. Acknowledgment. polyesters with more than one hundred different types of repeating units have been identified and characterized.
The purified enzyme is stable in aqueous solution and has been used for in vitro polymerization reactions of a wide variety of 3. Vol. Nevertheless.and 4-hydroxyalkanoate-CoA monomers. “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”. Karrer. especially on poly(3hydroxybutyrate-co-3-hydroxyhexanoate). Arabidopsis thaliana. 56 Had he known about PHB. 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. Morawetz discusses how organic chemists at that time considered starch. Bernard Hautecoeur and Archives of the Institut Pasteur provided historical background concerning Professor Maurice Lemoigne as surveyed by Rene Dujarric de la Riviere.56 A leading German organic chemist at that time. To reduce the latter limitation.53 Lenz and co-workers at the University of Massachusetts reported in 2000 that these in vitro polymerization reactions can form “living polymers”. PHAs still appear to be far removed from large scale production.
Figure 4. In soil burial or composting experiments. and the transgenic plant can then accumulate PHB granules. eutrophus can be inserted into a plant.
119. 45. research... J. H. (24) Stockdale. Doudoroff. (29) Haywood. 1964. pp 293-314. Willkinson. (56) Morawetz.. Genser. 198. (23) (a) Schlegel. M. Arch. Soga. C. 7. A. H. Macromolecules 1992. C. Bacteriol. 1987. 1958. Sci. White. 41... (25) Merrick. 54. M. M. A. W. 253. Maurice ´ ` ´ Lemoigne (1883-1967) C. pp 1-65... 770. H. R. Y. Nawrath. C. 245.. P. H. S. Abe. Bacteriol. M. N. C. 35. Witholt.. In Biopolymers: Polyesters II. A.. FEMS Microbiol. Eds: Reidel (Kluwer) Pub. Ribbons. L. P. 1964. M. S. Volge. 1927. G. 1925.. R. M. 2000. Bull. D. ReV. J. John Wiley: New York... (44) Merrick. (12) Marchessault. Soc. ¨ M. C. 2. Macromolecules 1988. J.. G. Dawes.. 9311. M. 25. K. Nature 1961.. Gallaher. N. C. D. EnViron. R. 1089... 1455. In DeVelopments in Crystalline Polymers Vol. H. Stadler. U. ReV. M. R. A.. 88.
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