artigo steinbuchel

Biochemical Engineering Journal 16 (2003) 81–96

Review

Metabolic engineering and pathway construction for biotechnologicalproduction of relevant polyhydroxyalkanoates in microorganisms

Alexander Steinbüchel∗, Tina Lütke-EverslohInstitut für Mikrobiologie der Westfälischen Wilhelms-Universität, Corrensstraße 3, D-48149 Münster, 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 insolubleinclusions in the cytoplasm for storage of carbon and energy. PHAs are considered for various technical applications due to interestingphysical and material properties. In order to establish economically feasible biotechnological production systems and to obtain PHAs fromcheap carbon sources with a preference from renewable resources, CO2 or residual materials, efforts are undertaken to engineer novelpathways in recombinant prokaryotic and eukaryotic organisms. This requires transfer of a PHA synthase structural gene, expression of anenzymatically active PHA synthase protein and in particular engineering of pathways that provide this key enzyme of PHA synthesis withsuitable substrates at sufficient concentrations. Only if all three aspects are well considered, a functional active PHA biosynthesis pathwaywill be expressed allowing PHA biosynthesis from central intermediates and therefore biotechnological production from renewable carbonsources or even CO2. This review will focus on the engineering of pathways resulting in the formation of PHAs containing 3-hydroxyvalericacid, 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 livingmatter. According to their chemical structures, the follow-ing classes of biopolymers can be distinguished: (i) nucleicacids, (ii) polyamides, (iii) polysaccharides, (iv) polyox-oesters, (v) polythioesters, (vi) polyanhydrides, (vii) poly-isoprenoides, and (viii) polyphenols[1]. This review willfocus on polyhydroxyalkanoates, PHA, from prokaryoticmicroorganisms, which represent the most important groupof natural polyoxoesters beside polymalic acid from eukary-otic microorganisms and cutin and suberin from plants[1].

PHAs comprise a rather large class of biopolymers withpoly(3-hydroxybutyric acid), poly(3HB), having most prob-ably the largest abundance and having studied in mostdetail. Poly(3HB) occurs as insoluble cytoplasmic inclu-sions exclusively in many eubacteria and also in someextremely halophilic archaea as storage compound forcarbon and energy; it can contribute up to about 90%(w/w) of the cellular dry mass[2–4]. In addition, it oc-

∗ Corresponding author. Tel.:+49-251-833-9821;fax: +49-251-8338388.E-mail address:[email protected] (A. Steinbüchel).

curs in prokaryotes and also in eukaryotes as so-calledcomplex-poly(3HB) contributing to only a very minor frac-tion of the cell mass. The functions of complex-poly(3HB)have not been revealed, yet[5]. Beside poly(3HB), storagepolyesters consisting of other hydroxyalkanoic acids asconstituents were also detected. Since a detailed review onPHA constituents in 1995[6] many additional interestingnew constituents have been detected. It is not in the scopeof this review to refer to all these new constituents; how-ever, at present approximately 150 different constituentsoccurring in PHAs alone as hompolyesters or in combina-tion as copolyesters are known. Biosynthesis of all thesePHAs is possible due to PHA synthases exhibiting extraor-dinary broad substrate ranges. Poly(3-hydroxybutyrate)and other PHAs consisting of short-carbon-chain lengthhydroxyalkanoic acids (HASCL) comprising three to fivecarbon atoms are distinguished from PHAs consistingof medium-carbon-chain-length hydroxyalkanoic acids(HAMCL) comprising six or more carbon atoms. It shouldbe emphasized that by employing the PHA synthase fromRalstonia eutropha, structurally related biopolymers con-taining mercaptoalkanoic acids as constituents were recentlyalso obtained. These sulfur-containing polythioesters withthioester 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

82 A. Steinbüchel, T. Lütke-Eversloh / Biochemical Engineering Journal 16 (2003) 81–96

backbone were synthesized if the cells were cultivatedwith 3-mercaptopropionic acid, 3-mercaptobutyric acid or3,3′-thiodipropionic acid as carbon sources[7–9].

PHAs share physical and material properties which rec-ommend them for applications in various areas. They arethermoplastic and/or elastomeric, insoluble in water, enan-tiomeric pure, non-toxic, biocompatible, piezoelectric andexhibit a high degree of polymerization and molecularweights of up to several million Da[4,10]. By far the mostimportant feature is the biodegradability of PHAs[11].Since poly(3HB), 3HB containing copolyesters and someother PHAs can be produced from renewable resources,they are considered as an alternative to non-biodegradableplastics produced from fossil oils. Intensive research onbiotechnological production of PHAs by high cell densitycultivation of microorganisms was performed[12,13], andseveral companies persued the development of processes forbiotechnological production of PHAs[14,15]. Meanwhile,many other applications for example in medicine and phar-macy are also investigated[16]. PHAs served certainly alsoas a model to develop other biodegradable polymers forplastic materials which can be either chemically producedor by a combination of biotechnological and syntheticprocesses (for review see[17]).

2. The key enzymes of PHA biosynthesis: PHAsynthases

The committed steps of the various PHA biosynthesispathways are catalyzed by PHA synthases. The naturalsubstrates of this key enzyme are coenzyme A thioestersof (R)-hydroxyalkanoic acid with the hydroxyl group atposition 3, 4, 5 or 6 of the acyl moiety, with various carbonchain length and also with a large variety of substituents[6]. In PHA accumulating cells, PHA synthases are boundto the surface of the PHA granules[18] together with otherproteins from which the phasins[19,20] and specific regu-lator proteins are probably most important and interesting[21–23]. Since biosynthesis of PHAs is independent froma template and since the processicity of the enzyme ishigh, polydisperse products with relatively high molecularweights 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 revealeddue to homology searches in prokaryotic genome sequencedata banks[25–27]. According to their subunit composition(only PhaC, or PhaC together with PhaE) and their substratespecificity (resulting in HASCL or HAMCL incorporation, re-spectively), three different well studied classes of PHA syn-thases (I, II and III) are distinguished. The PHA synthasesof R. eutropha, Pseudomonas aeruginosaand Allochro-matium vinosumrepresenting classes I, II, or III, respec-tively, are currently studied in much detail. From all PHAsynthases investigated so far, only the enzyme fromBacil-lus megateriumseems to be different and might represent

another class[28]. Site-directed mutagenesis of PHA syn-thases has revealed much knowledge regarding the bio-chemistry of PHA synthases, and much progress has beenmade to understand the catalytic mechanism of this poly-merase[29–34]. PHA synthases belong to the�/� hydro-lase superfamily possessing a catalytic triad comprising ahighly conserved cysteine from a ‘lipase box’, an asparticacid and a histidine[35]. The knowledge on PHA synthasesand efficient screening systems are now utilized to obtainmodified PHA synthases with new properties or enhancedin vivo or in vitro PHA synthase activity by in vivo randommutagenesis, in vitro site-directed mutagenesis and geneshuffling [36–38].

3. The necessity for pathway construction andmetabolic engineering

With relatively few exceptions most PHAs are only avail-able if precursor substrates are provided as carbon sourcesto the cells during microbial fermentations. This is becausethe number of potential PHA synthases substrates that canbe synthesized from common intermediates of the centralmetabolism is limited in naturally occurring PHA accumu-lating microorganisms.

3.1. Precursor carbon sources

Therefore, the unusual chemical structures of most knownPHA constituents, except poly(3HB) and a few others, cannot be produced from simple carbon sources or even CO2.The characteristics of precursor substrates are the structuralrelatedness of their carbon skeletons to those of the hydrox-yalkanoic acid to be incorporated into PHAs and that theposition of the hydroxyl group of the constituent is alreadydirectly or indirectly contained in the structure of the carbonsource. For example, the hydroxyl group may be replacedby an amino or a keto group, or a keto group may be pre-dictably incorporated by�-oxidation during the catabolismof the carbon source. Therefore, the cells do not need tosynthesize an unusual hydroxyacyl-coenzyme A thioester assubstrate for the PHA synthase from a central intermedi-ate; instead, the PHA substrate is directly formed during thecatabolism of the structurally related carbon source. How-ever, these precursor substrates are generally more expensivethan renewable resources, and they are often also toxic. Thelatter prevents that this precursor carbon source can be pro-vided at a high concentration in the medium, and thereforemore sophisticated process engineering for efficient PHAproduction during fermentation is required[39].

As a consequence, naturally occurring chemolithoau-totrophic bacteria such asR. eutrophaand photoautotrophicbacteria such as the anoxygenic phototrophic or thecyanobacteria, which were considered as suitable candidatesfor biotechnological production of PHAs from CO2 in thepast [40,41], are unsuitable for production of most PHAs

A. Steinbüchel, T. Lütke-Eversloh / Biochemical Engineering Journal 16 (2003) 81–96 83

not representing poly(3HB) from CO2. The same is truefor naturally occurring plants. On the other side, plants areconsidered as most suitable candidates for sustainable andeconomically feasible production of PHAs. Therefore, gen-eration of transgenic plants synthesizing poly(3HB) basedon heterologous expression of theR. eutrophaPHA biosyn-thesis genes or other PHAs by expression of other PHAsynthases plus additional foreign genes is currently beingintensively persued. Successful efforts to obtain poly(3HB)synthesis in transgenic plants has been already described 10years ago[42]. This achievement opened new perspectivesfor production of PHAs by agriculture[43,44].

3.2. Problems to be addressed during establishmentand engineering of functional active PHA biosynthesispathways

The inflow of nutrients and the outflow of products in anyliving cell are linked by a complex network of biochemicalreactions comprising cyclic and branched pathways[45]. IfPHA biosynthesis is to be established in a putative produc-tion organism, many aspects have to be considered (Fig. 1):(i) PHA synthase must be transferred into the consideredorganism and (ii) and must be functionally expressed, i.e.transcription and translation of the gene(s) must occurat sufficient rates resulting in the formation of an activeenzyme protein. Evidence for a post-transcriptional mod-ification of PHA synthase protein could not be confirmed[31] and seems not to be relevant. Formation of an activePHA synthase seems also not essentially dependant fromthe presence of phasins[20]; the latter may only modulatethe specific activity, if they have an influence at all[46].Expression of an enzymatically active PHA synthase seemsto be the easiest part and was successfully demonstrated formany different PHA synthase genes in many different het-erologous hosts[1]. The most difficult problem is to providethe PHA synthase in the cells with sufficient concentra-tions of substrate. It may be already a problem to obtainsimply sufficient (R)-3HB-CoA for poly(3HB) synthesis inparticular eukaryotic organisms; synthesis of (R)-HA-CoAthioesters for other PHAs is generally even more diffi-cult. (iii) Therefore, new pathways must be constructedwhich allow the withdrawal of central intermediates and the

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

conversion of the latter to a hydroxyacyl-CoA thioester thatcan be used by a PHA synthase as substrate. Only a few cen-tral pathways can be utilized for this[47]. (iv) Furthermore,these pathways must allow a sufficiently high flux of theseintermediates towards the PHA synthase. Metabolic fluxanalysis will be very helpful[48]. Only then a functional ac-tive pathway is available that allows biosynthesis and accu-mulation of a particular PHA from simple carbon sources oreven CO2. Choice of genes to establish a new pathway andattempts to modulate the flux must consider the physiologyof the organisms in which biosynthesis of a particular PHAis to be established. Elementary mode analysis as employedto establish poly(3HB) biosynthesis and accumulation inrecombinantSaccharomyces cerevisiae[49] might be veryhelpful. (v) If PHA has to occur in a eukaryotic organismlike a plant, further problems must be addressed. The func-tional active pathway must reflect the complex eukaryoticcell structure and also the different tissues if a higher eu-karyotic organism like a plant is engineered. The enzymesof the engineered pathway must be expressed in a suitablecompartment and organell and also in a suitable tissue[43,44]. (vi) Finally, and if all previously mentioned aspectshave been successfully addressed, the genetic informationmust be stably maintained in the production organism.

3.3. In vitro engineering and PHA biosynthesis

In principal metabolic pathways can be engineered orconstructed in vivo and in vitro. In vitro PHA biosynthesiscan be achieved using quite different polymerizing enzymesand different substrates. Enzymes that have been shown tosynthesize PHAs in vitro are PHA synthases, various lipases,proteinase K and also PHA depolymerase. However, onlyPHA synthases catalyze polymerization under physiologicalconditions. Successfully employed substrates were mainlyvarious lactones, other cyclic monomers, dicarboxylic es-ters, dicarboxylic acid derivatives plus glycols and acidanhydride derivatives (for review see[50,51]). If a polymer-izing enzyme is used, which catalyzes biosynthesis under‘physiological’ conditions, and if this enzyme is combinedwith additional enzymes that synthesize the substrates of thepolymerizing enzyme, more or less short pathways can beengineered (for review see[1]). These pathways should alsocontain enzymes which recycle the coenzyme A releasedby the PHA synthase and other coenzymes involved[52].Otherwise, coenzyme A has to be applied in stoichiometricamounts resulting in extraordinary high costs; in addition,polymerization will be inhibited due to the accumulation ofcoenzyme A released from the substrate during polymeriza-tion by the PHA synthase. The currently in vitro engineered‘pathways’ are far away from being suitable for biotech-nological production of PHAs; nevertheless sufficientlylarge amounts of PHA material can be obtained to revealsome basic properties of the polyesters such as meltingpoint, glass transition temperature, crystallinity, etc. Also,non-natural compounds can be applied as substrates of the


PHA synthase. Another advantage is that the functionalityof such pathways may be investigated and confirmed withina relatively short period before more time consuming in vivometabolic engineering is done. In one example (see below)an in vitro engineered pathway was successfully expressedin a functional active form inEscherichia coli[53].

4. Poly(3HB-co-3HV)

Biosynthesis of poly(3HB-co-3HV) requires beside 3HB-CoA also 3-hydroxyvaleryl-CoA, 3HV-CoA. The latteris also required if other copolyesters containing 3HV orpoly(3HV) homopolyester are to be synthesized. 3HV-CoAis obtained from condensation of acetyl-CoA and propionyl-CoA to 3-ketovaleryl-CoA and subsequent reduction of thecondensation product to 3HV-CoA. These two reactions arecatalyzed by�-ketothiolases and acetoacetyl-CoA reduc-tases, respectively. The�-ketothiolase BktB ofR. eutrophaand the acetoacetyl-CoA reductase of theR. eutrophaPHAbiosynthesis operon catalyze for example these reactions.It is important to know that the�-ketothiolase PhaA en-coded by the PHA biosynthesis operon ofR. eutrophadoesnot catalyse this reaction[54]. Whereas acetyl-CoA is anobligate central intermediate occurring in any organismand under any physiological condition, this is not the casefor propionyl-CoA, which is only synthesized under spe-cial physiological conditions and from only few substrates.Therefore, processes aiming at the biosynthesis of, e.g.poly(3HB-co-3HV) require formation and occurrence ofpropionyl-CoA in the cells. In the past, several exogenousand endogenous propionigenic substrates have been utilizedfor biosynthesis of this polyester (Fig. 2).

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

4.1. From propionic acid

The first example of poly(3HB-co-3HV) biosynthesisoccurring in an axenic bacterial culture is based on theuse of propionic acid as additional carbon source whenImperial Chemical Industries (ICI) in United Kingdom de-veloped a biotechnological process for production of thiscopolyester employing a strain ofR. eutropha[15,55]. Al-though propionic acid is the most widely used ‘precursorsubstrate’ for poly(3HB-co-3HV) biosynthesis, it has twomajor disadvantages: firstly, propionic acid is more expen-sive than simple carbon sources like for example glucose.Secondly, propionic acid is a highly toxic compound; thisfeature is utilized when propionic acid is applied as a con-servative in food. For this reason propionic acid must beco-fed at relatively low concentrations in order to avoid toohigh concentrations in the medium. In addition, propionicacid is not only converted to 3HV, it is also catabolizedto pyruvic acid or succinyl-CoA, which are intermediatesof the central metabolism. The methylcitric acid cycle(MCC) and the methylmalonyl-CoA pathway, respectively,are the major pathways initiating complete oxidation ofpropionic acid in aerobic bacteria (for overview see[53]).Consequently, a 2-methylcitric acid synthase mutant ofBurkholderia sacchari, a bacterium catabolizing propionicacid via the MCC and used for biotechnological productionof poly(3HB-co-3HV) in Brazil [57], accumulated PHAswith a higher 3HV content[58].

Propionic acid is converted to propionyl-CoA by dif-ferent enzymes (Fig. 2). Enzymes from the type of fattyacid thiokinases such as propionyl-CoA synthetase (PrpE)from Salmonella entericaserovar Typhimurium[59] or anacetyl-CoA synthetase (AcoE)[59,60] from R. eutrophaor


various coenzyme A transferases such as the propionyl-CoAtransferase fromClostridium propionicum [61,62] cancatalyze this conversion. Some of these enzymes wereused to engineerE. coli strains suitable for production ofpoly(3HB-co-3HV) from propionic acid alone or in combi-nation with other carbon sources. Propionate utilization andextent of 3HV incorporation in recombinantE. coli express-ing the PHA biosynthesis operon ofR. eutrophadependedstrongly on the expression of the synthetase[63,64].

4.2. From other aliphatic fatty acids

Aliphatic fatty acids with a higher carbon chain length andan odd number of carbon atoms like valeric acid, heptanoicacid, nonanoic acid, etc. are also propionigenic substratesbecause propionyl-CoA instead of acetyl-CoA remains af-ter the last turn of the�-oxidation cycle (Fig. 2). In addi-tion, intermediates of the�-oxidation cycle with five carbonatoms of the acyl chain may be converted into (R)-3HV ifthe respective enzymes are present (see below). Therefore,bacteria possessing a PHASCL synthase usually synthesizepoly(3HB-co-3HV) if cultivated on the fatty acids mentionedabove.

4.3. From levulinic acid

For bacteria possessing a PHASCL synthase levulinicacid (4-ketovaleric acid) is structurally the most closelyrelated precursor substrate that is available for biosynthesisof polyesters containing 4-hydroxyvaleric acid (4HV) asconstituent. PHAs accumulated by bacteria when cultivatedon levulinic acid usually contain beside 4HV also 3HBand 3HV as further constituents[65]. Since levulinic acidis a relatively cheap carbon source, which could be madeabundantly available from renewable resources throughchemical conversion[66], much efforts were undertakento scale up fermentation processes yielding 4HV contain-ing PHAs [67]. The presence of 3HB and 3HV indicatesthat intermediates of the ‘upper’ part of the levulinic acidcatabolic pathway, which has not be studied in detail, arefurther degraded by�-oxidation or enzymes catalyzing sim-ilar reactions releasing propionyl-CoA beside acetyl-CoA.The induction of the enzymes of the methyl citric acidcycle, which converts propionyl-CoA to pyruvate, duringcultivation on levulinic acid for example inR. eutrophaisconsistent with the formation of propionyl-CoA (Fig. 2,[54,68]).

4.4. From pentanol

The facultative methylotrophic bacteriumParacoccusdenitrificanssynthesized and accumulated poly(3HV) ho-mopolyester during cultivation onn-pentanol as sole carbonsource[69]. This alkane is oxidized via valeraldehyde to va-leric acid and subsequently converted to valeryl-CoA. Theauthors suggested that 3-ketovaleryl-CoA, which is formed

during�-oxidation, is reduced to (R)-3-hydroxyvaleryl-CoAand is subsequently polymerized.

4.5. From amino acids

The catabolism of some amino acids is another impor-tant source of propionyl-CoA. Valine, isoleucine, threo-nine and methionine are therefore precursor substrates for3HV containing PHAs because they are catabolized viapropionyl-CoA (Fig. 2). The use of these amino acids as pre-cursor substrates during fermentation will be certainly notfeasible from an economic point of view due to their highcosts and from process engineering due to the low solubilityof some of them. However, intracellular generation or over-production of these amino acids or related intermediateswith subsequent degradation allows intracellular generationof propionyl-CoA from renewable resources and ammonia.

One first example for this was a spontaneous revertantto prototrophy of an isoleucine-auxotrophic mutant ofR.eutropha. This mutant overexpressed the enzyme aceto-lactate synthase in order to compensate for a defectivethreonine 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 carbonsource was provided in excess, poly(3HB-co-3HV) was ac-cumulated from various simple carbon sources without theneed to fed propionic acid[70]. It was concluded that themethyl-branched 2-ketofatty acids corresponding to the re-spective amino acids were intracellularly overproduced andconsecutively degraded to propionyl-CoA in the cells underconditions permissive for PHA accumulation (Fig. 2).

Another important example for establishing poly(3HB-co-3HV) biosynthesis from an unrelated carbon source wasbased on another interesting approach applied to plants.Poly(3HB-co-3HV) production inArabidopsis thalianaandBrassica napuswas achieved by expressing the threninedeaminase gene fromE. coli (ilvA) beside other genes al-ready described above[71]. Theonine deaminase convertsthreonine to 2-ketobutyric acid, which is then oxidized anddecarboxylated to propionyl-CoA by the pyruvate dehy-drogenase of the plants to propionyl-CoA (Fig. 2). Thelatter is converted to (R)-3HV-CoA by the bktB-encoded�-ketothiolase B and thephaB-endoded acetoacetyl-CoAreductase ofR. eutrophaand subsequently copolymerizedwith 3HB-CoA to poly(3HB-co-3HV) by thephaC-encodedPHA synthase also fromR. eutropha.

4.6. From citric acid cycle via methylmalonyl-CoApathway

In addition to the methylcitric acid cycle, the coenzymeB12-dependent methylmalonyl-CoA pathway is another im-portant pathway for the catabolism of propionyl-CoA de-rived from propionic acid or other propionigenic carbonsources to succinyl-CoA (for overview see[56]). Since the


enzymes of this pathway are reversible, the pathway pro-vides the possibility to link via the citric acid cycle renew-able resources and CO2 with biosynthesis of 3HV containingPHAs (Fig. 2).

The only wild-type bacteria, which naturally synthe-size poly(3HB-co-3HV) with very high contents of 3HV(>75 mol%) from unrelated carbon sources like glucoseand which have been investigated in detail, are variousspecies belonging to the Gram-positive generaNocardiaorRhodococcus[72–74]. These bacteria degrade glucose viathe 2-keto-3-deoxy-6-phosphogluconate pathway to pyru-vate; the latter is probably carboxylated to oxaloacetateand subsequently converted to succinyl-CoA by reversereactions of the citric acid cycle. Succinyl-CoA is then con-verted via methylmalonyl-CoA to propionyl-CoA (Fig. 2)as revealed forN. corallina and R. ruber [74–76]. Thesebacteria provide a very good example for endogenous gen-eration of propionyl-CoA from simple carbon sources. Thisbecame also obvious by the high content of odd-numberedfatty acids that occur in the triacylglycerols, which area second important storage compound of these bacteria.Employing inhibitors of the fatty acid de novo synthesis(cerulenin) and of the�-oxidation/�-ketothiolase (acrylicacid) the competition of the enzymes of PHA biosynthesisenzymes and TAG biosynthesis as well as the influenceof these inhibitors on the availability of 3HB-CoA and3HV-CoA for the PHA synthase could be nicely demon-strated. Whereas cerulenin caused a decrease of the molar3HV content in the accumulated copolyester, acrylic acidcaused an increase of 3HV versus 3HB[74].

An interesting pathway for poly(3HB-co-3HV) biosyn-thesis was recently engineered in recombinantS. enter-ica serovar Typhimurium[77]. The authors expressed thegenes for (2R)-methylmalonyl-CoA mutase (sbm) and a(2R)-methylmalonyl-CoA decarboxylase (ygfG) from E.coli in this Salmonellastrain, thus enabling the conversionof succinyl-CoA to propionyl-CoA. In addition, they in-serted into the gene for 2-methylcitric acid synthase (prpC)of this bacterium the PHA biosynthesis operon ofActi-nobacter(phaCAB) thereby establishing PHA biosynthesisand disrupting propionate utilization. This recombinantSalmonellasp. synthesized poly(3HB-co-3HV) with up to31 mol% 3HV via succinyl-CoA (Fig. 2) if the cells weresimply cultivated on glycerol as carbon source.

5. Poly(3HAMCL)

A polyester consisting mainly of 3-hydroxyoctanoic acidis synthesized byPseudomonas oleovoransand was thefirst example for PHAs consisting of medium-chain-lengthhydroxyalkanoic acids, PHAMCL obtained from an axenicculture [78]. Meanwhile it is obvious that all pseudomon-ads sensu strictu with only a few exceptions are capableto synthesize poly(3HAMCL) not only when they are culti-vated on various aliphatic alkanes or aliphatic fatty acids

but also from glucose and many other structurally unrelatedcarbon sources[79,80]. For example,Psuedomonas putidaaccumulates during cultivation on octanoic acid as carbonsource a copolyester consisting of (R)-3-hydroxyoctanoicacid and (R)-3-hydroxyhexanoic acid as main and minorconstituents, respectively, whereas with gluconate as carbonsource a copolyester consisting of (R)-3-hydroxydecanoateas main constituent and (R)-3-hydroxydodecanoate and(R)-3-hydroxyoctanoate as minor constituents is accumu-lated. This seems to be an inherent feature of the speciesof this genus. This capability implies that the fatty acidmetabolism of these bacteria, i.e. fatty acid de novo biosyn-thesis and fatty acid�-oxidation, is linked with PHA biosyn-thesis (Fig. 3). Meanwhile, several different metabolic linksbetween fatty acid metabolism and PHAMCL biosynthe-sis have been revealed (see below). Knowledge of thesemetabolic links and the circumstance that fatty acid de novosynthesis and�-oxidation occur in almost any organismwere utilized to establish PHAMCL biosynthesis in variousnon-PHA producing organisms.

5.1. From alkanes

P. oleovoransutilizes various aliphatic alkanes as carbonsource. Oxidation of for example octane proceeds via oc-tanol and octaldehyde to octanoic acid and is catalyzed byalkane hydroxylase, alcohol dehydrogenase and aldehydedehydrogenase which are mostly membrane bound and en-coded by the OCT plasmid[81,82]. The resulting octanoicacid is converted by a thiokinase to octanoyl-CoA, which isthen oxidized by fatty acid�-oxidation to acetyl-CoA. In-termediates of the�-oxidation pathway are obviously alsowithdrawn and not degraded to acetyl-CoA but convertedto (R)-3-hydroxyoctanoyl-CoA and subsequently polymer-ized by one of two or both PHAMCL synthases detected inthis bacterium if the cultivation conditions are permissivefor PHA biosynthesis[83]. Details are described below (seeSection 5.3).

The oxidation of octane and other alkanes and its biocon-version to octanoic acid is achieved by two-liquid phase fer-mentation. It is also of interest that theP. oleovoransalkaneoxidizing system was functionally expressed in other bacte-ria like P. putidaandE. coli [78].

5.2. From acyl alcohols

Instead of octane alson-octanol or other acyl alcohols canbe utilized byP. oleovoransand some other pseudomonads.After oxidation by a dehydrogenase, the resulting octanoicacid is metabolized as mentioned above.

5.3. From fatty acids

For routine analysis in the laboratory it is convenientthat instead of aliphatic alkanes also the corresponding fattyacids are utilized by PHAMCL accumulating pseudomonads.


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

This enabled fermentation in only one phase and allowsPHA biosynthesis, which is independent from the alkaneoxidizing enzyme system. Furthermore, it was shown, thatthe carbon chain length of the 3-hydroxyalkanoic acids in-corporated into PHAs is related to the carbon chain lengthof the fatty acid used as carbon source[84]. Furthermore,this opened the perspective to use fatty acids from oils andfats of plants or from animal sources as carbon source forbiotechnological production of PHAMCl , and therefore fromagricultural resources and residual products[85].

As mentioned above (seeSection 5.1) the fatty acids areconverted to the corresponding acyl-CoA thioesters and ox-idized by fatty acid�-oxidation via trans-2-enoyl-CoA and(S)-3-hydroxyacyl-CoA to 3-ketoacyl-CoA which is cleavedby a �-ketothiolase to acetyl-CoA and acyl-CoA compris-ing two less carbon atoms as compared to the acyl-CoAthat entered the first cycle. Further cycles follow until theoriginal acyl-CoA is completely converted to acetyl-CoAin case of fatty acids having an even number of carbonatoms or acetyl-CoA plus propionyl-CoA having an oddnumber of carbon atoms. This is the case for saturated andnon-substituted aliphatic fatty acids. If the fatty acid con-tains functional groups, alkyl side chain as branches ornon-saturated carbon to carbon bonds, the degradative path-way 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 synthesisand accumulation of PHAMCL, the fatty acids are not com-pletely degraded to acetyl-CoA, and intermediates of the

�-oxidation cycle are partially or completely withdrawn andconverted into these polyesters. However, none of the inter-mediates is accepted as substrate by the PHAMCL synthases.Therefore, a metabolic link is required which converts oneof the intermediates into (R)-3-hydroxyacyl-CoA. Threedifferent enzyme activities establish this link (Fig. 3). Fora particular organisms capable to convert fatty acids intoPHAMCL it has to be investigated which enzyme is relevant.

One candidate are epimerases converting the (S)-isomerof 3-hydroxyacyl-CoA into the (R)-isomer. When PHAMCLbiosynthesis was established for the first time in a recom-binantE. coli, a PHAMCL synthase fromP. aeruginosawasexpressed in thefadB mutant LS1298 ofE. coli. Whereasthe dehydrogenase function of FadB is defective in thismutant, the epimerase and the hydratase function remainedactive. It was concluded that (S)-3-hydroxyacyl-CoA accu-mulates in the cytoplasm of this mutant to a sufficiently highlevel allowing effective conversion of the (S)-stereoisomerinto the (R)-stereoisomer and subsequent formation ofPHAMCL [86,87]. Addition of acrylic acid, which isan inhibitor of �-ketothiolases, to the medium allowedphaCMCL-dependant PHAMCL accumulation also inE. colistrains with intactfadB [88] demonstrating that like in thefadBmutant routing of (S)-3-hydroxyacyl-CoA to PHAMCLsynthesis occurred. Others achieved stable PHAMCL pro-duction inE. coli by inserting a PHAMCL synthases fromP.oleovoransinto the chromosome of afadBmutant[89].

Another candidates are hydratases converting trans-2-enoyl-CoA into (R)-3-hydroxyacyl-CoA. In Aeromonascaviaea (R)-specific enoyl-CoA hydratase encoded byphaJ


is responsible for the conversion of trans-2-enoyl-CoA to(R)-3-hydroxyacyl-CoA during the cultivation of this bac-terium on, e.g. hexanoate[90]. Two (R)-specific enoyl-CoAhydratase genes were also identified inP. aeruginosa[91].

Further candidates are enzymes exhibiting 3-ketoacyl-CoAreductase activity and which reduce 3-ketoacyl-CoA to(R)-3-hydroxyacyl-CoA. Studies in recombinant strains ofE. coli provided evidence that FabG of fatty acid de novosynthesis, which is a 3-ketoacyl-ACP reductase, is unspe-cific and exhibits also activities with the correspondingCoA-thioesters and is therefore capable to provide this link[92,93]. On the other side it was shown, that recombinantstrains ofE. coli could also use their own�-ketothiolase(FadA) in combination with the acetoacetyl-CoA reductase(PhaB) of R. eutrophafor conversion of fatty acids intopoly(3HAMCL) [94].

5.4. From glucose and other non-fatty acid substrates

Finding of PHAMCL biosynthesis from substrates likeglucose or gluconate in pseudomonads indicated particip-itation of fatty acid de novo synthesis[79,80]. Detailedstudies confirmed that this route contributes to the byfar major extent to PHAMCL biosynthesis in these bacte-ria [95,96]. One of the most interesting question was themetabolic link between fatty acid de novo and PHAMCLbiosynthesis. However, beside fatty acid de novo synthesis,also other routes contributed to PHAMCL synthesis to a mi-nor extent but could not be neglected[95,96]. Knowledgeof these routes and the availability of the relevant genesof the metabolic link enabled metabolic engineering to ob-tain organisms suitable for biotechnological production ofPHAMCL from carbohydrates.

5.4.1. Chain elongationProvision of substrates for PHAMCL synthases by chain

elongation of acyl-CoA derived from fatty acids used ascarbon sources or from cell lipids through�-oxidation aresignificant but contribute to only a minor fraction of thetotal constituents of PHA accumulated in the cells ofP.putida [96]. With respect to PHAMCL biosynthesis it isprobably only relevant for organisms, which overproducealso other cell components consisting of fatty acids. In thiscase,�-ketothiolase mediated chain elongation may indi-rectly contribute to some extent to PHAMCL biosynthesis.

5.4.2. De novo fatty acid biosynthesis and PhaGAs outlined above, clear experimental evidence was ob-

tained that the fatty acid de novo synthesis pathway is themajor pathway for provision of the 3-hydroxyacyl moietiesin P. putidacontributing to approximately 90% of the con-stituents of the accumulated PHAMCL [95,96] in P. putidaand probably also in all other pseudomonads. The missinglink between fatty acid de novo synthesis and PHAMCL syn-thesis is provided by an acyltransferase which inP. putida[97], P. aeruginosa[98], Pseudomonassp. 61-3[99] and

other pseudomonads[100] transfers the hydroxyacylmoietyfrom (R)-3-hydroxydecanoyl-acyl carrier protein to coen-zyme A thus forming (R)-3-hydroxydecanoyl-CoA, which isa substrate of the two PHAMCL synthases in this bacterium(Fig. 3). This key enzyme is encoded by thephaG gene,and it links fatty acid de novo synthesis and poly(3HAMCL)biosynthesis in these bacteria. InP. oleovoransthe genefor the acyltransferase is cryptic explaining why this bac-terium is unable to synthesize PHAMCL from carbohydrates[100]. In one study, it was demonstrated that the contentof PHAMCL obtained from gluconate as carbon source inP. putida could be increased if the isocitrate lyase gene(aceA) in this bacterium was disrupted by transposon inser-tion [101].

In subsequent experiments,phaG from P. putida wastransferred to and expressed inP. oleovoranswhich is ableto synthesize poly(3HAMCL) from alkanes or fatty acidsbut not from gluconate or fructose and exhibits thereforea similar phenotype as thephaGmutants ofP. putida(seeabove). Establishment ofphaG-mediated PHAMCL biosyn-thesis could be demonstrated in recombinantP. oleovorans[102]. Furthermore,phaGfrom P. aeruginosawas togetherwith the PHA synthase gene fromP. aeruginosatrans-ferred to the non-PHA accumulating bacteriumP. fragi, andboth enzymes were heterologously expressed in this host(Fig. 7)[102]. When the recombinant strains were cultivatedwith gluconate as carbon source under conditions permis-sive for the accumulation of PHAs, the cells accumulatedpoly(3HAMCL) contributing to approximately 50% of thecell dry matter in the case ofP. oleovoransand 10% in thecase ofP. fragi, respectively, whereas in the parent strainor in a recombinant strain harboring only the vector, PHAswere not detected. These two studies demonstrated that thecloned transacylase is functionally active and can be usedto establish poly(3HAMCL) synthesis in other bacteria.

5.4.3. Thioesterase mediated pathwayGenetically engineeredfad mutants of E. coli (see

Section 5.3) expressing foreign acyl–acyl carrier protein(ACP) thioesterases in addition to a class II PHA synthasesynthesized and accumulated small amounts of PHAMCLfrom gluconate[103,104]. In these strains the fatty acidsrequired for other cell constituents were obviously partiallyreleased as free fatty acid during fatty acid de novo synthe-sis and then directed to fatty acid�-oxidation from whichintermediates were withdrawn and converted to a PHAMCLsynthase substrate. Whereas in one study a thioesterase fromthe plantUmbellularia californicaplus a PHAMCL synthasefrom P. aeruginosawere used[103], the other study usedthe cytosolic thioesterase I (encoded bytesA) of E. coli plusa PHAMCL synthase fromP. oleovorans[104]. Althoughthe amounts of accumulated PHAMCL were low, these stud-ies established a new PHAMCL biosynthesis pathway inE.coli and showed a new strategy how to obtain PHAMCLfrom unrelated carbon sources and in particular from car-bohydrates. Application of this strategy will certainly yield


higher PHAMCL contents of the cells, if the affinity towardsthe substrates or the activity of the thioesterase could beincreased and if the metabolic flow rate from acetyl-CoAtowards fatty acids could be increased.

5.5. PHAMCL biosynthesis in recombinant eukaryoticorganisms

PHAMCL synthases from pseudomonads were also suc-cessfully applied to establish PHAMCL biosynthesis intransgenicA. thaliana[105] as well as in the yeastsS. cere-visae [106] and Pichia pastoris[107]. In these eukaryoticorganism it was necessary to pay special attention to thecompartimented lipid metabolism.

6. PHAs containing 4HB and poly(4HB)homopolyester

Several bacteria possessing a PHASCL synthase are capa-ble to incorporate 4-hydroxybutyric acid (4HB) into PHAs.However, the incorporation of 4HB strongly depends onthe use of precursor substrates as carbon sources (Fig. 4).This is also true for all other PHAs that contain non-3HAconstituents, which are normally only synthesized if precur-sor substrates are used. No wild-type strain has so far beendescribed which synthesizes 4HB-containing PHAs fromunrelated carbon sources. However, there is a reasonableperspective that 4HB containing PHAs can be also obtainedfrom simple carbon sources in the future employing engi-neered organisms, because a few important central interme-diates of the metabolism can be converted into 4HB-CoA.

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

Intensive efforts are going on to achieve biotechnologicalproduction of poly(4HB) from cheap carbon sources becausethis polyester has interesting properties[108]. Furthermore,poly(4HB) is not only hydrolyzed by PHA depolymerasesbut also by lipases and esterases since no alkyl side chainsoccur as pendant groups attached to the polyester backbone[109,110]. Therefore, these polyesters are considered forbiomedical and also pharmaceutical applications[16].

6.1. From 4-hydroxybutyric acid

PHAs containing 4HB as constituents were discoveredwhenR. eutrophawas cultivated on 4-hydroxybutyric acidas carbon source[111,112]. Later, biosynthesis of suchPHAs was also achieved inComamonas acidovorans[113],Hydrogenophaga pseudoflava[114], Ralstonia metallidu-rans (formerly R. eutropha) [115] and in many other bacte-ria possessing a PHASCL synthase. After uptake in the cells,4-hydroxybutyric acid is converted into 4HB-CoA eitherby a transferase or a thiokinase. HB-CoA is then used as asubstrate by the PHASCL synthase ofR. eutropha(Fig. 4).However, in most cases the bacteria incorporated also 3HBand therefore synthesized poly(3HB-co-4HB) copolyesters,at least if the accumulated PHAs contributed to a higherfraction of the total cellular dry matter. Incorporationof the comonomer 3HB resulted from the catabolism of4-hydroxybutyric acid leading to intermediates from which3-hydroxybutyryl-CoA was synthesized. The catabolism of4HB was studied in detail inR. eutrophastrain H16; themain catabolic pathway for 4HB is probably via succinicacid semialdehyde and succinic acid catalyzed by 4HB de-hydrogenase and succinic acid semialdehyde dehydrogenase


[116,117]. Most other precursor substrates for 4HB are firstconverted into 4HB-CoA.

When a 4-hydroxybutyric acid-CoA:CoA transferase genefrom Clostridium kluyveri(OrfZ) and theR. eutrophaH16PHA synthase gene were expressed inE. coli, and when therecombinant strain was cultivated in a mineral salts mediumor LB complex medium containing 4-hydroxybutyric acidplus glucose as carbon sources, a poly(4HB) homopolyesterwas synthesized and accumulated[118,119]. Poly(4HB) ho-mopolyester was also synthesized from 4-hydroxybutyricacid and accumulated in a recombinant strain ofC. acidovo-ranscontaining additional copies of its PHA synthase gene[113].

6.2. Fromγ-butyrolactone

It was shown that�-butyrolactone is another suitableprecursor carbon source for biosynthesis of 4HB con-taining PHAs in R. eutrophaand some other bacteria[111,112,114,116]. The lactone is hydrolytically cleaved to4-hydroxybutyric acid. Esterases or lactonases may catalyzethis step (Fig. 4). One esterase (EstA) capable to cleave�-butyrolactone was recently cloned fromR. metalliduransstrain CH34[115]. The resulting 4HB is than converted to4HB-CoA as mentioned above (seeSection 6.1) but alsocatabolized further, again resulting in the incorporation ofalso 3HB. A three-step cultivation scheme was found forH. pseudoflavawhich allowed biosynthesis of poly(4HB)homopolyester from�-butyrolactone [114]; the authorsobtained evidence that under these cultivation conditionscatabolism of 4-hydroxybutyric acid to acetyl-CoA couldnot occur due to a very low activity of the 4HB-DH whichinitiates the degradation of 4HB.

6.3. From 1,4-butanediol and otherω-alkanediols

Another suitable precursor substrate is 1,4-butanediol[111,120]. This �-alkanediol is oxidized in two subse-quent enzymatic reactions to 4-hydroxybutyric acid whichis converted to 4HB-CoA as described above (Fig. 4, seeSection 6.1). �-Alkanediols with a greater carbon chainlength but an even number of carbon atoms are also suit-able precursor substrates[120]. They are obviously alsofirst oxidized to the corresponding�-hydroxyfatty acid,which is then converted into to an coenzyme A thioesterand subjected to�-oxidation until 4HB-CoA occurs. Since4HB-CoA is in contrast to 3HB-CoA not a chiral interme-diate, it can be directly polymerized by the PHA synthase.

6.4. From 4-chlorobutyric acid

Another suitable precursor for 4HB containing PHAsand R. eutrophaH16 is 4-chlorobutyric acid[114]. Thepathway of this compound has not been studied inR.eutropha. 4-Chlorobutyric acid is probably converted to4-hydroxybutyric acid by a haloalkane dehalogenase which

employs a hydrolytic mechanism (for review see[121]).The product is than converted to 4HB-CoA as mentionedabove (Fig. 4, seeSection 6.1).

6.5. From glucose

An interesting approach was used to engineer a novelpathway to produce 4HB containing PHAs inE. coli fromglucose [122]. A recombinant strain ofE. coli, whichexpressed succinic acid semialdehyde dehydrogenase,4HB-DH and 4-hydroxybutyric acid-CoA:CoA transferasegene fromC. kluyveriin addition to the PHA synthase fromR. eutropha, synthesized from glucose poly(3HB-co-4HB)containing up to 2 mol% 4HB (Fig. 4). Although the molarfraction of 4HB was very low, this study provided clear ev-idence that the citric acid cycle intermediate succinyl-CoAcan be converted via succinic acid semialdehyde and4-hydroxybutyric acid to 4HB-CoA in a genetically en-gineered organism (Fig. 4). If the metabolic flux fromsuccinyl-CoA to 4HB-CoA could be increased, the contentof 4HB in the copolyester should increase.

6.6. From amino acids

The engineered pathway described above (seeSection 6.5)could be extended to other interesting carbon sources andintermediates of metabolism. It was shown that glutamicacid and�-aminobutyric acid can be converted to 4HB con-taining PHAs in recombinant strains ofE. coli if a glutamicacid:succinic acid semialdehyde transaminase (gabT) fromE. coli and a glutamic acid decarboxylase (gadA) from E.coli or A. thalianawere expressed inE. coli (Fig. 4, [123]).Although the molar fractions of 4HB in the accumulatedpolyesters were again relatively low, this study demonstratedthat two amino acids, which are also synthesized by manybacteria and plants, can be converted in such PHAs. Thesestudies demonstrated the usefullness of enzymes which arein strictly anaerobic bacteria involved in the fermentation of4HB and glutamate[124,125]. It is now necessary to over-produce these amino acids from glucose and ammonium inthe recombinantE. coli strains.

7. Poly(3HASCL-co-3HAMCL)

Biosynthesis of PHAs, in which 3HASCL and 3HAMCLare covalently linked in the same polyester molecules, i.e.real copolyesters of 3HASCL and 3HAMCL requires thepresence of a PHA synthase exhibiting a substrate rangecombining those of PHASCL and PHAMCL synthases. Itrequires of course also the provision of this PHASMCL syn-thase with coenzyme A thioesters of 3HAs with a carbonchain length ranging from SCL to MCL. Simultaneouspresence of a PHASCL synthase plus a PHAMCL syn-thase will only result in the formation of a blend of poly-(3HASCL) and poly(3HAMCL) rather than in the formation


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

of poly(3HASCL-co-3HAMCL) (Fig. 5). This was shown fora recombinant strain ofP. oleovoransexpressing beside itsown class II PHA synthase the class I PHA synthase ofR.eutropha[126,127].

The same will certainly be true for any other copolyestercontaining constituents that are incorporated into PHAs byPHA synthases having an excluding substrate range, for ex-ample one for X-HA-CoA and the other for Y-HA-CoA. Aslong as two different PHA synthases are present, the oneusing X-HA-CoA and the other Y-HA-CoA, blends of twodifferent types of PHAs will be synthesized. Only if the cellspossess one PHA synthase that uses X-HA-CoA as well asY-HA-CoA, a true copolyester is synthesized.

First evidences for the existence of PHA synthases suit-able for production of poly(3HASCL-co-3HAMCL) wereobtained when the PHA synthase cloned fromThiocapsapfennigii was cloned and heterologously expressed inPHA-negative mutants ofR. eutrophaand P. putida. Un-der suitable cultivation conditions and from fatty acids, therecombinant strains synthesized copolyesters of 3HB with3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoicacid (3HO); copolyesters of 3HB with non-3HAMCL couldbe also obtained[128]. There are only a few wild-typebacteria which synthesize and accumulate poly(3HASCL-co-3HAMCL). Strains ofA. caviae[129] and Aeromonas hy-drophila [130] suitable for production of PHAs consistingof poly(3HB-co-3HHx) were most interesting besidePseu-domonassp. 61-3 from which copolyesters of 3HB with3HAMCL of various chain length could be obtained[131].These strains are studied in detail and possess PHA syn-thases with an unusual broad substrate range.

Recently, it was found that the PHASCL or class I synthaseof R. eutrophais not exclusively restricted to HASCL-CoAthioesters as substrates. Although this was the conclusionobtained from in vitro measurements of the enzyme activity[18], theR. eutrophaPHA synthase could be forced in vivo toincorporate also 3HAMCL (from C6 to C12) beside 3HB intoPHA copolyesters[132–134]. For this cells ofR. eutrophahad to cultivated on fatty acids (e.g. octanoic acid) as carbonsource in the presence of the�-ketothiolase inhibitor acrylicacid [132], or its own genes for acetoacetyl-CoA reductase

and PHA synthase were expressed in a PHA-negative mu-tant [133]. Alternatively, a recombinant strain ofE. coli ex-pressing the PHA synthase ofR. eutrophawas cultivatedon fatty acids (e.g. octanoic acid, decanoic acid, dodecanoicacid) as carbon source[134].

Bacterial systems suitable for biosynthesis of poly(3HB-co-3HAMCL) and for biotechnological production of thesecopolyesters are now intensively studied and engineered be-cause they exhibit interesting material properties[135,136].

8. A new versatile composed, non-natural PHAbiosynthesis pathway

A new, non-natural pathway comprising the butyrate ki-nase (Buk) and phosphotransbutyrylase (Ptb) fromClostrid-ium acetobutylicum, which are in this anaerobic bacteriuminvolved in butyric acid formation[137], and the PHAsynthase fromT. pfennigiior A. vinosumwas recently con-structed (Fig. 6). In previous experiments, it was shown thatthe two enzymes fromC. acetobutylicumexhibited a lowsubstrate specificity not only using the substrates occurringduring butyric acid synthesis but also substrates relevant forpoly(3HB) biosynthesis or biosynthesis of other interesting

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


PHAs. Therefore, these enzymes used substrates structurallysignificantly different from the natural substrates. The puri-fied enzymes were then combined (Fig. 6) and successfullyexploited for in vitro synthesis of a wide range of differenthomo- and copolyesters demonstrating that this engineeredpathway was quite versatile[138]. Depending on the pre-cursor carbon sources used, various homo- and copolyestersof 3HB, 4HB and 4HV were obtained. The genes for thesethree enzymes were then functionally expressed inE. coli,and in vivo synthesis of PHAs very similar to those syn-thesized in vitro were obtained[53]. One advantage ofthis E. coli system is the possibility to produce poly(4HB)or poly(4HV) homopolyesters because�-ketothiolase andacetoacetyl-CoA reductase were not active under these con-ditions; therefore 3HB could not be synthesized. Later it wasshown that this engineered pathway synthesizes not onlythe PHAs mentioned above but also polythioesters (TinaLütke-Eversloh and Alexander Steinbüchel, unpublishedresults). Therefore, this pathway is rather versatile.

Part of this metabolic pathway, i.e. the phosphotransbu-tyrylase and the butyrate kinase fromC. acetobutylicumwere used in reverse direction as above together with the�-ketothiolase (PhaA) and the acetoacetyl-CoA reductase(PhaB) fromR. eutrophato engineer a pathway enabling re-combinantE. coli to produce (R)-3-hydroxybutyrate. Whena recombinant strain ofE. coli harboringphaA, phaB, ptband buk, which functionally expressed the correspondingenzymes in the absence of a PHA synthase, was cultivatedin the presence of glucose as carbon source, it excreted(R)-3-hydroxybutyrate into the medium to a concentrationof up to 35 g/l[139].

9. Outlook and further perspectives

Since cloning of the first PHA synthase gene, many sig-nificant achievements and breakthroughs were made fornovel processes for biotechnological production of PHAsduring the last years. It is expected that one or the othertype of PHA will be commercially produced in the near fu-ture for technical applications. Any production system willmost probably rely on genetically engineered organisms.High cell density cultivations with bacteria were much im-proved[12], and bacteria are at present the best and almostonly source for PHAs consisting not only of 3HB. Dueto various constrains, we personally assume that bacterialPHAs different from poly(3HB) and most probably alsodifferent from poly(3HB-co-3HV) will be in the future pro-duced for special non-bulk application and possibly also forsome niche applications. For bulk applications, PHAs mustbe cheap. The production costs for PHAs were recentlyestimated for processes using microbial high cell densitycultivations at a large scale[12,140]. Production costs canbe only really low and competitive with current bulk plas-tics, if PHAs are produced in transgenic plants. The costsmay be similar low as those for sucrose, starch or plant

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

Acknowledgements

During the last 15 years the research of the correspond-ing author’s laboratory on PHAs was generously supportedby the Bundesministerium für Bildung und Forschung, theBundesministerium für Verbraucherschutz, Ernährung undLandwirtschaft, the Deutsche Forschungsgemeinschaft, theDeutscher Akademischer Austauschdienst, the Fonds derChemischen Industrie, the Max-Buchner Forschungsstiftungand various partners from the chemical industry. This finan-cial support enabled detailed basic and applied research onthe subject, gave many undergraduate and graduate studentsas well as PostDocs and guest scientists a perspective andis gratefully acknowledged.

References

[1] A. Steinbüchel, Perspectives for biotechnological production andutilization of biopolymers: metabolic engineering of polyhydroxyal-kanoate biosynthesis pathways as a successful example, Macromol.Biosci. 1 (2001) 1–24.

[2] A.J. Anderson, E.A. Dawes, Occurrence, metabolism, metabolicrole, and industrial uses of bacterial polyhydroxyalkanoates,Microbiol. Rev. 54 (1990) 450–472.

[3] Y. Doi, Microbial Polyesters, first ed., VCH Publishers, New York,1990.

[4] J. Hocking, R.H. Marchessault, Biopolyesters, in: G.F.L. Griffin(Ed.), Chemistry and Technology of Biodegradable Polymers,Chapman and Hall, London, 1994, pp. 48–96.

[5] R.N. Reusch, Non-storage poly(R)-3-hydroxyalkanoates (complexedPHAs) in prokaryotes and eukaryotes, in: Y. Doi, A. Steinbüchel(Eds.), Biopolymers, vol. 3a, Wiley/VCH, Weinheim, 2002,pp. 123–171.

[6] A. Steinbüchel, H.E. Valentin, Diversity of bacterial polyhydro-xyalkanoic acids, FEMS Microbiol. Lett. 128 (1995) 219–228.

[7] T. Lütke-Eversloh, K. Bergander, H. Luftmann, A. Steinbüchel,Identification of a new class of biopolymer: bacterial synthesis ofa sulfur-containing polymer with thioester linkages, Microbiol. UK147 (2001) 11–19.

[8] T. Lütke-Eversloh, K. Bergander, H. Luftmann, A. Steinbüchel,Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) asa sulfur analogue to poly(3-hydroxybutyrate) (PHB), Biomacro-molecules 2 (2001) 1061–1065.

[9] T. Lütke-Eversloh, J. Kawada, R.H. Marchessault, A. Steinbüchel,Characterization of microbial polythioesters: physical properties ofnovel copolymers synthesized byRalstonia eutropha, Biomacro-molecules 3 (2002) 159–166.

[10] H.M. Müller, D. Seebach, Poly(hydroxyfettsäureester), eine fünfteKlasse von physiologisch bedeutsamen organischen biopolymeren?Angew. Chem. 105 (1993) 483–509.

[11] D. Jendrossek, A. Schirmer, H.G. Schlegel, Biodegradation ofpolyhydroxyalkanoic acids, Appl. Microbiol. Biotechnol. 46 (1996)451–463.


[12] S.Y. Lee, S.J. Park, Fermentative production of SCL-PHAs, in:Y. Doi, A. Steinbüchel (Eds.), Biopolymers, vol. 3a, Wiley/VCH,Weinheim, 2002, pp. 263–290.

[13] R.A. Weusthuis, B. Kessler, M.P.M. Dielissen, B. Witholt, G.Eggink, Fermentative production of medium-chain-length poly(3-hydroxyalkanoate), in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers,vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 291–316.

[14] J. Asrar, K.J. Gruys, Biodegradable polymer (Biopol®), in: Y. Doi,A. Steinbüchel (Eds.), Biopolymers, vol. 3a, Wiley/VCH, Weinheim,2002, pp. 53–90.

[15] D. Byrom, Production of poly-�-hydroxybutyrate: poly-�-hydro-xyvalerate copolymers, FEMS Microbiol. Lett. 103 (1992) 247–250.

[16] S.F. Williams, D.P. Martin, Applications of PHAs in medicine andpharmacy, in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers, vol. 3a,Wiley/VCH, Weinheim, 2002, pp. 91–127.

[17] A. Steinbüchel, New degradable resins, in: H.J. Rehm, G. Reed, A.Pühler, P. Stadler (Eds.), Biotechnology, second ed., Wiley/VCH,Weinheim, 2001, pp. 423–438.

[18] G.W. Haywood, A.J. Anderson, E.A. Dawes, The importanceof PHB-synthase substrate specificity in polyhydroxyalkanoatesynthesis inAlcaligenes eutrophus, FEMS Microbiol. Lett. 57(1989) 1–6.

[19] U. Pieper-Fürst, M.H. Madkour, F. Mayer, A. Steinbüchel,Purification and characterization of a 14-kilodalton protein thatis bound to the surface of polyhydroxyalkanoic acid granules inRhodococcus ruber, J. Bacteriol. 176 (1994) 4328–4337.

[20] A. Steinbüchel, K. Aerts, W. Babel, C. Föllner, M. Liebergesell,M.H. Madkour, F. Mayer, U. Pieper-Fürst, A. Pries, H.E. Valentin,R. Wieczorek, Considerations of the structure and biochemistry ofbacterial polyhydroxyalkanoic acid inclusions, Can. J. Microbiol.41 (Suppl. 1) (1995) 94–105.

[21] A. Maehara, Y. Doi, Z. Nishiyama, Y. Takagi, S. Ueda, H. Nakano,T. Yamane, PhaR: a protein of unknown function conserved amongshort-chain-length polyhydroxyalkanoic acids producing bacteria,is a DNA-binding protein and repressesParacoccus denitrificansphaPexpression in vitro, FEMS Microbiol. Lett. 200 (2001) 9–15.

[22] G.M. York, J. Stubbe, A.J. Sinskey, TheRalstonia eutrophaPhaRprotein couples synthesis of the PhaP phasin to the presence ofpolyhydroxybutyrate in cells and promotes polyhydroxybutyrateproduction, J. Bacteriol. 184 (2002) 59–66.

[23] M. Pötter, M.H. Madkour, F. Mayer, A. Steinbüchel, Regulationof phasin expression and polyhydroxyalkanoate (PHA) granuleformation in Ralstonia eutrophaH16, Microbiol. UK 148 (2002)2405–2412.

[24] R.H. Marchessault, K. Okamura, C.J. Su, Macromolecules 3 (1970)735–740.

[25] B.H.A. Rehm, A. Steinbüchel, Biochemical and genetic analysis ofPHA synthases and other proteins required for PHA synthesis, Int.J. Biol. Macromol. 25 (1999) 3–19.

[26] A. Steinbüchel, S. Hein, Biochemical and molecular basis ofpolyhydroxyalkanoic acids in microorganisms, in: A. Steinbüchel,W. Babel (Eds.), Biopolyesters, Adv. Biochem. Eng. Biotechnol.71 (2001) 81–123.

[27] B.H.A. Rehm, A. Steinbüchel, PHA synthases: the key enzymesof PHA synthesis, in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers,vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 173–215.

[28] G.J. McCool, M.C. Cannon, PhaC and PhaR are required forpolyhydroxyalkanoic acid synthase activity inBacillus megaterium,J. Bacteriol. 183 (2001) 4235–4243.

[29] S. Kalousek, D. Dennis, W. Lubitz, Genetic engineering of PHBsynthase fromAlcaligenes eutrophusH16, FEMS Microbiol. Rev.103 (1992) 426–427.

[30] T.U. Gerngross, K.D. Snell, O.P. Peoples, A.J. Sinskey, E. Csuhai,S. Masamune, J. Stubbe, Overexpression and purification of thesoluble polyhydroxyalkanoate synthase fromAlcaligenes eutrophus:evidence for a required post-translational modification for catalyticactivity, Biochemistry 33 (1994) 9311–9320.

[31] A. Hoppensack, B.H.A. Rehm, A. Steinbüchel, Analysis of4-phosphopantetheinylation of polyhydroxybutyrate synthase fromRalstonia eutropha: generation of �-alanine auxotrophic Tn5mutants and cloning of thepanD gene region, J. Bacteriol. 181(1999) 1429–1435.

[32] U. Müh, A.J. Sinskey, D.P. Kirby, W.S. Lane, J. Stubbe, PHAsynthase fromChromatium vinosum: cysteine 149 is involved incovalent catalysis, Biochemistry 38 (1999) 826–837.

[33] S. Taguchi, A. Maehara, K. Takase, M. Nakahara, A. Nakamura,Y. Doi, Analysis of mutational effects of a polyhydroxybutyrate(PHB) polymerase on bacterial PHB accumulation using an in vivoassay system, FEMS Microbiol. Lett. 198 (2001) 65–71.

[34] Y. Jia, W. Yuan, J. Wodzinska, C. Park, A.J. Sinskey, J. Stubbe,Mechanistic studies on class I polyhydroxybutyrate (PHB) synthasefrom Ralstonia eutropha: classes I and III synthases share a similarcatalytic mechanism, Biochemistry 40 (2001) 1011–1019.

[35] Y. Jia, T.J. Kappock, T. Frick, A.J. Sinskey, J. Stubbe, Lipasesprovide a new mechanistic model for polyhydroxybutyrate (PHB)synthases: characterization of the functional residues inChromatiumvinosumPHB synthase, Biochemistry 39 (2000) 3927–3936.

[36] A.A. Amara, A. Steinbüchel, B.H.A. Rehm, In vivo evolution ofthe Aeromonas punctatapolyhydroxyalkanoate (PHA) synthase:isolation and characterization of modified PHA synthase withenhanced activity, Appl. Microbiol. Biotechnol. 59 (2002) 477–482.

[37] T. Kichise, S. Taguchi, Y. Doi, Enhanced accumulation and changedmonomer composition in polyhydroxyalkanoate (PHA) copolyesterby in vitro evolution of Aeromonas caviaePHA synthase, Appl.Environ. Microbiol. 68 (2002) 2411–2419.

[38] D.K.Y. Solaiman, R.D. Ashby, T.A. Foglia, Synthesis ofpoly(hydroxyalkanoates) byEscherichia coli expressing mutatedand chimeric PHA synthase genes, Biotechnol. Lett. 24 (2002)1011–1016.

[39] K. Taguchi, S. Taguchi, K. Sudesh, A. Maehara, T. Tsuge, Y.Doi, Metabolic pathways and engineering of PHA biosynthesis, in:Y. Doi, A. Steinbüchel (Eds.), Biopolymers, vol. 3a, Wiley/VCH,Weinheim, 2002, pp. 217–247.

[40] A. Ishizaki, K. Tanaka, N. Taga, Microbial production ofpoly-d-3-hydroxybutyrate from CO2, Appl. Microbiol. Biotechnol.57 (1991) 6–12.

[41] M. Miyake, K. Takase, M. Narato, E. Khatipov, J. Schnackenberg,M. Shirai, R. Kurane, Y. Asada, Polyhydroxybutyrate productionfrom carbon dioxide by cyanobacteria, Appl. Biochem. Biotechnol.84 (2000) 991–1002.

[42] Y. Poirier, D.E. Dennis, K. Klomparens, C. Sommerville,Polyhydroxybutyrate: biodegradable thermoplastic, produced intransgenic plants, Science 256 (1992) 520–523.

[43] Poirier, Y., Gruys, K.J., Production of polyhydroxyalkanoates intransgenic plants, in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers,vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 401–435.

[44] K.D. Snell, O. Peoples, Polyhydroxyalkanoate polymers and theirproduction in transgenic plants, Metabol. Eng. 4 (2002) 29–40.

[45] L. Rohlin, M.K. Oh, J.C. Liao, Microbial pathway engineeringfor industrial processes: evolution, combinatorial biosynthesis andrational design, Curr. Opin. Microbiol. 4 (2001) 330–335.

[46] G.M. York, B.H. Junker, J. Stubbe, A.J. Sinskey, Accumulation ofthe PhaP phasin ofRalstonia eutrophais dependent on productionof polyhydroxybutyrate in cells, J. Bacteriol. 183 (2001) 4217–4226.

[47] A. Steinbüchel, B. Füchtenbusch, Bacterial and other biologicalsystems for polyester production, Trends Biotechnol. 16 (1998)419–427.

[48] S.Y. Lee, S.H. Hong, S.J. Park, R. van Wegen, A.P.J.Middelberg, Metabolic flux analysis of the production ofpoly(3-hydroxybutyrate), in: Y. Doi, A. Steinbüchel (Eds.),Biopolymers, vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 249–261.

[49] R. Carlson, D. Fell, F. Srienc, Metabolic pathway analysis ofa recombinant yeast for rational strain development, Biotechnol.Bioeng. 79 (2002) 121–134.


[50] S. Kobayashi, H. Uyama, Enzymatic polymerization to polyesters,in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers, vol. 3a, Wiley/VCH,Weinheim, 2002, pp. 373–400.

[51] S. Zhang, R.W. Lenz, S. Goodwin, In vitro biosynthesisof polyhydroxyalkanoates, in: Y. Doi, A. Steinbüchel (Eds.),Biopolymers, vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 353–372.

[52] R. Jossek, A. Steinbüchel, In vitro synthesis of poly(3-hydro-xybutyric acid) by using an enzymatic coenzyme A recyclingsystem, FEMS Microbiol. Lett. 168 (1998) 319–324.

[53] S.-J. Liu, A. Steinbüchel, A novel genetically engineered pathwayfor synthesis of poly(hydroxyalkanoic acids) inEscherichia coli,Appl. Environ. Microbiol. 66 (2000) 739–743.

[54] S. Slater, K.L. Houmiel, M. Tran, T.A. Mitsky, N.B. Taylor,S.R. Padgette, K.J. Gruys, Multiple�-ketothiolaes mediatepoly(�-hydroxyalkanoate) copolymer synthesis inRalstoniaeutropha, J. Bacteriol. 180 (1998) 1979–1987.

[55] P.A. Holmes, L.F. Wright, S.H. Collins, Eur. Pat. Appl. EP 052.459(1981).

[56] S. Textor, V.F. Wendisch, A.A. De Graaf, U. Müller, M.I. Linder,W. Buckel, Propionate oxidation inEscherichia coli: evidence foroperation of a methylcitrate cycle in bacteria, Arch. Microbiol. 168(1997) 428–436.

[57] R.V. Noanto, P.E. Mantelatto, C.E.V. Rossell, Integrated productionof biodegradable plastic, sugar and ethanol, Appl. Microbiol.Biotechnol. 57 (2001) 1–5.

[58] C.O. Brämer, L.F. Silva, J.G.C. Gomez, H. Priefert, A. Steinbüchel,Identification of the 2-methylcitrate pathway involved in thecatabolism of propionate in the polyhydroxyalkanoate-producingstrain Burkholderia sacchariIPT101(T) and analysis of a mutantaccumulating a copolyester with higher 3-hydroxyvalerate content,Appl. Environ. Microbiol. 68 (2002) 271–279.

[59] H.E. Valentin, T.A. Mitsky, D.A. Mahadeo, M. Tran, K.J.Gruys, Application of a propionyl coenzyme A synthetase forpoly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation inrecombinantEscherichia coli, Appl. Environ. Microbiol. 66 (2000)5253–5258.

[60] H. Priefert, A. Steinbüchel, Identification and molecularcharacterization of the acetyl-coenzyme A synthetase gene (acoE)of Alcaligenes eutrophus, J. Bacteriol. 174 (1992) 6590–6599.

[61] G. Schweiger, W. Buckel, On the dehydration of (R)-lactate in thefermentation of alanine to propionate byClostridium propionicum,FEBS Lett. 171 (1984) 79–84.

[62] H.E. Valentin, A. Steinbüchel, Application of enzymaticallysynthesized short-chain-length hydroxy fatty acid coenzyme Athioesters for assay of polyhydroxyalkanoic acid synthases, Appl.Microbiol. Biotechnol. 40 (1994) 699–709.

[63] S. Slater, T. Gallaher, D. Dennis, Production of poly-(3-hydroxy-butyrate-co-3-hydroxyvalerate) in a recombinantEscherichia colistrain, Appl. Environ. Microbiol. 58 (1992) 1089–1094.

[64] H.G. Rhie, D. Dennis, Role offadR and atoC (Con) mutationsin poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis inrecombinantpha+ Escherichia coli, Appl. Environ. Microbiol. 61(1995) 2487–2492.

[65] H.E. Valentin, A. Steinbüchel, Accumulation of poly(3-hydroxy-butyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) bymutants and recombinant strains ofAlcaligenes eutrophus, J.Environ. Polym. Degrad. 3 (1995) 169–175.

[66] J.J. Bozell, L. Moens, D.C. Elliott, Y. Wang, G.G. Neuenscwander,S.W. Fitzpatrick, R.J. Bilski, J.L. Jarnefeld, Production of levulinicacid and use as a platform chemical for derived products, Res.Conserv. Recycl. 28 (2000) 227–239.

[67] V. Gorenflo, G. Schmack, R. Vogel, A. Steinbüchel, Developmentof a process for the biotechnological large-scale production of4-hydroxyvalerate-containing polyesters and characterization oftheir physical and mechanical properties, Biomacromolecules 2(2001) 45–57.

[68] C.O. Brämer, A. Steinbüchel, The methylcitric acid pathway inRalstonia eutropha: new genes identified in propionate metabolism,Microbiol. UK 147 (2001) 2203–2214.

[69] T. Yamane, X.-F. Chen, S. Ueda, Growth-associated productionof poly(3-hydroxyvalerate) fromn-pentanol by a methylotrophicbacterium,Paracoccus denitrificans, Appl. Environ. Microbiol. 62(1996) 380–384.

[70] A. Steinbüchel, U. Pieper, Production of a copolyester of3-hydroxybutyric acid and 3-hydroxyvaleric acid from singleunrelated carbon sources by a mutant ofAlcaligenes eutrophus,Appl. Microbiol. Biotechnol. 37 (1992) 1–6.

[71] S. Slater, T.A. Mitsky, K.L. Houmiel, M. Hao, S.E. Reiser, N.B.Taylor, M. Tran, H.E. Valentin, D.J. Rodriguez, D.A. Stone,S.R. Padgette, G. Kishore, K.J. Gruys, Metabolic engineering ofArabidopsisand Brassica for poly(3-hydroxybutyrate-co-3-hydro-xyvalterate) copolymer production, Nat. Biotechnol. 17 (1999)1011–1016.

[72] G. Haywood, A.J. Anderson, D.R. Williams, E.A. Dawes, D.F.Ewing, Accumulation of a poly(hydroxyalkanoate) copolymercontaining primarily 3-hydroxyvalerate from simple carbohydratesubstrates byRhodococcussp. NCIMB 40126, Int. J. Biol.Macromol. 13 (1991) 83–88.

[73] A.J. Anderson, D.R. Williams, E.A. Dawes, D.F. Ewing,Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) inRhodococcus ruber, Can. J. Microbiol. 41 (Suppl. 1) (1995) 4–13.

[74] H.M. Alvarez, R. Kalscheuer, A. Steinbüchel, Accumulation ofstorage lipids in species ofRhodococcusandNocardiaand effect ofinhibitors and polyethylene glycol, Fett-Lipid 99 (1997) 239–246.

[75] R. Williams, A.J. Anderson, E.A. Dawes, D.F. Ewing, Productionof a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvalericacid from succinic acid byRhodococcus rubber—biosyntheticconsiderations, Appl. Microbiol. Biotechnol. 40 (1994) 717–723.

[76] H.E. Valentin, D. Dennis, Metabolic pathway for poly(3-hydroxy-butyrate-co-3-hydroxyvalerate) formation inNocardia corallina:inactivation of mutB by chromosomal integration of a kanamycinresistance gene, Appl. Environ. Microbiol. 62 (1996) 372–379.

[77] I.S. Aldor, S.-W. Kim, K.L. Jones Prather, J.D. Keasling,Metabolic engineering of a novel propionate-independent pathwayfor the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)in recombinantSalmonella entericaserovar Typhimurium, Appl.Environ. Microbiol. 68 (2002) 3848–3854.

[78] M.J. De Smet, G. Eggink, B. Witholt, J. Kingma, H. Wynberg,Characterization of intracellular inclusions formed byPseudomonasoleovoransduring growth on octane, J. Bacteriol. 154 (1983) 870–878.

[79] G.W. Haywood, A.J. Anderson, D.F. Ewing, E.A. Dawes,Accumulation of a polyhydroxyalkanoate containing primarily3-hydroxydecanoate from simple carbohydrate substrates byPseudomonassp. strain NCIMB 40135, Appl. Environ. Microbiol.56 (1990) 3354–3359.

[80] A. Timm, A. Steinbüchel, Formation of polyesters consisting ofmedium-chain-length 3-hydroxyalkanoic acids from gluconate byPseudomonas aeruginosaand other fluorescent pseudomonads,Appl. Environ. Microbiol. 56 (1990) 3360–3367.

[81] G. Eggink, R.G. Lageveen, B. Altenburg, B. Witholt, Controlledand functional expression of thePseudomonas oleovoransalkaneutilizing system inPseudomonas putidaand Escherichia coli, J.Biol. Chem. 262 (1987) 17712–17718.

[82] O. Favre-Bulle, T. Schouten, J. Kingma, B. Witholt, Bioconversionof N-octane to octanoic acid by a recombinantEscherichia colicultured in a two-liquid phase bioreactor, BioTechnology 9 (1991)367–371.

[83] G.W. Huisman, E. Wonink, R. Meima, B. Kazemier, P. Terpstra,B. Witholt, Metabolism of poly(3-hydroxyalkanoates) (PHAs) byPseudomonas oleovorans, J. Biol. Chem. 266 (1991) 2191–2198.

[84] R.A. Gross, C. DeMello, R.W. Lenz, H. Brandl, R.C. Fuller,Biosynthesis and characterization of poly(�-hydroxyalkanoates)


produced byPseudomonas oleovorans, Macromolecules 22 (1989)1106–1115.

[85] R.D. Ashby, T.A. Foglia, Poly(hydroxyalkanoate) biosynthesis fromtriglyceride substrates, Appl. Microbiol. Biotechnol. 49 (1998) 431–437.

[86] S. Langenbach, B.H.A. Rehm, A. Steinbüchel, Functionalexpression of the PHA synthase gene PhaC1 fromPseudomonasaeruginosain Escherichia coliresults in poly(3-hydroxyalkanoate)synthesis, FEMS Microbiol. Lett. 150 (1997) 303–309.

[87] Q.S. Qi, B.H.A. Rehm, A. Steinbüchel, Synthesis ofpoly(3-hydroxyalkanoates) inEscherichia coliexpressing the PHAsynthase genephaC2 from Pseudomonas aeruginosa; comparisonof PhaC1 and PhaC2, FEMS Microbiol. Lett. 157 (1997) 155–162.

[88] Q.S. Qi, A. Steinbüchel, B.H.A. Rehm, Metabolic routing towardspolyhydroxyalkanoic acid synthesis in recombinantEscherichia coli(fadR): inhibition of fatty acid beta-oxidation by acrylic acid, FEMSMicrobiol. Lett. 167 (1998) 89–94.

[89] M.A. Prieto, M.B. Kellerhals, G.B. Bozzato, D. Radnovic, B.Witholt, B. Kessler, Engineering of stable recombinant bacteriafor production of chiral medium-chain-length poly-3-hydroxy-alkanoates, Appl. Environ. Microbiol. 65 (1999) 3265–3271.

[90] T. Fukui, Y. Doi, Cloning and analysis of the poly(3-hydroxy-butyrate-co-3-hydroxyhexanoate) biosynthesis genes ofAeromonascaviae, J. Bacteriol. 179 (1997) 4821–4830.

[91] T. Tsuge, T. Fukui, H. Matsusaki, S. Taguchi, G. Kobayashi, A.Ishizaki, Y. Doi, Molecular cloning of two (R)-specific enoyl-CoAhydratase genes fromPseudomonas aeruginosaand their use forpolyhydroxyalkanoate synthesis, FEMS Microbiol. Lett. 184 (2000)193–198.

[92] K. Taguchi, Y. Aoyagi, H. Matsusaki, T. Fukui, Y. Doi, Co-expres-sion of 3-ketoacyl-ACP reductase and polyhydroxyalkanoatesynthase genes induces PHA produces inEscherichia coliHB101strain, FEMS Microbiol. Lett. 176 (1999) 183–190.

[93] Q. Ren, N. Sierro, B. Witholt, B. Kessler, FabG, an NADPH-dependent 3-ketoacyl reductase ofPseudomonas aeruginosa,provides precursors for medium-chain-length poly-3-hydroxyal-kanoate biosynthesis inEscherichia coli, J. Bacteriol. 182 (2000)2978–2981.

[94] Q. Ren, N. Sierro, M. Kellerhals, B. Kessler, B. Witholt, Propertiesof engineered poly-3-hydroxyalkanoates produced in recombinantEscherichia colistrains, Appl. Environ. Microbiol. 66 (2000) 1311–1320.

[95] G.N.M. Huijberts, G. Eggink, P. De Waard, G.W. Huisman,B. Witholt, Pseudomonas putidaKT2442 cultivated on glucoseaccumulates poly(3-hydroxyalkanoates) consisting of saturated andunsaturated monomers, Appl. Environ. Microbiol. 58 (1992) 536–544.

[96] G.N.M. Huijberts, T.C. De Rijk, P. De Waard, G. Eggink,13C nuclear magnetic resonance studies ofPseudomonas putidafatty acid metabolic routes involved in poly(3-hydroxyalkanoate)synthesis, J. Bacteriol. 176 (1994) 1661–1666.

[97] B.H.A. Rehm, N. Krüger, A. Steinbüchel, A new metabolic linkbetween fatty acid de novo synthesis and polyhydroxyalkanoicacid synthesis—thephaG gene from Pseudomonas putidaKT2440 encodes a 3-hydroxyacyl-acyl carrier protein coenzyme Atransferase, J. Biol. Chem. 273 (1998) 24044–24051.

[98] N. Hoffmann, A. Steinbüchel, B.H.A. Rehm, ThePseudomonasaeruginosa phaGgene product is involved in the synthesisof polyhydroxyalkanoic acids consisting of medium-chain-lengthconstituents from non-related carbon sources, FEMS Microbiol.Lett. 184 (2000) 253–260.

[99] K. Matsumoto, H. Matsusaki, S. Taguchi, M. Seki, Y. Doi, Cloningand characterization of thePseudomonassp. 61-3 phaG geneinvolved in polyhydroxyalkanoate biosynthesis, Biomacromolecules2 (2001) 142–147.

[100] N. Hoffmann, A. Steinbüchel, B.H.A. Rehm, Homologousfunctional expression of crypticphaG from Pseudomonas

oleovorans establishes the transacylase-mediated polyhydroxy-alkanoate biosynthetic pathway, Appl. Microbiol. Biotechnol. 54(2000) 665–670.

[101] S. Klinke, M. Dauner, G. Scott, B. Kessler, B. Witholt,Inactivation of isocitrate lyase leads to increased production ofmedium-chain-length poly(3-hydroxyalkanoates) inPseudomonasputida, Appl. Environ. Microbiol. 66 (2000) 909–913.

[102] S. Fiedler, A. Steinbüchel, B.H.A. Rehm, PhaG-mediated synthesisof poly(3-hydroxyalkanoates) consisting of medium-chain-lengthconstituents form non-related carbon sources in recombinantPseudomonas fragi, Appl. Environ. Microbiol. 66 (2000) 2117–2124.

[103] B.H.A. Rehm, A. Steinbüchel, Heterologous expression ofthe acyl–acyl carrier protein thioesterase gene from theplant Umbellularia californica mediates polyhydroxyalkanoatebiosynthesis in recombinantEscherichia coli, Appl. Microbiol.Biotechnol. 55 (2001) 205–209.

[104] S. Klinke, Q. Ren, B. Witholt, B. Kessler, Production ofmedium-chain-length poly(3-hydroxyalkanoates) from gluconate byrecombinantEscherichia coli, Appl. Environ. Microbiol. 65 (1999)540–548.

[105] V. Mittendorf, E.J. Robertson, R.M. Leech, N. Kruger,A. Steinbüchel, Y. Poirier, Synthesis of medium-chain-lengthpolyhydroxyalkanoates inArabidopsis thalianausing intermediatesof peroxisomal fatty acid beta-oxidation, Proc. Natl. Acad. Sci.U.S.A. 95 (1998) 13397–13402.

[106] Y. Poirier, N. Erard, J.M.C. Petetot, Synthesis of polyhydro-xyalkanoate in the peroxisome ofSaccharomyces cerevisiaebyusing intermediates of fatty acid beta-oxidation, Appl. Environ.Microbiol. 67 (2001) 5254–5260.

[107] Y. Poirier, N. Erard, J.M.C. Petetot, Synthesis of polyhydro-xyalkanoate in the peroxisome ofPichia pastoris, FEMS Microbiol.Lett. 207 (2002) 97–102.

[108] Y. Saito, Y. Doi, Microbial synthesis and propertiesof poly(3-hydroxybutyrate-co-4-hydroxybutyrate) inComamonasacidovorans, Int. J. Biol. Macromol. 16 (1994) 99–104.

[109] K. Mukai, Y. Doi, Y. Sema, K. Tomita, Substrate specificitiesin hydrolysis of polyhydroxyalkanoates by microbial esterases,Biotechnol. Lett. 15 (1993) 601–604.

[110] K.E. Jaeger, A. Steinbüchel, D. Jendrossek, Substrate specificities ofbacterial polyhydroxyalkanoate depolymerases and lipases: bacteriallipases hydrolyse poly(�-hydroxyalkanoates), Appl. Environ.Microbiol. 61 (1995) 3113–3118.

[111] M. Kunioka, Y. Nakamura, Y. Doi, New bacterial copolyestersproduced in Alcaligenes eutrophusfrom organic acids, Polym.Commun. 29 (1988) 174–176.

[112] Y. Doi, A. Segawa, M. Kunioka, Biosynthesis and characterizationof poly(3-hydroxybutyrate-co-4-hydroxybutyrate) inA. eutrophus,Int. J. Biol. Macromol. 12 (1990) 1012–1106.

[113] K. Sudesh, T. Fukui, K. Taguchi, T. Iwata, Y. Doi, Improvedproduction of poly(4-hydroxybutyrate) byComamonas acidovoransand its freeze-fracture morphology, Int. J. Biol. Macromol. 25 (1999)79–85.

[114] M.H. Choi, S.C. Yoon, R.W. Lenz, Production of poly(3-hydro-xybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydro-xybutyric acid) with subsequent degradation byHydrogenophagapseudoflava, Appl. Environ. Microbiol. 65 (1999) 1570–1577.

[115] S. Song, S. Hein, A. Steinbüchel, Cloning, nucleotide sequenceand primary biochemical characterization of esterase EstA fromRalstonia eutrophaCH34, Biotechnol. Lett. 22 (2000) 443–449.

[116] H.E. Valentin, G. Zwingmann, A. Schönebaum, A. Steinbüchel,Metabolic pathway for biosynthesis of poly(3-hydroxybut-yrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate byAlcaligeneseutrophus, Eur. J. Biochem. 227 (1995) 43–609.

[117] T. Lütke-Eversloh, A. Steinbüchel, Biochemical and molecularcharacterization of a succinate semialdehyde dehydrogenaseinvolved in the catabolism of 4-hydroxybutyric acid inRalstoniaeutropha, FEMS Microbiol. Lett. 181 (1999) 63–71.


[118] S. Hein, B. Söhling, G. Gottschalk, A. Steinbüchel, Biosynthesis ofpoly(4-hydroxybutyric acid) by recombinant strains ofEscherichiacoli, FEMS Microbiol. Lett. 153 (1997) 411–418.

[119] S. Song, S. Hein, A. Steinbüchel, Production of poly(4-hydro-xybutyric acid) by fed-batch cultures of recombinant strains ofEscherichia coli, Biotechnol. Lett. 21 (1999) 193–197.

[120] Y. Doi, A. Segawa, S. Nakamura, M. Kunioka, 1990. Productionof biodegradable copolyesters byAlcaligenes eutrophus, in: E.A.Dawes (Ed.), Novel Biodegradable Microbial Polymers, KluwerAcademic Press, Dordrecht, pp. 37–48.

[121] S. Fetzner, Bacterial dehalogenation, Appl. Microbiol. Biotechnol.50 (1998) 633–657.

[122] H.E. Valentin, D. Dennis, Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinantEscherichia coligrown onglucose, J. Biotechnol. 58 (1997) 33–38.

[123] H.E. Valentin, S. Reiser, K.J. Gruys, Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate andglutamate, Biotechnol. Bioeng. 67 (2000) 291–299.

[124] W. Buckel, Unusual enzymes involved in five pathways of glutamatefermentation, Appl. Microbiol. Biotechnol. 57 (2001) 263–273.

[125] A. Gerhardt, I. Cinkaya, D. Linder, G. Huisman, W. Buckel,Fermentation of 4-aminobutyrate byClostridium aminobutyricum:cloning of two genes involved in the formation and dehydration of4-hydroxybutyryl-CoA, Arch. Microbiol. 174 (2000) 189–199.

[126] A. Timm, D. Byrom, A. Steinbüchel, Formation of blends ofvarious poly(3-hydroxyalkanoic acids) by a recombinant strain ofPseudomonas oleovorans, Appl. Microbiol. Biotechnol. 33 (1990)296–301.

[127] H. Preusting, J. Kingma, G. Huisman, A. Steinbüchel, B.Witholt, Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: different poly(3-hydroxyalkanoates) arestored in separate granules, J. Environ. Polym. Degrad. 1 (1983)45–53.

[128] M. Liebergesell, F. Mayer, A. Steinbüchel, Analysis ofpolyhydroxyalkanoic acid biosynthesis genes of anoxygenic photo-trophic bacteria reveals synthesis of a polyester exhibiting anunusual composition, Appl. Microbiol. Biotechnol. 40 (1993) 292–300.

[129] T. Fukui, Y. Doi, Cloning and analysis of the poly(3-hydro-xybutyrate-co-3-hydroxyhexanoate) biosynthesis genes ofAero-monas caviae, J. Bacteriol. 179 (1997) 4821–4830.

[130] S.H. Lee, D.H. Oh, W.S. Ahn, Y. Lee, J. Choi, S.Y. Lee,Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) byhigh-cell-density cultivation ofAeromonas hydrophila, Biotechnol.Bioeng. 67 (2000) 240–244.

[131] M. Kato, H.J. Bao, C.K. Kang, T. Fukui, Y. Doi, Production ofa novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids byPseudomonassp. 61-3 fromsugars, Appl. Microbiol. Biotechnol. 45 (1996) 363–370.

[132] P.R. Green, J. Kemper, L. Schechtman, L. Guo, M. Satkowski,S. Fiedler, A. Steinbüchel, B.H.A. Rehm, Formation ofshort chain length/medium-chain-length polyhydroxyalkanoatecopolymers by fatty acid�-oxidation inhibitedRalstonia eutropha,BioMacromolecules 3 (2002) 208–213.

[133] D. Dennis, M. McCoy, A. Stangl, H.E. Valentin, Z. Wu,Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) byPHA synthase fromRalstonia eutropha, J. Biotechnol. 64 (1998)177–186.

[134] R.V. Antonio, A. Steinbüchel, B.H.A. Rehm, Analysis of in vitrosubstrate specificity of the PHA synthase fromRalstonia eutropha:formation of novel copolyesters in recombinantEscherichia coli,FEMS Microbiol. Lett. 182 (2000) 111–117.

[135] G.Q. Chen, G. Zhang, S.J. Park, S.Y. Lee, Industrial scaleproduction of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),Appl. Microbiol. Biotechnol. 57 (2001) 50–55.

[136] S.Y. Lee, S.J. Park, Biosynthesis and fermentative production ofSCL-MCL-PHAs, in: Y. Doi, A. Steinbüchel (Eds.), Biopolymers,vol. 3a, Wiley/VCH, Weinheim, 2002, pp. 317–336.

[137] P. Dürre, New insights and novel developments in clostridial ace-tone/butanol/isopropanol fermentation, Appl. Microbiol. Biotechnol.49 (1998) 639–648.

[138] S.-J. Liu, A. Steinbüchel, Exploitation of butyrate kinase andphosphotransbutyrylase fromClostridium acetobutylicumfor the invitro biosynthesis of poly(hydroxyalkanoic acid), Appl. Microbiol.Biotechnol. 53 (2000) 545–552.

[139] H.-J. Gao, W.U. Qiong, G.-Q. Chen, Enhanced production ofd-(−)-3-hydroxybutyric acid by recombinantEscherichia coli,FEMS Microbiol. Lett. 213 (2002) 59–65.

[140] J. Choi, S.Y. Lee, Economic consideration in the productionof poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterialfermentation, Appl. Microbiol. Biotechnol. 53 (2001) 646–649.

artigo steinbuchel

Documents