Biochemical Engineering Journal 16 (2003) 8196

Review

Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms
Alexander Steinbchel , Tina Ltke-Eversloh
Institut fr Mikrobiologie der Westflischen Wilhelms-Universitt, Corrensstrae 3, D-48149 Mnster, Germany Received 9 September 2002; accepted after revision 23 September 2002

Abstract Prokaryotes synthesize a wide range of different polyhydroxyalkanoic acids (PHA) and accumulate these polyesters as insoluble inclusions in the cytoplasm for storage of carbon and energy. PHAs are considered for various technical applications due to interesting physical and material properties. In order to establish economically feasible biotechnological production systems and to obtain PHAs from cheap carbon sources with a preference from renewable resources, CO2 or residual materials, efforts are undertaken to engineer novel pathways in recombinant prokaryotic and eukaryotic organisms. This requires transfer of a PHA synthase structural gene, expression of an enzymatically active PHA synthase protein and in particular engineering of pathways that provide this key enzyme of PHA synthesis with suitable substrates at sufcient concentrations. Only if all three aspects are well considered, a functional active PHA biosynthesis pathway will be expressed allowing PHA biosynthesis from central intermediates and therefore biotechnological production from renewable carbon sources or even CO2 . This review will focus on the engineering of pathways resulting in the formation of PHAs containing 3-hydroxyvaleric acid, medium-chain-length 3-hydroxyalkanoic acids or 4-hydroxybutyric acid as constituents. 2003 Elsevier Science B.V. All rights reserved.
Keywords: Biodegradable polymers; Bioplastics; Biopolymer production; Metabolic engineering; Microbial polyesters; Pathway construction; Polyhydroxyalkanoates; PHA; PHB; PHA synthase; Poly(3HB)

1. Introduction A large variety of polymers is synthesized by the living matter. According to their chemical structures, the following classes of biopolymers can be distinguished: (i) nucleic acids, (ii) polyamides, (iii) polysaccharides, (iv) polyoxoesters, (v) polythioesters, (vi) polyanhydrides, (vii) polyisoprenoides, and (viii) polyphenols [1]. This review will focus on polyhydroxyalkanoates, PHA, from prokaryotic microorganisms, which represent the most important group of natural polyoxoesters beside polymalic acid from eukaryotic microorganisms and cutin and suberin from plants [1]. PHAs comprise a rather large class of biopolymers with poly(3-hydroxybutyric acid), poly(3HB), having most probably the largest abundance and having studied in most detail. Poly(3HB) occurs as insoluble cytoplasmic inclusions exclusively in many eubacteria and also in some extremely halophilic archaea as storage compound for carbon and energy; it can contribute up to about 90% (w/w) of the cellular dry mass [24]. In addition, it ocCorresponding author. Tel.: +49-251-833-9821; fax: +49-251-8338388. E-mail address: steinbu@uni-muenster.de (A. Steinbchel).

curs in prokaryotes and also in eukaryotes as so-called complex-poly(3HB) contributing to only a very minor fraction of the cell mass. The functions of complex-poly(3HB) have not been revealed, yet [5]. Beside poly(3HB), storage polyesters consisting of other hydroxyalkanoic acids as constituents were also detected. Since a detailed review on PHA constituents in 1995 [6] many additional interesting new constituents have been detected. It is not in the scope of this review to refer to all these new constituents; however, at present approximately 150 different constituents occurring in PHAs alone as hompolyesters or in combination as copolyesters are known. Biosynthesis of all these PHAs is possible due to PHA synthases exhibiting extraordinary broad substrate ranges. Poly(3-hydroxybutyrate) and other PHAs consisting of short-carbon-chain length hydroxyalkanoic acids (HASCL ) comprising three to ve carbon atoms are distinguished from PHAs consisting of medium-carbon-chain-length hydroxyalkanoic acids (HAMCL ) comprising six or more carbon atoms. It should be emphasized that by employing the PHA synthase from Ralstonia eutropha, structurally related biopolymers containing mercaptoalkanoic acids as constituents were recently also obtained. These sulfur-containing polythioesters with thioester linkages instead of oxoester linkages in the polymer

1369-703X/$ see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1369-703X(03)00036-6

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backbone were synthesized if the cells were cultivated with 3-mercaptopropionic acid, 3-mercaptobutyric acid or 3,3 -thiodipropionic acid as carbon sources [79]. PHAs share physical and material properties which recommend them for applications in various areas. They are thermoplastic and/or elastomeric, insoluble in water, enantiomeric pure, non-toxic, biocompatible, piezoelectric and exhibit a high degree of polymerization and molecular weights of up to several million Da [4,10]. By far the most important feature is the biodegradability of PHAs [11]. Since poly(3HB), 3HB containing copolyesters and some other PHAs can be produced from renewable resources, they are considered as an alternative to non-biodegradable plastics produced from fossil oils. Intensive research on biotechnological production of PHAs by high cell density cultivation of microorganisms was performed [12,13], and several companies persued the development of processes for biotechnological production of PHAs [14,15]. Meanwhile, many other applications for example in medicine and pharmacy are also investigated [16]. PHAs served certainly also as a model to develop other biodegradable polymers for plastic materials which can be either chemically produced or by a combination of biotechnological and synthetic processes (for review see [17]).

another class [28]. Site-directed mutagenesis of PHA synthases has revealed much knowledge regarding the biochemistry of PHA synthases, and much progress has been made to understand the catalytic mechanism of this polymerase [2934]. PHA synthases belong to the / hydrolase superfamily possessing a catalytic triad comprising a highly conserved cysteine from a lipase box, an aspartic acid and a histidine [35]. The knowledge on PHA synthases and efcient screening systems are now utilized to obtain modied PHA synthases with new properties or enhanced in vivo or in vitro PHA synthase activity by in vivo random mutagenesis, in vitro site-directed mutagenesis and gene shufing [3638].

3. The necessity for pathway construction and metabolic engineering With relatively few exceptions most PHAs are only available if precursor substrates are provided as carbon sources to the cells during microbial fermentations. This is because the number of potential PHA synthases substrates that can be synthesized from common intermediates of the central metabolism is limited in naturally occurring PHA accumulating microorganisms. 3.1. Precursor carbon sources Therefore, the unusual chemical structures of most known PHA constituents, except poly(3HB) and a few others, can not be produced from simple carbon sources or even CO2 . The characteristics of precursor substrates are the structural relatedness of their carbon skeletons to those of the hydroxyalkanoic acid to be incorporated into PHAs and that the position of the hydroxyl group of the constituent is already directly or indirectly contained in the structure of the carbon source. For example, the hydroxyl group may be replaced by an amino or a keto group, or a keto group may be predictably incorporated by -oxidation during the catabolism of the carbon source. Therefore, the cells do not need to synthesize an unusual hydroxyacyl-coenzyme A thioester as substrate for the PHA synthase from a central intermediate; instead, the PHA substrate is directly formed during the catabolism of the structurally related carbon source. However, these precursor substrates are generally more expensive than renewable resources, and they are often also toxic. The latter prevents that this precursor carbon source can be provided at a high concentration in the medium, and therefore more sophisticated process engineering for efcient PHA production during fermentation is required [39]. As a consequence, naturally occurring chemolithoautotrophic bacteria such as R. eutropha and photoautotrophic bacteria such as the anoxygenic phototrophic or the cyanobacteria, which were considered as suitable candidates for biotechnological production of PHAs from CO2 in the past [40,41], are unsuitable for production of most PHAs

2. The key enzymes of PHA biosynthesis: PHA synthases The committed steps of the various PHA biosynthesis pathways are catalyzed by PHA synthases. The natural substrates of this key enzyme are coenzyme A thioesters of (R)-hydroxyalkanoic acid with the hydroxyl group at position 3, 4, 5 or 6 of the acyl moiety, with various carbon chain length and also with a large variety of substituents [6]. In PHA accumulating cells, PHA synthases are bound to the surface of the PHA granules [18] together with other proteins from which the phasins [19,20] and specic regulator proteins are probably most important and interesting [2123]. Since biosynthesis of PHAs is independent from a template and since the processicity of the enzyme is high, polydisperse products with relatively high molecular weights are formed [24]. More than 60 PHA synthase genes (phaC) from eubacteria have been cloned and sequenced, and many more PHA synthase sequences have been revealed due to homology searches in prokaryotic genome sequence data banks [2527]. According to their subunit composition (only PhaC, or PhaC together with PhaE) and their substrate specicity (resulting in HASCL or HAMCL incorporation, respectively), three different well studied classes of PHA synthases (I, II and III) are distinguished. The PHA synthases of R. eutropha, Pseudomonas aeruginosa and Allochromatium vinosum representing classes I, II, or III, respectively, are currently studied in much detail. From all PHA synthases investigated so far, only the enzyme from Bacillus megaterium seems to be different and might represent

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not representing poly(3HB) from CO2 . The same is true for naturally occurring plants. On the other side, plants are considered as most suitable candidates for sustainable and economically feasible production of PHAs. Therefore, generation of transgenic plants synthesizing poly(3HB) based on heterologous expression of the R. eutropha PHA biosynthesis genes or other PHAs by expression of other PHA synthases plus additional foreign genes is currently being intensively persued. Successful efforts to obtain poly(3HB) synthesis in transgenic plants has been already described 10 years ago [42]. This achievement opened new perspectives for production of PHAs by agriculture [43,44]. 3.2. Problems to be addressed during establishment and engineering of functional active PHA biosynthesis pathways The inow of nutrients and the outow of products in any living cell are linked by a complex network of biochemical reactions comprising cyclic and branched pathways [45]. If PHA biosynthesis is to be established in a putative production organism, many aspects have to be considered (Fig. 1): (i) PHA synthase must be transferred into the considered organism and (ii) and must be functionally expressed, i.e. transcription and translation of the gene(s) must occur at sufcient rates resulting in the formation of an active enzyme protein. Evidence for a post-transcriptional modication of PHA synthase protein could not be conrmed [31] and seems not to be relevant. Formation of an active PHA synthase seems also not essentially dependant from the presence of phasins [20]; the latter may only modulate the specic activity, if they have an inuence at all [46]. Expression of an enzymatically active PHA synthase seems to be the easiest part and was successfully demonstrated for many different PHA synthase genes in many different heterologous hosts [1]. The most difcult problem is to provide the PHA synthase in the cells with sufcient concentrations of substrate. It may be already a problem to obtain simply sufcient (R)-3HB-CoA for poly(3HB) synthesis in particular eukaryotic organisms; synthesis of (R)-HA-CoA thioesters for other PHAs is generally even more difcult. (iii) Therefore, new pathways must be constructed which allow the withdrawal of central intermediates and the

conversion of the latter to a hydroxyacyl-CoA thioester that can be used by a PHA synthase as substrate. Only a few central pathways can be utilized for this [47]. (iv) Furthermore, these pathways must allow a sufciently high ux of these intermediates towards the PHA synthase. Metabolic ux analysis will be very helpful [48]. Only then a functional active pathway is available that allows biosynthesis and accumulation of a particular PHA from simple carbon sources or even CO2 . Choice of genes to establish a new pathway and attempts to modulate the ux must consider the physiology of the organisms in which biosynthesis of a particular PHA is to be established. Elementary mode analysis as employed to establish poly(3HB) biosynthesis and accumulation in recombinant Saccharomyces cerevisiae [49] might be very helpful. (v) If PHA has to occur in a eukaryotic organism like a plant, further problems must be addressed. The functional active pathway must reect the complex eukaryotic cell structure and also the different tissues if a higher eukaryotic organism like a plant is engineered. The enzymes of the engineered pathway must be expressed in a suitable compartment and organell and also in a suitable tissue [43,44]. (vi) Finally, and if all previously mentioned aspects have been successfully addressed, the genetic information must be stably maintained in the production organism. 3.3. In vitro engineering and PHA biosynthesis In principal metabolic pathways can be engineered or constructed in vivo and in vitro. In vitro PHA biosynthesis can be achieved using quite different polymerizing enzymes and different substrates. Enzymes that have been shown to synthesize PHAs in vitro are PHA synthases, various lipases, proteinase K and also PHA depolymerase. However, only PHA synthases catalyze polymerization under physiological conditions. Successfully employed substrates were mainly various lactones, other cyclic monomers, dicarboxylic esters, dicarboxylic acid derivatives plus glycols and acid anhydride derivatives (for review see [50,51]). If a polymerizing enzyme is used, which catalyzes biosynthesis under physiological conditions, and if this enzyme is combined with additional enzymes that synthesize the substrates of the polymerizing enzyme, more or less short pathways can be engineered (for review see [1]). These pathways should also contain enzymes which recycle the coenzyme A released by the PHA synthase and other coenzymes involved [52]. Otherwise, coenzyme A has to be applied in stoichiometric amounts resulting in extraordinary high costs; in addition, polymerization will be inhibited due to the accumulation of coenzyme A released from the substrate during polymerization by the PHA synthase. The currently in vitro engineered pathways are far away from being suitable for biotechnological production of PHAs; nevertheless sufciently large amounts of PHA material can be obtained to reveal some basic properties of the polyesters such as melting point, glass transition temperature, crystallinity, etc. Also, non-natural compounds can be applied as substrates of the

Fig. 1. Key measures to establish PHA biosynthesis in non-PHA producing organisms.

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PHA synthase. Another advantage is that the functionality of such pathways may be investigated and conrmed within a relatively short period before more time consuming in vivo metabolic engineering is done. In one example (see below) an in vitro engineered pathway was successfully expressed in a functional active form in Escherichia coli [53].

4.1. From propionic acid The rst example of poly(3HB-co-3HV) biosynthesis occurring in an axenic bacterial culture is based on the use of propionic acid as additional carbon source when Imperial Chemical Industries (ICI) in United Kingdom developed a biotechnological process for production of this copolyester employing a strain of R. eutropha [15,55]. Although propionic acid is the most widely used precursor substrate for poly(3HB-co-3HV) biosynthesis, it has two major disadvantages: rstly, propionic acid is more expensive than simple carbon sources like for example glucose. Secondly, propionic acid is a highly toxic compound; this feature is utilized when propionic acid is applied as a conservative in food. For this reason propionic acid must be co-fed at relatively low concentrations in order to avoid too high concentrations in the medium. In addition, propionic acid is not only converted to 3HV, it is also catabolized to pyruvic acid or succinyl-CoA, which are intermediates of the central metabolism. The methylcitric acid cycle (MCC) and the methylmalonyl-CoA pathway, respectively, are the major pathways initiating complete oxidation of propionic acid in aerobic bacteria (for overview see [53]). Consequently, a 2-methylcitric acid synthase mutant of Burkholderia sacchari, a bacterium catabolizing propionic acid via the MCC and used for biotechnological production of poly(3HB-co-3HV) in Brazil [57], accumulated PHAs with a higher 3HV content [58]. Propionic acid is converted to propionyl-CoA by different enzymes (Fig. 2). Enzymes from the type of fatty acid thiokinases such as propionyl-CoA synthetase (PrpE) from Salmonella enterica serovar Typhimurium [59] or an acetyl-CoA synthetase (AcoE) [59,60] from R. eutropha or

4. Poly(3HB-co-3HV) Biosynthesis of poly(3HB-co-3HV) requires beside 3HBCoA also 3-hydroxyvaleryl-CoA, 3HV-CoA. The latter is also required if other copolyesters containing 3HV or poly(3HV) homopolyester are to be synthesized. 3HV-CoA is obtained from condensation of acetyl-CoA and propionylCoA to 3-ketovaleryl-CoA and subsequent reduction of the condensation product to 3HV-CoA. These two reactions are catalyzed by -ketothiolases and acetoacetyl-CoA reductases, respectively. The -ketothiolase BktB of R. eutropha and the acetoacetyl-CoA reductase of the R. eutropha PHA biosynthesis operon catalyze for example these reactions. It is important to know that the -ketothiolase PhaA encoded by the PHA biosynthesis operon of R. eutropha does not catalyse this reaction [54]. Whereas acetyl-CoA is an obligate central intermediate occurring in any organism and under any physiological condition, this is not the case for propionyl-CoA, which is only synthesized under special physiological conditions and from only few substrates. Therefore, processes aiming at the biosynthesis of, e.g. poly(3HB-co-3HV) require formation and occurrence of propionyl-CoA in the cells. In the past, several exogenous and endogenous propionigenic substrates have been utilized for biosynthesis of this polyester (Fig. 2).

Fig. 2. Sources of propionyl-CoA for biosynthesis of PHAs containing 3HV as constituent.

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various coenzyme A transferases such as the propionyl-CoA transferase from Clostridium propionicum [61,62] can catalyze this conversion. Some of these enzymes were used to engineer E. coli strains suitable for production of poly(3HB-co-3HV) from propionic acid alone or in combination with other carbon sources. Propionate utilization and extent of 3HV incorporation in recombinant E. coli expressing the PHA biosynthesis operon of R. eutropha depended strongly on the expression of the synthetase [63,64]. 4.2. From other aliphatic fatty acids Aliphatic fatty acids with a higher carbon chain length and an odd number of carbon atoms like valeric acid, heptanoic acid, nonanoic acid, etc. are also propionigenic substrates because propionyl-CoA instead of acetyl-CoA remains after the last turn of the -oxidation cycle (Fig. 2). In addition, intermediates of the -oxidation cycle with ve carbon atoms of the acyl chain may be converted into (R)-3HV if the respective enzymes are present (see below). Therefore, bacteria possessing a PHASCL synthase usually synthesize poly(3HB-co-3HV) if cultivated on the fatty acids mentioned above. 4.3. From levulinic acid For bacteria possessing a PHASCL synthase levulinic acid (4-ketovaleric acid) is structurally the most closely related precursor substrate that is available for biosynthesis of polyesters containing 4-hydroxyvaleric acid (4HV) as constituent. PHAs accumulated by bacteria when cultivated on levulinic acid usually contain beside 4HV also 3HB and 3HV as further constituents [65]. Since levulinic acid is a relatively cheap carbon source, which could be made abundantly available from renewable resources through chemical conversion [66], much efforts were undertaken to scale up fermentation processes yielding 4HV containing PHAs [67]. The presence of 3HB and 3HV indicates that intermediates of the upper part of the levulinic acid catabolic pathway, which has not be studied in detail, are further degraded by -oxidation or enzymes catalyzing similar reactions releasing propionyl-CoA beside acetyl-CoA. The induction of the enzymes of the methyl citric acid cycle, which converts propionyl-CoA to pyruvate, during cultivation on levulinic acid for example in R. eutropha is consistent with the formation of propionyl-CoA (Fig. 2, [54,68]). 4.4. From pentanol The facultative methylotrophic bacterium Paracoccus denitricans synthesized and accumulated poly(3HV) homopolyester during cultivation on n-pentanol as sole carbon source [69]. This alkane is oxidized via valeraldehyde to valeric acid and subsequently converted to valeryl-CoA. The authors suggested that 3-ketovaleryl-CoA, which is formed

during -oxidation, is reduced to (R)-3-hydroxyvaleryl-CoA and is subsequently polymerized. 4.5. From amino acids The catabolism of some amino acids is another important source of propionyl-CoA. Valine, isoleucine, threonine and methionine are therefore precursor substrates for 3HV containing PHAs because they are catabolized via propionyl-CoA (Fig. 2). The use of these amino acids as precursor substrates during fermentation will be certainly not feasible from an economic point of view due to their high costs and from process engineering due to the low solubility of some of them. However, intracellular generation or overproduction of these amino acids or related intermediates with subsequent degradation allows intracellular generation of propionyl-CoA from renewable resources and ammonia. One rst example for this was a spontaneous revertant to prototrophy of an isoleucine-auxotrophic mutant of R. eutropha. This mutant overexpressed the enzyme acetolactate synthase in order to compensate for a defective threonine dehydratase causing the auxotrophic phenotype. It excreted valine, leucine and isoleucine into the medium, when the nitrogen source in the medium was not limiting; however, when ammonium was limiting and when a carbon source was provided in excess, poly(3HB-co-3HV) was accumulated from various simple carbon sources without the need to fed propionic acid [70]. It was concluded that the methyl-branched 2-ketofatty acids corresponding to the respective amino acids were intracellularly overproduced and consecutively degraded to propionyl-CoA in the cells under conditions permissive for PHA accumulation (Fig. 2). Another important example for establishing poly(3HBco-3HV) biosynthesis from an unrelated carbon source was based on another interesting approach applied to plants. Poly(3HB-co-3HV) production in Arabidopsis thaliana and Brassica napus was achieved by expressing the threnine deaminase gene from E. coli (ilvA) beside other genes already described above [71]. Theonine deaminase converts threonine to 2-ketobutyric acid, which is then oxidized and decarboxylated to propionyl-CoA by the pyruvate dehydrogenase of the plants to propionyl-CoA (Fig. 2). The latter is converted to (R)-3HV-CoA by the bktB-encoded -ketothiolase B and the phaB-endoded acetoacetyl-CoA reductase of R. eutropha and subsequently copolymerized with 3HB-CoA to poly(3HB-co-3HV) by the phaC-encoded PHA synthase also from R. eutropha. 4.6. From citric acid cycle via methylmalonyl-CoA pathway In addition to the methylcitric acid cycle, the coenzyme B12-dependent methylmalonyl-CoA pathway is another important pathway for the catabolism of propionyl-CoA derived from propionic acid or other propionigenic carbon sources to succinyl-CoA (for overview see [56]). Since the

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enzymes of this pathway are reversible, the pathway provides the possibility to link via the citric acid cycle renewable resources and CO2 with biosynthesis of 3HV containing PHAs (Fig. 2). The only wild-type bacteria, which naturally synthesize poly(3HB-co-3HV) with very high contents of 3HV (>75 mol%) from unrelated carbon sources like glucose and which have been investigated in detail, are various species belonging to the Gram-positive genera Nocardia or Rhodococcus [7274]. These bacteria degrade glucose via the 2-keto-3-deoxy-6-phosphogluconate pathway to pyruvate; the latter is probably carboxylated to oxaloacetate and subsequently converted to succinyl-CoA by reverse reactions of the citric acid cycle. Succinyl-CoA is then converted via methylmalonyl-CoA to propionyl-CoA (Fig. 2) as revealed for N. corallina and R. ruber [7476]. These bacteria provide a very good example for endogenous generation of propionyl-CoA from simple carbon sources. This became also obvious by the high content of odd-numbered fatty acids that occur in the triacylglycerols, which are a second important storage compound of these bacteria. Employing inhibitors of the fatty acid de novo synthesis (cerulenin) and of the -oxidation/ -ketothiolase (acrylic acid) the competition of the enzymes of PHA biosynthesis enzymes and TAG biosynthesis as well as the inuence of these inhibitors on the availability of 3HB-CoA and 3HV-CoA for the PHA synthase could be nicely demonstrated. Whereas cerulenin caused a decrease of the molar 3HV content in the accumulated copolyester, acrylic acid caused an increase of 3HV versus 3HB [74]. An interesting pathway for poly(3HB-co-3HV) biosynthesis was recently engineered in recombinant S. enterica serovar Typhimurium [77]. The authors expressed the genes for (2R)-methylmalonyl-CoA mutase (sbm) and a (2R)-methylmalonyl-CoA decarboxylase (ygfG) from E. coli in this Salmonella strain, thus enabling the conversion of succinyl-CoA to propionyl-CoA. In addition, they inserted into the gene for 2-methylcitric acid synthase (prpC) of this bacterium the PHA biosynthesis operon of Actinobacter (phaCAB) thereby establishing PHA biosynthesis and disrupting propionate utilization. This recombinant Salmonella sp. synthesized poly(3HB-co-3HV) with up to 31 mol% 3HV via succinyl-CoA (Fig. 2) if the cells were simply cultivated on glycerol as carbon source.

but also from glucose and many other structurally unrelated carbon sources [79,80]. For example, Psuedomonas putida accumulates during cultivation on octanoic acid as carbon source a copolyester consisting of (R)-3-hydroxyoctanoic acid and (R)-3-hydroxyhexanoic acid as main and minor constituents, respectively, whereas with gluconate as carbon source a copolyester consisting of (R)-3-hydroxydecanoate as main constituent and (R)-3-hydroxydodecanoate and (R)-3-hydroxyoctanoate as minor constituents is accumulated. This seems to be an inherent feature of the species of this genus. This capability implies that the fatty acid metabolism of these bacteria, i.e. fatty acid de novo biosynthesis and fatty acid -oxidation, is linked with PHA biosynthesis (Fig. 3). Meanwhile, several different metabolic links between fatty acid metabolism and PHAMCL biosynthesis have been revealed (see below). Knowledge of these metabolic links and the circumstance that fatty acid de novo synthesis and -oxidation occur in almost any organism were utilized to establish PHAMCL biosynthesis in various non-PHA producing organisms. 5.1. From alkanes P. oleovorans utilizes various aliphatic alkanes as carbon source. Oxidation of for example octane proceeds via octanol and octaldehyde to octanoic acid and is catalyzed by alkane hydroxylase, alcohol dehydrogenase and aldehyde dehydrogenase which are mostly membrane bound and encoded by the OCT plasmid [81,82]. The resulting octanoic acid is converted by a thiokinase to octanoyl-CoA, which is then oxidized by fatty acid -oxidation to acetyl-CoA. Intermediates of the -oxidation pathway are obviously also withdrawn and not degraded to acetyl-CoA but converted to (R)-3-hydroxyoctanoyl-CoA and subsequently polymerized by one of two or both PHAMCL synthases detected in this bacterium if the cultivation conditions are permissive for PHA biosynthesis [83]. Details are described below (see Section 5.3). The oxidation of octane and other alkanes and its bioconversion to octanoic acid is achieved by two-liquid phase fermentation. It is also of interest that the P. oleovorans alkane oxidizing system was functionally expressed in other bacteria like P. putida and E. coli [78]. 5.2. From acyl alcohols

5. Poly(3HAMCL ) A polyester consisting mainly of 3-hydroxyoctanoic acid is synthesized by Pseudomonas oleovorans and was the rst example for PHAs consisting of medium-chain-length hydroxyalkanoic acids, PHAMCL obtained from an axenic culture [78]. Meanwhile it is obvious that all pseudomonads sensu strictu with only a few exceptions are capable to synthesize poly(3HAMCL ) not only when they are cultivated on various aliphatic alkanes or aliphatic fatty acids

Instead of octane also n-octanol or other acyl alcohols can be utilized by P. oleovorans and some other pseudomonads. After oxidation by a dehydrogenase, the resulting octanoic acid is metabolized as mentioned above. 5.3. From fatty acids For routine analysis in the laboratory it is convenient that instead of aliphatic alkanes also the corresponding fatty acids are utilized by PHAMCL accumulating pseudomonads.

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Fig. 3. Linkages between fatty acid metabolism and PHAMCL biosynthesis.

This enabled fermentation in only one phase and allows PHA biosynthesis, which is independent from the alkane oxidizing enzyme system. Furthermore, it was shown, that the carbon chain length of the 3-hydroxyalkanoic acids incorporated into PHAs is related to the carbon chain length of the fatty acid used as carbon source [84]. Furthermore, this opened the perspective to use fatty acids from oils and fats of plants or from animal sources as carbon source for biotechnological production of PHAMCl , and therefore from agricultural resources and residual products [85]. As mentioned above (see Section 5.1) the fatty acids are converted to the corresponding acyl-CoA thioesters and oxidized by fatty acid -oxidation via trans-2-enoyl-CoA and (S)-3-hydroxyacyl-CoA to 3-ketoacyl-CoA which is cleaved by a -ketothiolase to acetyl-CoA and acyl-CoA comprising two less carbon atoms as compared to the acyl-CoA that entered the rst cycle. Further cycles follow until the original acyl-CoA is completely converted to acetyl-CoA in case of fatty acids having an even number of carbon atoms or acetyl-CoA plus propionyl-CoA having an odd number of carbon atoms. This is the case for saturated and non-substituted aliphatic fatty acids. If the fatty acid contains functional groups, alkyl side chain as branches or non-saturated carbon to carbon bonds, the degradative pathway may be different and may require additional enzymes. Furthermore, in addition to acetyl-CoA (and propionyl-CoA) other products may occur. Under physiological conditions permissive for synthesis and accumulation of PHAMCL , the fatty acids are not completely degraded to acetyl-CoA, and intermediates of the

-oxidation cycle are partially or completely withdrawn and converted into these polyesters. However, none of the intermediates is accepted as substrate by the PHAMCL synthases. Therefore, a metabolic link is required which converts one of the intermediates into (R)-3-hydroxyacyl-CoA. Three different enzyme activities establish this link (Fig. 3). For a particular organisms capable to convert fatty acids into PHAMCL it has to be investigated which enzyme is relevant. One candidate are epimerases converting the (S)-isomer of 3-hydroxyacyl-CoA into the (R)-isomer. When PHAMCL biosynthesis was established for the rst time in a recombinant E. coli, a PHAMCL synthase from P. aeruginosa was expressed in the fadB mutant LS1298 of E. coli. Whereas the dehydrogenase function of FadB is defective in this mutant, the epimerase and the hydratase function remained active. It was concluded that (S)-3-hydroxyacyl-CoA accumulates in the cytoplasm of this mutant to a sufciently high level allowing effective conversion of the (S)-stereoisomer into the (R)-stereoisomer and subsequent formation of PHAMCL [86,87]. Addition of acrylic acid, which is an inhibitor of -ketothiolases, to the medium allowed phaCMCL -dependant PHAMCL accumulation also in E. coli strains with intact fadB [88] demonstrating that like in the fadB mutant routing of (S)-3-hydroxyacyl-CoA to PHAMCL synthesis occurred. Others achieved stable PHAMCL production in E. coli by inserting a PHAMCL synthases from P. oleovorans into the chromosome of a fadB mutant [89]. Another candidates are hydratases converting trans-2enoyl-CoA into (R)-3-hydroxyacyl-CoA. In Aeromonas caviae a (R)-specic enoyl-CoA hydratase encoded by phaJ

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is responsible for the conversion of trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA during the cultivation of this bacterium on, e.g. hexanoate [90]. Two (R)-specic enoyl-CoA hydratase genes were also identied in P. aeruginosa [91]. Further candidates are enzymes exhibiting 3-ketoacyl-CoA reductase activity and which reduce 3-ketoacyl-CoA to (R)-3-hydroxyacyl-CoA. Studies in recombinant strains of E. coli provided evidence that FabG of fatty acid de novo synthesis, which is a 3-ketoacyl-ACP reductase, is unspecic and exhibits also activities with the corresponding CoA-thioesters and is therefore capable to provide this link [92,93]. On the other side it was shown, that recombinant strains of E. coli could also use their own -ketothiolase (FadA) in combination with the acetoacetyl-CoA reductase (PhaB) of R. eutropha for conversion of fatty acids into poly(3HAMCL ) [94]. 5.4. From glucose and other non-fatty acid substrates Finding of PHAMCL biosynthesis from substrates like glucose or gluconate in pseudomonads indicated participitation of fatty acid de novo synthesis [79,80]. Detailed studies conrmed that this route contributes to the by far major extent to PHAMCL biosynthesis in these bacteria [95,96]. One of the most interesting question was the metabolic link between fatty acid de novo and PHAMCL biosynthesis. However, beside fatty acid de novo synthesis, also other routes contributed to PHAMCL synthesis to a minor extent but could not be neglected [95,96]. Knowledge of these routes and the availability of the relevant genes of the metabolic link enabled metabolic engineering to obtain organisms suitable for biotechnological production of PHAMCL from carbohydrates. 5.4.1. Chain elongation Provision of substrates for PHAMCL synthases by chain elongation of acyl-CoA derived from fatty acids used as carbon sources or from cell lipids through -oxidation are signicant but contribute to only a minor fraction of the total constituents of PHA accumulated in the cells of P. putida [96]. With respect to PHAMCL biosynthesis it is probably only relevant for organisms, which overproduce also other cell components consisting of fatty acids. In this case, -ketothiolase mediated chain elongation may indirectly contribute to some extent to PHAMCL biosynthesis. 5.4.2. De novo fatty acid biosynthesis and PhaG As outlined above, clear experimental evidence was obtained that the fatty acid de novo synthesis pathway is the major pathway for provision of the 3-hydroxyacyl moieties in P. putida contributing to approximately 90% of the constituents of the accumulated PHAMCL [95,96] in P. putida and probably also in all other pseudomonads. The missing link between fatty acid de novo synthesis and PHAMCL synthesis is provided by an acyltransferase which in P. putida [97], P. aeruginosa [98], Pseudomonas sp. 61-3 [99] and

other pseudomonads [100] transfers the hydroxyacylmoiety from (R)-3-hydroxydecanoyl-acyl carrier protein to coenzyme A thus forming (R)-3-hydroxydecanoyl-CoA, which is a substrate of the two PHAMCL synthases in this bacterium (Fig. 3). This key enzyme is encoded by the phaG gene, and it links fatty acid de novo synthesis and poly(3HAMCL ) biosynthesis in these bacteria. In P. oleovorans the gene for the acyltransferase is cryptic explaining why this bacterium is unable to synthesize PHAMCL from carbohydrates [100]. In one study, it was demonstrated that the content of PHAMCL obtained from gluconate as carbon source in P. putida could be increased if the isocitrate lyase gene (aceA) in this bacterium was disrupted by transposon insertion [101]. In subsequent experiments, phaG from P. putida was transferred to and expressed in P. oleovorans which is able to synthesize poly(3HAMCL ) from alkanes or fatty acids but not from gluconate or fructose and exhibits therefore a similar phenotype as the phaG mutants of P. putida (see above). Establishment of phaG-mediated PHAMCL biosynthesis could be demonstrated in recombinant P. oleovorans [102]. Furthermore, phaG from P. aeruginosa was together with the PHA synthase gene from P. aeruginosa transferred to the non-PHA accumulating bacterium P. fragi, and both enzymes were heterologously expressed in this host (Fig. 7) [102]. When the recombinant strains were cultivated with gluconate as carbon source under conditions permissive for the accumulation of PHAs, the cells accumulated poly(3HAMCL ) contributing to approximately 50% of the cell dry matter in the case of P. oleovorans and 10% in the case of P. fragi, respectively, whereas in the parent strain or in a recombinant strain harboring only the vector, PHAs were not detected. These two studies demonstrated that the cloned transacylase is functionally active and can be used to establish poly(3HAMCL ) synthesis in other bacteria. 5.4.3. Thioesterase mediated pathway Genetically engineered fad mutants of E. coli (see Section 5.3) expressing foreign acylacyl carrier protein (ACP) thioesterases in addition to a class II PHA synthase synthesized and accumulated small amounts of PHAMCL from gluconate [103,104]. In these strains the fatty acids required for other cell constituents were obviously partially released as free fatty acid during fatty acid de novo synthesis and then directed to fatty acid -oxidation from which intermediates were withdrawn and converted to a PHAMCL synthase substrate. Whereas in one study a thioesterase from the plant Umbellularia californica plus a PHAMCL synthase from P. aeruginosa were used [103], the other study used the cytosolic thioesterase I (encoded by tesA) of E. coli plus a PHAMCL synthase from P. oleovorans [104]. Although the amounts of accumulated PHAMCL were low, these studies established a new PHAMCL biosynthesis pathway in E. coli and showed a new strategy how to obtain PHAMCL from unrelated carbon sources and in particular from carbohydrates. Application of this strategy will certainly yield

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higher PHAMCL contents of the cells, if the afnity towards the substrates or the activity of the thioesterase could be increased and if the metabolic ow rate from acetyl-CoA towards fatty acids could be increased. 5.5. PHAMCL biosynthesis in recombinant eukaryotic organisms PHAMCL synthases from pseudomonads were also successfully applied to establish PHAMCL biosynthesis in transgenic A. thaliana [105] as well as in the yeasts S. cerevisae [106] and Pichia pastoris [107]. In these eukaryotic organism it was necessary to pay special attention to the compartimented lipid metabolism.

Intensive efforts are going on to achieve biotechnological production of poly(4HB) from cheap carbon sources because this polyester has interesting properties [108]. Furthermore, poly(4HB) is not only hydrolyzed by PHA depolymerases but also by lipases and esterases since no alkyl side chains occur as pendant groups attached to the polyester backbone [109,110]. Therefore, these polyesters are considered for biomedical and also pharmaceutical applications [16]. 6.1. From 4-hydroxybutyric acid PHAs containing 4HB as constituents were discovered when R. eutropha was cultivated on 4-hydroxybutyric acid as carbon source [111,112]. Later, biosynthesis of such PHAs was also achieved in Comamonas acidovorans [113], Hydrogenophaga pseudoava [114], Ralstonia metallidurans (formerly R. eutropha) [115] and in many other bacteria possessing a PHASCL synthase. After uptake in the cells, 4-hydroxybutyric acid is converted into 4HB-CoA either by a transferase or a thiokinase. HB-CoA is then used as a substrate by the PHASCL synthase of R. eutropha (Fig. 4). However, in most cases the bacteria incorporated also 3HB and therefore synthesized poly(3HB-co-4HB) copolyesters, at least if the accumulated PHAs contributed to a higher fraction of the total cellular dry matter. Incorporation of the comonomer 3HB resulted from the catabolism of 4-hydroxybutyric acid leading to intermediates from which 3-hydroxybutyryl-CoA was synthesized. The catabolism of 4HB was studied in detail in R. eutropha strain H16; the main catabolic pathway for 4HB is probably via succinic acid semialdehyde and succinic acid catalyzed by 4HB dehydrogenase and succinic acid semialdehyde dehydrogenase

6. PHAs containing 4HB and poly(4HB) homopolyester Several bacteria possessing a PHASCL synthase are capable to incorporate 4-hydroxybutyric acid (4HB) into PHAs. However, the incorporation of 4HB strongly depends on the use of precursor substrates as carbon sources (Fig. 4). This is also true for all other PHAs that contain non-3HA constituents, which are normally only synthesized if precursor substrates are used. No wild-type strain has so far been described which synthesizes 4HB-containing PHAs from unrelated carbon sources. However, there is a reasonable perspective that 4HB containing PHAs can be also obtained from simple carbon sources in the future employing engineered organisms, because a few important central intermediates of the metabolism can be converted into 4HB-CoA.

Fig. 4. Sources of 4-hydroxybutyryl-CoA for biosynthesis of PHAs containing 4HB as constituent.

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[116,117]. Most other precursor substrates for 4HB are rst converted into 4HB-CoA. When a 4-hydroxybutyric acid-CoA:CoA transferase gene from Clostridium kluyveri (OrfZ) and the R. eutropha H16 PHA synthase gene were expressed in E. coli, and when the recombinant strain was cultivated in a mineral salts medium or LB complex medium containing 4-hydroxybutyric acid plus glucose as carbon sources, a poly(4HB) homopolyester was synthesized and accumulated [118,119]. Poly(4HB) homopolyester was also synthesized from 4-hydroxybutyric acid and accumulated in a recombinant strain of C. acidovorans containing additional copies of its PHA synthase gene [113]. 6.2. From -butyrolactone It was shown that -butyrolactone is another suitable precursor carbon source for biosynthesis of 4HB containing PHAs in R. eutropha and some other bacteria [111,112,114,116]. The lactone is hydrolytically cleaved to 4-hydroxybutyric acid. Esterases or lactonases may catalyze this step (Fig. 4). One esterase (EstA) capable to cleave -butyrolactone was recently cloned from R. metallidurans strain CH34 [115]. The resulting 4HB is than converted to 4HB-CoA as mentioned above (see Section 6.1) but also catabolized further, again resulting in the incorporation of also 3HB. A three-step cultivation scheme was found for H. pseudoava which allowed biosynthesis of poly(4HB) homopolyester from -butyrolactone [114]; the authors obtained evidence that under these cultivation conditions catabolism of 4-hydroxybutyric acid to acetyl-CoA could not occur due to a very low activity of the 4HB-DH which initiates the degradation of 4HB. 6.3. From 1,4-butanediol and other -alkanediols Another suitable precursor substrate is 1,4-butanediol [111,120]. This -alkanediol is oxidized in two subsequent enzymatic reactions to 4-hydroxybutyric acid which is converted to 4HB-CoA as described above (Fig. 4, see Section 6.1). -Alkanediols with a greater carbon chain length but an even number of carbon atoms are also suitable precursor substrates [120]. They are obviously also rst oxidized to the corresponding -hydroxyfatty acid, which is then converted into to an coenzyme A thioester and subjected to -oxidation until 4HB-CoA occurs. Since 4HB-CoA is in contrast to 3HB-CoA not a chiral intermediate, it can be directly polymerized by the PHA synthase. 6.4. From 4-chlorobutyric acid Another suitable precursor for 4HB containing PHAs and R. eutropha H16 is 4-chlorobutyric acid [114]. The pathway of this compound has not been studied in R. eutropha. 4-Chlorobutyric acid is probably converted to 4-hydroxybutyric acid by a haloalkane dehalogenase which

employs a hydrolytic mechanism (for review see [121]). The product is than converted to 4HB-CoA as mentioned above (Fig. 4, see Section 6.1). 6.5. From glucose An interesting approach was used to engineer a novel pathway to produce 4HB containing PHAs in E. coli from glucose [122]. A recombinant strain of E. coli, which expressed succinic acid semialdehyde dehydrogenase, 4HB-DH and 4-hydroxybutyric acid-CoA:CoA transferase gene from C. kluyveri in addition to the PHA synthase from R. eutropha, synthesized from glucose poly(3HB-co-4HB) containing up to 2 mol% 4HB (Fig. 4). Although the molar fraction of 4HB was very low, this study provided clear evidence that the citric acid cycle intermediate succinyl-CoA can be converted via succinic acid semialdehyde and 4-hydroxybutyric acid to 4HB-CoA in a genetically engineered organism (Fig. 4). If the metabolic ux from succinyl-CoA to 4HB-CoA could be increased, the content of 4HB in the copolyester should increase. 6.6. From amino acids The engineered pathway described above (see Section 6.5) could be extended to other interesting carbon sources and intermediates of metabolism. It was shown that glutamic acid and -aminobutyric acid can be converted to 4HB containing PHAs in recombinant strains of E. coli if a glutamic acid:succinic acid semialdehyde transaminase (gabT) from E. coli and a glutamic acid decarboxylase (gadA) from E. coli or A. thaliana were expressed in E. coli (Fig. 4, [123]). Although the molar fractions of 4HB in the accumulated polyesters were again relatively low, this study demonstrated that two amino acids, which are also synthesized by many bacteria and plants, can be converted in such PHAs. These studies demonstrated the usefullness of enzymes which are in strictly anaerobic bacteria involved in the fermentation of 4HB and glutamate [124,125]. It is now necessary to overproduce these amino acids from glucose and ammonium in the recombinant E. coli strains.

7. Poly(3HASCL -co-3HAMCL ) Biosynthesis of PHAs, in which 3HASCL and 3HAMCL are covalently linked in the same polyester molecules, i.e. real copolyesters of 3HASCL and 3HAMCL requires the presence of a PHA synthase exhibiting a substrate range combining those of PHASCL and PHAMCL synthases. It requires of course also the provision of this PHASMCL synthase with coenzyme A thioesters of 3HAs with a carbon chain length ranging from SCL to MCL. Simultaneous presence of a PHASCL synthase plus a PHAMCL synthase will only result in the formation of a blend of poly(3HASCL ) and poly(3HAMCL ) rather than in the formation

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Fig. 5. Routes for biosynthesis of poly(3HASCL -co-3HAMCL ).

of poly(3HASCL -co-3HAMCL ) (Fig. 5). This was shown for a recombinant strain of P. oleovorans expressing beside its own class II PHA synthase the class I PHA synthase of R. eutropha [126,127]. The same will certainly be true for any other copolyester containing constituents that are incorporated into PHAs by PHA synthases having an excluding substrate range, for example one for X-HA-CoA and the other for Y-HA-CoA. As long as two different PHA synthases are present, the one using X-HA-CoA and the other Y-HA-CoA, blends of two different types of PHAs will be synthesized. Only if the cells possess one PHA synthase that uses X-HA-CoA as well as Y-HA-CoA, a true copolyester is synthesized. First evidences for the existence of PHA synthases suitable for production of poly(3HASCL -co-3HAMCL ) were obtained when the PHA synthase cloned from Thiocapsa pfennigii was cloned and heterologously expressed in PHA-negative mutants of R. eutropha and P. putida. Under suitable cultivation conditions and from fatty acids, the recombinant strains synthesized copolyesters of 3HB with 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO); copolyesters of 3HB with non-3HAMCL could be also obtained [128]. There are only a few wild-type bacteria which synthesize and accumulate poly(3HASCL -co3HAMCL ). Strains of A. caviae [129] and Aeromonas hydrophila [130] suitable for production of PHAs consisting of poly(3HB-co-3HHx) were most interesting beside Pseudomonas sp. 61-3 from which copolyesters of 3HB with 3HAMCL of various chain length could be obtained [131]. These strains are studied in detail and possess PHA synthases with an unusual broad substrate range. Recently, it was found that the PHASCL or class I synthase of R. eutropha is not exclusively restricted to HASCL -CoA thioesters as substrates. Although this was the conclusion obtained from in vitro measurements of the enzyme activity [18], the R. eutropha PHA synthase could be forced in vivo to incorporate also 3HAMCL (from C6 to C12) beside 3HB into PHA copolyesters [132134]. For this cells of R. eutropha had to cultivated on fatty acids (e.g. octanoic acid) as carbon source in the presence of the -ketothiolase inhibitor acrylic acid [132], or its own genes for acetoacetyl-CoA reductase

and PHA synthase were expressed in a PHA-negative mutant [133]. Alternatively, a recombinant strain of E. coli expressing the PHA synthase of R. eutropha was cultivated on fatty acids (e.g. octanoic acid, decanoic acid, dodecanoic acid) as carbon source [134]. Bacterial systems suitable for biosynthesis of poly(3HBco-3HAMCL ) and for biotechnological production of these copolyesters are now intensively studied and engineered because they exhibit interesting material properties [135,136].

8. A new versatile composed, non-natural PHA biosynthesis pathway A new, non-natural pathway comprising the butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) from Clostridium acetobutylicum, which are in this anaerobic bacterium involved in butyric acid formation [137], and the PHA synthase from T. pfennigii or A. vinosum was recently constructed (Fig. 6). In previous experiments, it was shown that the two enzymes from C. acetobutylicum exhibited a low substrate specicity not only using the substrates occurring during butyric acid synthesis but also substrates relevant for poly(3HB) biosynthesis or biosynthesis of other interesting

Fig. 6. A versatile non-natural PHASCL biosynthesis pathway.

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PHAs. Therefore, these enzymes used substrates structurally signicantly different from the natural substrates. The puried enzymes were then combined (Fig. 6) and successfully exploited for in vitro synthesis of a wide range of different homo- and copolyesters demonstrating that this engineered pathway was quite versatile [138]. Depending on the precursor carbon sources used, various homo- and copolyesters of 3HB, 4HB and 4HV were obtained. The genes for these three enzymes were then functionally expressed in E. coli, and in vivo synthesis of PHAs very similar to those synthesized in vitro were obtained [53]. One advantage of this E. coli system is the possibility to produce poly(4HB) or poly(4HV) homopolyesters because -ketothiolase and acetoacetyl-CoA reductase were not active under these conditions; therefore 3HB could not be synthesized. Later it was shown that this engineered pathway synthesizes not only the PHAs mentioned above but also polythioesters (Tina Ltke-Eversloh and Alexander Steinbchel, unpublished results). Therefore, this pathway is rather versatile. Part of this metabolic pathway, i.e. the phosphotransbutyrylase and the butyrate kinase from C. acetobutylicum were used in reverse direction as above together with the -ketothiolase (PhaA) and the acetoacetyl-CoA reductase (PhaB) from R. eutropha to engineer a pathway enabling recombinant E. coli to produce (R)-3-hydroxybutyrate. When a recombinant strain of E. coli harboring phaA, phaB, ptb and buk, which functionally expressed the corresponding enzymes in the absence of a PHA synthase, was cultivated in the presence of glucose as carbon source, it excreted (R)-3-hydroxybutyrate into the medium to a concentration of up to 35 g/l [139].

oil, i.e. even lower than for conventional synthetic plastics [47]. Beside poly(3HB), only a few other PHAs such as most probably poly(3HB-co-3HV), poly(3HB-co-4HB) and poly(3HAMCL ) will be available from plants. In any case there will be a strong competition with conventional plastics and novel plastics. For the latter, polylactide is so most advanced and most competing material.

Acknowledgements During the last 15 years the research of the corresponding authors laboratory on PHAs was generously supported by the Bundesministerium fr Bildung und Forschung, the Bundesministerium fr Verbraucherschutz, Ernhrung und Landwirtschaft, the Deutsche Forschungsgemeinschaft, the Deutscher Akademischer Austauschdienst, the Fonds der Chemischen Industrie, the Max-Buchner Forschungsstiftung and various partners from the chemical industry. This nancial support enabled detailed basic and applied research on the subject, gave many undergraduate and graduate students as well as PostDocs and guest scientists a perspective and is gratefully acknowledged. References
[1] A. Steinbchel, Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example, Macromol. Biosci. 1 (2001) 124. [2] A.J. Anderson, E.A. Dawes, Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates, Microbiol. Rev. 54 (1990) 450472. [3] Y. Doi, Microbial Polyesters, rst ed., VCH Publishers, New York, 1990. [4] J. Hocking, R.H. Marchessault, Biopolyesters, in: G.F.L. Grifn (Ed.), Chemistry and Technology of Biodegradable Polymers, Chapman and Hall, London, 1994, pp. 4896. [5] R.N. Reusch, Non-storage poly(R)-3-hydroxyalkanoates (complexed PHAs) in prokaryotes and eukaryotes, in: Y. Doi, A. Steinbchel (Eds.), Biopolymers, vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 123171. [6] A. Steinbchel, H.E. Valentin, Diversity of bacterial polyhydroxyalkanoic acids, FEMS Microbiol. Lett. 128 (1995) 219228. [7] T. Ltke-Eversloh, K. Bergander, H. Luftmann, A. Steinbchel, Identication of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages, Microbiol. UK 147 (2001) 1119. [8] T. Ltke-Eversloh, K. Bergander, H. Luftmann, A. Steinbchel, Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB), Biomacromolecules 2 (2001) 10611065. [9] T. Ltke-Eversloh, J. Kawada, R.H. Marchessault, A. Steinbchel, Characterization of microbial polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha, Biomacromolecules 3 (2002) 159166. [10] H.M. Mller, D. Seebach, Poly(hydroxyfettsureester), eine fnfte Klasse von physiologisch bedeutsamen organischen biopolymeren? Angew. Chem. 105 (1993) 483509. [11] D. Jendrossek, A. Schirmer, H.G. Schlegel, Biodegradation of polyhydroxyalkanoic acids, Appl. Microbiol. Biotechnol. 46 (1996) 451463.

9. Outlook and further perspectives Since cloning of the rst PHA synthase gene, many signicant achievements and breakthroughs were made for novel processes for biotechnological production of PHAs during the last years. It is expected that one or the other type of PHA will be commercially produced in the near future for technical applications. Any production system will most probably rely on genetically engineered organisms. High cell density cultivations with bacteria were much improved [12], and bacteria are at present the best and almost only source for PHAs consisting not only of 3HB. Due to various constrains, we personally assume that bacterial PHAs different from poly(3HB) and most probably also different from poly(3HB-co-3HV) will be in the future produced for special non-bulk application and possibly also for some niche applications. For bulk applications, PHAs must be cheap. The production costs for PHAs were recently estimated for processes using microbial high cell density cultivations at a large scale [12,140]. Production costs can be only really low and competitive with current bulk plastics, if PHAs are produced in transgenic plants. The costs may be similar low as those for sucrose, starch or plant

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