Bioresource Technology 98 (2007) 2313–2320

Review

The production of polyhydroxyalkanoates in recombinantEscherichia coli

Rui Li, Hanxing Zhang, Qingsheng Qi *

State Key Lab of Microbial Technology, Life Science School, Shandong University, 250100 Jinan, PR China

Received 26 June 2006; received in revised form 25 August 2006; accepted 5 September 2006Available online 9 November 2006

Abstract

Polyhydroxyalkanoates, the natural polyester that many microorganisms accumulate to store carbon and reducing equivalents, havebeen considered as a future alternative of traditional plastic due to their special properties. In Escherichia coli, a previous non-poly-hydroxyalkanoates producer, pathway engineering has been developed as a very powerful approach to set up microbial productionprocess through the introduction of direct genetic changes by recombinant DNA technology. Various metabolic pathways leading tothe polyhydroxyalkanoates accumulation with desirable properties at low-cost and high-productivity have been developed. At the sametime, high density fermentation technology of E. coli provides an efficient polyhydroxyalkanoates production strategy. This reviewfocused on metabolic engineering, fermentation and downstream process aiming to polyhydroxyalkanoates production in E. coli.

� 2006 Elsevier Ltd. All rights reserved.

Keywords: Escherichia coli; Polyhydroxyalkanoates; Metabolic engineering; Polyester; Production

1. Introduction

Polyhydroxyalkanoates are storage compounds for car-bon and energy produced by many eubacteria and somearchaea (Steinbuchel et al., 1995). PHAs can be mainlydivided into three types based on the sizes of the monomersincorporated into the polymer. Short-chain-length PHAs(PHASCL) consist of monomer units of C3–C5; medium-chain-length PHAs (PHAMCL) consist of monomer unitsof C6–C14, and SCL–MCL PHAs (PHASCL–MCL) consistof monomers ranging in size from C3 to C14. PHAs arebiodegradable thermoplastics that have a wide variety ofphysical properties depending on the length of the pendant

0960-8524/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2006.09.014

Abbreviations: PHAs, polyhydroxyalkanoates; PHASCL, Short-chain-length PHAs; PHAMCL, medium-chain-length PHAs; PHASCL–MCL,SCL–MCL PHAs; PHB, polyhydroxybutyrate; 3HB, 3-hydroxybutyrate;3HHx, 3-hydroxyhexanoate; 3HV, 3-hydroxyvaleryrate; 4HB,4-hydroxybutyrate.

* Corresponding author. Tel.: +86 531 88365628; fax: +86 53188565610.

E-mail address: qiqingsheng@sdu.edu.cn (Q. Qi).

groups of the monomer units in the polymer. PHASCL areoften stiff and brittle, whereas PHAMCL are elastomeric innature. PHASCL–MCL copolymers have properties betweenthe two states, depending on the ratio of SCL and MCLmonomers in the copolymer. Particular physical and mate-rial properties of PHAs enable them for applications invarious areas, including medicine and pharmacy. Theyare thermoplastic and/or elastomeric, insoluble in water,enantiomeric pure, non-toxic, biocompatible, piezoelectric,and exhibit a high degree of polymerization and molecularweights of up to several million Da (Steinbuchel and Hein,2001).

The natural metabolic production of PHAs can beclassified into two groups depending on their monomercompositions. The first group, represented by Ralstonia

eutropha (formally Alcaligenes eutrophus), is synthesizedvia acetoacetyl-CoA to 3-hydroxybutyryl-CoA giving riseto polyhydroxybutyrate (PHB). With regard to other con-stituents in PHASCL, such as 3-hydroxyvalerate, additionof precursors or substrates and conversion towards theircorresponding coenzyme A thioester are required. The sec-ond group, represented by Pseudomonas aeruginosa and till

2314 R. Li et al. / Bioresource Technology 98 (2007) 2313–2320

now only found in Pseudomonas, is synthesized via fattyacid metabolism, either fatty acid beta-oxidation or fattyacid de novo biosynthesis (Steinbuchel and Lutke-Eversloh,2003). Since PHAs can be produced from renewableresources, they are often considered as alternatives tonon-biodegradable plastics produced from fossil oils. It isdesirable to find out new and low-cost ways to producePHAs with superior qualities (Aldor and Keasling, 2003)(Table 1).

Many bacteria have been tried for their capability asPHA producers cultured with various substrates. Forinstance, Comamonas testosteroni was found to be ableto produce PHAMCL by utilizing vegetable oil (Thakoret al., 2005). Some strains was found to synthesis PHAsby photosynthesis, such as Nostoc muscorum, Synechocystis

sp. (Wu et al., 2001; Sharma et al., in press). Even so, pro-duction of PHAs employing Escherichia coli has manyunexampled advantages over other bacteria: a variety ofaspects of E. coli including genome have been extensivelystudied. The culturing technology and downstream processare relatively mature and simple.

E. coli is considered an ideal host for the production ofPHAs. PHB; one kind of PHASCL, has been produced inE. coli to about 90% (w/w) of the cellular dry weight bygenetic engineering of the PHB synthase genes (Lee andChang, 1995). The pathway links between fatty acid meta-bolism and PHAMCL biosynthesis were identified inE. coli several years ago (Langenbach et al., 1997; Qiet al., 1997). Copolyesters such as poly(3HB-co-3HV) andpoly(3HB-co-4HB) can also be produced in recombinantE. coli by adding corresponding precursors and engineeringcorresponding metabolic pathways (Rhie and Dennis,1995; Valentin and Dennis, 1997). The successful econom-ically feasible biotechnological production systems requiretransfer of a PHA synthase structural gene, expression ofan enzymatically active PHA synthase protein and in par-

Table 1Comparison of several PHAs production process in recombinant E. coli

Strain Culturemode

PHAs biosynthesisgenes source

Type ofPHAs

Major su

Escherichia

coli XL1-Blue

Fed-batch Alcaligenes latus P(3HB) Glucose

Escherichia

coli

HMS174

Fed-batch Ralstonia eutropha P(3HB) Molasses

Escherichia

coli

GCSC4401

Cell recyclefed-batch

Alcaligenes latus P(3HB) Whey (la

Escherichia

coli XL1-Blue

Fed-batch Alcaligenes latus P(3HB-co-3HV)

Glucoseacid supp

Escherichia

coli XL1-Blue

Fed-batch Ralstonia eutropha,Clostridium kluyveri

P(4HB) Glucose,4-hydrox

Escherichia

coli RS3097Fed-batch Pseudomonas

aeruginosa

PHAMCL Decanoic

ticular engineering of pathways that provide this keyenzyme of PHA synthesis with suitable substrates at suffi-cient concentrations (Steinbuchel and Lutke-Eversloh,2003; Stubbe et al., 2005). Many important factors con-cerning PHA production process have been reviewed(Kessler and Witholt, 2001; Steinbuchel and Hein, 2001;Aldor and Keasling, 2003; Steinbuchel and Lutke-Evers-loh, 2003; Salehizadeh and Van Loosdrecht, 2004). Thisreview, however, only focused on the production of PHAsin recombinant E. coli.

2. Pathway engineering towards the formation

of PHASCL

The PHB biosynthesis pathway of R. eutropha has beenstudied in detail for many years (Schubert et al., 1988). Inthis bacterium, the PHB biosynthetic process is initiatedby the condensation of two acetyl-CoA molecules to pro-duce acetoacetyl-CoA, which is catalyzed by the enzymeb-ketothiolase (phbA). Acetoacetyl-CoA is then reducedto (R)-3-hydroxybutyryl-CoA by the NADPH-dependentacetoacetyl-CoA reductase (phbB). Finally, PHB is synthe-sized with the function of PHB synthase (phbC). Theseenzymes have also been found and studied in several otherPHB-accumulating bacteria (Rehm and Steinbuchel, 1999).Accumulation of other types of PHASCL depends on theavail-ability of different precursors.

Two key genes phbA and phbB, which encode b-keto-thiolase and acetoacetyl-CoA reductase, respectively, asindicated above, are involved in formation of 3-hydroxya-cyl-CoA with short side chain. To reproduce this metabolicpathway in E. coli, both of the genes together with phbC,which encodes PHB synthase, have to be functionallyexpressed in the host. The first metabolic pathway towardsthe PHASCL formation in E. coli was established simply bycloning the whole phb gene operon into E. coli (Schubert

bstrate Cell conc.(g L�1)

PHAcontent(%)

Productivity(g L�1 h�1)

References

194.1 73 4.63 Choi et al.(1998)

39.5 80 1 Liu et al.(1998)

ctose) 194 87 4.6 Ahn et al.(2001)

propioni; oleiclementation

203.1 78.2 2.88 Choi andLee (1999b)

ybutyrate12.6 36 0.07 Song et al.

(1999)

acid 2.6 38 0.06 Qi et al.(1998)

R. Li et al. / Bioresource Technology 98 (2007) 2313–2320 2315

et al., 1988). Since then, many phb gene operons or PHBbiosynthesis genes from various bacteria have been identi-fied and functionally expressed in E. coli, which led toPHASCL formation (Peoples and Sinskey, 1989; Valentinand Steinbuchel, 1993; Tombolini et al., 1995; Hein et al.,1998; Qi and Rehm, 2001; Ramachander and Rawal,2005). Synthesis of PHB can also be realized through fattyacid de novo biosynthesis pathway. By over-expression ofmalonyl-CoA-ACP transacylase gene (fabD), or 3-keto-acyl-ACP synthase III gene (fabH) with Aeromonas caviae

polyhydroxyalkanoate synthase gene (phaCAc), PHB wasobtained (Taguchi et al., 1999b). Transacylating proteins,FabD and FabH, play the role as monomer supplier forPHB biosynthesis.

Biosynthesis of PHB in E. coli is not only a matter ofpathway construction, but is also affected by many otherfactors. For example, acetyl-CoA is an essential centralintermediate, which can directly increase 3-hydroxybuty-ryl-CoA formation and cell growth. By inactivation ofthe pta gene, which encodes a phosphotransacetylase,E. coli will accumulate more PHB than wild type E. coli

(Miyake et al., 2000). Another important factor concerningthis process is NADPH, which is necessary for PHB pro-duction. By knocking out the phosphoglucose isomerase(pgi) gene, more NADPH will be produced from pentosephosphate pathway (PP), and eventually the PHB produc-tion will be enhanced (Kabir and Shimizu, 2003). Trans-ketolase (tkt) was also used to improve the metabolismin non-oxidative pentose phosphate pathway (Jung et al.,2004). Enforcing glucose-6-phosphate dehydrogenase(zwf) and 6-phosphogluconate dehydrogenase (gnd) willincrease the PHB production in E. coli by increasedNADPH (Lim et al., 2002).

PHASCL synthase has a broad substrate specificity,which can incorporate precursors like 3-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, 3-hydroxyvaleryl-CoAas well as 4-hydroxybutyryl-CoA into the end polymer.When the metabolic pathways leading to the formationof 3-hydroxyvaleryl-CoA are available, copolymer poly(3HB-co-3HV) will be formed. This can be simply achievedby adding the precursor substrate–propionate (Slater et al.,1992; Eschenlauer et al., 1996). The 3-HB and 3-HV ratiosin the copolymer can be manipulated by altering the propi-onate concentration and/or the glucose concentration inthe culture (Law et al., 2004). Based on the same idea,copolymer containing 3HP units can also be obtained inthe presence of propionate by co-expression of prpE, whichencodes a propionyl coenzyme A synthetase. Copolymercontaining 4-HB unit can be obtained by co-expressionof orfZ gene, which encodes a 4-hydroxybutyryl-CoAtransferase, from Clostridium kluyveri (Hein et al., 1997;Valentin et al., 2000). No wild type strain has so far beendescribed to synthesize 4HB-containing PHA fromunrelated carbon sources. The metabolic formation of4-hydroxybutyryl-CoA in E. coli and other bacteriahas been reviewed by Steinbuchel and Lutke-Eversloh(2003).

3. Pathway engineering towards the formation of

PHAMCL

PHAMCL are synthesized and accumulated in largeamounts by a variety of Pseudomonas (Steinbuchel et al.,1992). The co-monomer composition of PHAMCL dependsmainly on the carbon source, the cultivation conditions,and the metabolic routes leading to PHA formation (Stein-buchel et al., 1995). Data on PHA synthesis in P. oleovoran

and P. putida, growing on various MCL fatty acids,strongly suggest that 3-hydroxyacyl coenzyme A (acyl-CoA) intermediates of the beta-oxidation route were chan-neled to PHA synthesis. In addition, most Pseudomonads

belonging to this group, except P. oleovorans, are capableof PHAMCL synthesis from acetyl-CoA during cultivationon other simple non-related carbon sources, such as gluco-nate. Thus intermediates or derivatives of the de novo fattyacid biosynthesis are incorporated into PHAMCL.

The first successful metabolic production of PHAMCL inE. coli was achieved by the employment of fatty acid oxida-tion deficient strain, E. coli fadB mutant (Langenbach et al.,1997). The PHAMCL biosynthesis gene operon, includingtwo PHAMCL synthase genes, a PHA depolymerase geneand two proposed regulator genes, from P. aeruginosa

was already assigned in 1992 (Timm and Steinbuchel,1992), however, expression of PHAMCL synthase genescould not lead to the accumulation of PHAMCL in E. coli,though it was supposed that E. coli possessed the necessaryenzyme activities towards the formation of 3-hydroxyacyl-CoA. Until 1997, Steinbuchel group successfully synthesizePHAMCL in E. coli by employing the fadB mutant with thepartially blocked fatty acid oxidation pathway (Langen-bach et al., 1997; Qi et al., 1997). Later, Prieto et al. con-structed a stable PHAMCL producer by cloning the phaC1

on chromosome of the host cell (Prieto et al., 1999). Expres-sion of fabG from E. coli and the rhlG from P. aeruginosa

will further increase PHA production from fatty acid path-way (Park et al., 2002a).

However, the enzymes responsible for channeling thebeta-oxidation intermediates to PHAMCL biosynthesis inE. coli fadB mutant were not fully elucidated at that time.Only recently, two enzymes encoded by yfcX and maoC

have been found to be partially responsible for this (Snellet al., 2002; Park and Yup Lee, 2004). E. coli fadB strainswith an inactivated yfcX gene in the chromosome and inthe presence of PHAMCL synthase gene were unable toproduce PHAMCL from fatty acid. MaoC from E. coli,which is homologous to P. aeruginosa R-specific enoyl-CoA hydratase, was also found to be important for PHAbiosynthesis in E. coli fadB mutant (Park and Lee, 2003).Coexpression of the paaG, paaF, and ydbU genes fromE. coli resulted in as almost double PHA production as thatobtained with E. coli fadB mutant expressing only phaC2

gene (Park and Yup Lee, 2004). Introduction of the phbBgene from R. eutropha causes both PHAMCL content andmonomer composition to change in E. coli (Ren et al.,2000a).

2316 R. Li et al. / Bioresource Technology 98 (2007) 2313–2320

Many genes, which seem to be unrelated to PHAMCL

production, were actually found to be linked with or ableto improve the PHA formation in recombinant E. coli.fabG gene encodes a 3-ketoacyl-ACP reductase, which isa key enzyme in fatty acid de novo biosynthesis pathwayand responsible for the reduction of 3-ketoacyl-ACP to3-hydroxyacyl-ACP. E. coli fadB or fadA mutants express-ing fabG from P. aeruginosa accumulated more PHAMCL

compared with those E. coli carrying phaC alone (Renet al., 2000b). Over-expression of E. coli or P. aeruginosa

3-ketoacyl-ACP reductase gene established the metaboliclink between fatty acid biosynthesis and PHAMCL produc-tion (Taguchi et al., 1999a). But production of PHAMCL

from unrelated carbon source was still at a low level com-pare with the wild type bacteria. Further improvement isrequired, and further gene responsible for this links hasto be investigated.

In addition, manipulated metabolic links betweenfatty acid de novo biosynthesis, fatty acid oxidationand PHAMCL production were established by employingPHAMCL synthase and a thioesterase (Klinke et al., 1999;Rehm and Steinbuchel, 2001). During this process, thio-esterase hydrolyzes acyl-ACPs, producing enhanced intra-cellular levels of free fatty acids, which can then bechanneled into the b-oxidation pathway.

4. Formation of copolyester, poly(HASCL-co-HAMCL)

As discussed above, PHA synthase has broad substratespecificity. PHA synthases from A. caviae, Aeromonashydrophila, Pseudomonas sp. 61-3, P. stuzteri and N. coral-

lina are able to incorporate 3-hydroxyacyl-CoA with bothshort side and medium side chain (Hall et al., 1998; Matsu-saki et al., 1998; Chen et al., 2004). To provide both kindsof precursor for co-polyester biosynthesis in E. coli, meta-bolic pathways leading to the formation of these precursorswere engineered. phaJ, encoding (R)-specific enoyl-CoAhydratase, from Aeromonas, was demonstrated to supply3-hydroxyacyl-CoA of C4–C6 for PHA biosynthesis viabeta-oxidation pathway (Fukui et al., 1998; Lu et al.,2004). Later, it was found that four PhaJs with differentsubstrate specificities from P. aeruginosa were responsiblefor the transfer of enoyl-CoA to 3-hdroxyacyl-CoA (Tsugeet al., 2000, 2003). Recently, it was demonstrated thatfabG, encoding a 3-ketoacyl-ACP reductase, was also ableto convert 3-ketoacyl-CoA to 3-hydroxyacyl-CoA with abroad substrate range. Coexpression of this gene with phaC

in E. coli led to the PHA accumulation with monomercomposition containing C4, C6, C8, and C10 from un-related carbon source (Nomura et al., 2005). When do-decanoic acid plus odd carbon number fatty acid wasprovided, recombinant E. coli strain harboring phaC fromA. hydrophila could produce a terpolymer of Poly(3HB-co-3HV-co-3HHx). orf1 gene of this strain was found to play acritical role in assimilating the 3HV monomer and in regu-lating the monomer fraction in the terpolymer (Park et al.,2001).

Providing R. eutropha phaAB genes and phaC gene fromPseudomonas sp. 61-3 in E. coli fadA and/or fadB mutant,E. coli strains could synthesize PHA consisting of C4, C6,C8, and C10 monomer units (Park and Lee, 2004). Themodified 3-ketoacyl-acyl carrier protein synthase III gene(fabH) at the point of encoding amino acid 87 fromE. coli was found to produce PHAMCL–SCL copolymerfrom glucose when co-expressed with A. caviae or Pseudo-

monas sp. 61-3 PHA synthase gene (Nomura et al., 2004a).Again additional phbA, phbB from R. eutropha were able toenhance the PHASCL–MCL copolymers production fromglucose in recombinant E. coli. The cumulative effect oftwo monomer-supplying pathways and genetically engi-neered enzyme resulted in higher accumulated amountsof PHASCL–MCL copolymer from glucose (Nomura et al.,2004b). This was a representative work of combinationbetween pathway engineering and protein engineering forthe production of desired compounds. Further exampleslike this are expected.

5. Fermentation process

Many efforts have been made for the production ofPHA since the first report appeared in 1994 on fed-batchculturing of E. coli containing the R. eutropha PHB biosyn-thetic genes with glucose in which Lee et al. (1994) achieveda relative high PHB productivity of 2.08 g L�1 h�1. E. coli

harboring the A. latus PHB biosynthesis genes accumu-lated PHB more efficiently than those of harboring the R.

eutropha genes (Choi et al., 1998). With a pH-stat fed-batchculture, a high-productivity of 4.63 g L�1 h�1was obtained.Later, it was found that one of the reasons for the reducedPHB production was severe filamentation of cells. Toovercome this, an essential cell division protein, FtsZ, wasover-expressed in recombinant E. coli, This filamentation-suppressed strain reached a high PHB productivity of3.4 g L�1 h�1 by the pH-stat fed-batch culture (Wang andLee, 1997). This indicated that fermentation optimizationof the natural process in genetic level would lead PHA pro-duction towards a more economical process. Furthermore,improvement of cell respiratory capacity can benefit thebacterial growth and PHA production. This was also con-firmed by expression of Vitreoscilla globin gene (vgb) inE. coli. The expression of vgb can induce the parent promo-tion effect on cell growth and PHB accumulation, especiallyunder low DO conditions during the fermentation process(Yu et al., 2002). arcA mutant, which confers high respira-tory capacity of the host under microaerobic conditions,gave rise to higher polymer accumulation than normalstrains (Nikel et al., 2006).

Production of PHAs from cheap substrates attractedmore attention of biologists. Cheese whey, a co-productof dairy industry, is considered as a suitable substrate forPHAs production (Solaiman et al., 2006). Supplied withwhey as substrate, recombinant E. coli accumulated PHBat a productivity of 4.6 g L�1 h�1 (Wong and Lee, 1998;Ahn et al., 2000, 2001). This process has been realized in

R. Li et al. / Bioresource Technology 98 (2007) 2313–2320 2317

a scale of 300 L fermenter (Park et al., 2002b). Further-more, by limiting the maximum agitation speed and theoxygen, PHB content achieved 80% without removingwhey culture broth (Kim, 2000). Statistical analysis wasalso employed in this area for enhancing production andoffering industrial production more useful information.By changing one or more variables and examining theeffects of cultivation process in E. coli, Nikel et al. obtainedoptimal medium concentrations for maximal biomass andbiopolymer production (Nikel et al., 2005). Molasses, aco-product of crop refining industries, was also used as asubstrate for PHB production by E. coli. Employing beetmolasses as a sole carbon source, recombinant E. coli pro-duced PHB at a productivity of 1 g L�1 h�1. The PHB con-tent in the cell reached 80% (Liu et al., 1998). The use oflow-cost substrates including agricultural feedstock andtheir product could improve the economics of microbialPHA production. Furthermore, the production of high-valuable PHAs with various monomer compositions isimportant.

However, reports on E. coli production of other types ofPHAs were quite few. In 1999, Choi and Lee (1999b) devel-oped fermentation strategies, including improved nutrientfeeding, acetic acid induction, and oleic acid supplementa-tion, for the production of high concentrations of P(3HB-co-3HV) by recombinant E. coli. In 2002, they finishedpilot-scale fed-batch fermentation in 300L fermentor (Choiet al., 2002). At the same time, production of P(4HB) andPHAMCL by recombinant E. coli was also tried. After fed-batch culture in M9 mineral salts medium containingglucose and 4-hydroxybutyric acid as carbon sources,recombinant E. coli accumulated up to 36% of cell dryweight P(4HB), while recombinant E. coli accumulated34% of cell dry weight PHAMCL in a batch fermentationprocess (Qi et al., 1998; Song et al., 1999). But both pro-cesses are not optimized.

6. Downstream processes

It is known that the recovery/purification cost forms ahuge part of the total production cost in bioprocesses forthe production of PHB (Ling et al., 1997b; Jung et al.,2005). A few methods have been reported for this, whichfall into two categories: systems employing chemicals, orother additives, and systems employing the strains whichare able to spontaneously liberate PHAs from cells. Theextraction of PHAs with organic solvents and sodiumhypochlorite were employed at the beginning (Bergeret al., 1989). This method is based on two strategies, PHAssolubilization and non-PHA cell mass (NPCM) dissolu-tion. However, both systems have inherent drawbacks:the former one has been reported to be able to extractPHB with a high purity, but it requires toxic solvents; thelatter one is relatively simpler and more effective at theexpense of the fact that degradation of PHB into a lowermolecular weight. In addition, these methods were not eco-nomically efficient and not designed for E. coli but other

PHAs producers, like R. eutropha. Thus, many less expen-sive chemicals were tried to recover PHAs by digestion ofNPCM. Among these chemicals, NaOH and KOH werefound to be the ideal choices for PHB recovery, becausethey are cheaper and efficient with both high PHB yieldand high purity (Choi and Lee, 1999a). Furthermore,PHB with lower endotoxin level was achieved by NaOHdigestion, which is more suitable for biomedical use (Leeet al., 1999). Recently, a new recovery and purification sys-tem of PHAs by selective dissolution of cell mass wasdeveloped, which was expected to make the cost even lower(Yu and Chen, 2006). An enzymatic digestion of NPCMwas developed to recover PHAs as a non-pollution method(Holmes and Lim, 1990). However, the high cost ofenzymes prevents the further development of this method.

At the same time, development of systems with auto-matic PHAs liberating capabilities began to attract people’sattention. This system was initially and specially developedfor E. coli production. Phage lysis genes were used. In com-bination with the phaCAB genes, expression of lysis gene E

of bacteriophage PhiX174 from plasmid pSH2 was used torelease PHB granules in E. coli. In this method, a thermo-sensitive expression system was used to control the expres-sion of lysis gene E (Resch et al., 1998). In another method,lytic genes of phage k with S amber mutation (S-RRz) andPHB biosynthetic genes (phbCAB) were employed. EDTA/Tris buffer would cause lysis of the PHAs containing cells(Yu et al., 2000). Interestingly, Lae Jung et al., found thatby manipulating the initial inoculum size and the composi-tion of the medium, E. coli strain MG1655/pTZ18U-PHBcould produce PHB and undergo lysis, thereby releasingthe PHB granules (Jung et al., 2005).

In addition, a few physical methods have also beendeveloped as supplement of above systems, or as indepen-dent systems. Coupled with hypochlorite treatment, PHBwas recovered by homogenization and centrifugation fromrecombinant E. coli. High fractional cell debris removal(94%) was achieved with this method (Ling et al., 1997a).This may help manufactory of PHB reducing its recoverycost (Ling et al., 1997b).

7. Conclusion and perspective

Cost effectiveness, one of the main factors, has preventedthe use of PHAs as a biodegradable commodity plastic.Broader use of PHA in packaging and disposable productsas a potential solution to a significant environmental prob-lem depends heavily on further reducing the cost and estab-lishing a novel PHA production strategy. Methods forgenetic engineering in E. coli for improved production havebeen developed. The production cost is also highly sensitiveto fermentation process and recovery strategy, and moder-ately sensitive to medium cost and cell growth yield on glu-cose. By using agro-industrial residues as alternativessubstrates, the cost could be reduced. Further efficient pro-cesses based on lower cost carbon feedstock are neededincludes: improving substrate uptake system of E. coli;

2318 R. Li et al. / Bioresource Technology 98 (2007) 2313–2320

setting up complex metabolic pathways; optimizing thesolution to the balance of carbon, redox and energy withrespect to fermentation and other downstream process.

Acknowledgements

We thank the National Natural Science Foundation ofChina for their financial support (30470049).

References

Ahn, W.S., Park, S.J., Lee, S.Y., 2000. Production of poly(3-hydroxybu-tyrate) by fed-batch culture of recombinant Escherichia coli with ahighly concentrated whey solution. Appl. Environ. Microbiol. 66,3624–3627.

Ahn, W.S., Park, S.J., Lee, S.Y., 2001. Production of poly(3-hydroxy-butyrate) from whey by cell recycle fed-batch culture of recombinantEscherichia coli. Biotechnol. Lett. 23, 235–240.

Aldor, I.S., Keasling, J.D., 2003. Process design for microbial plasticfactories: metabolic engineering of polyhydroxyalkanoates. Curr.Opin. Biotechnol. 14, 475–483.

Berger, E., Ramsay, B.A., Ramsay, J.A., Chavarie, C., Braunegg, G.,1989. PHB recovery by hypochlorite digestion of non-PHB biomass.Biotechnol. Tech. 3, 227–232.

Chen, J.Y., Liu, T., Zheng, Z., Chen, J.C., Chen, G.Q., 2004. Polyhy-droxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas

stutzeri 1317 had different substrate specificities. FEMS Microbiol.Lett. 234, 231–237.

Choi, J., Lee, S.Y., 1999a. Efficient and economical recovery of poly(3-hydroxybutyrate) from recombinant Escherichia coli by simple diges-tion with chemicals. Biotechnol. Bioeng. 62, 546–553.

Choi, J.I., Lee, S.Y., 1999b. High-level production of poly(3-hydroxy-butyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinantEscherichia coli. Appl. Environ. Microbiol. 65, 4363–4368.

Choi, J.I., Lee, S.Y., Han, K., 1998. Cloning of the Alcaligenes latus

polyhydroxyalkanoate biosynthesis genes and use of these genes forenhanced production of poly(3-hydroxybutyrate) in Escherichia coli.Appl. Environ. Microbiol. 64, 4897–4903.

Choi, J. et al., 2002. Pilot scale production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia

coli. Biotechnol. Bioprocess. Eng. 7, 371–374.Eschenlauer, A.C., Stoup, S.K., Srienc, F., Somers, D.A., 1996. Produc-

tion of heteropolymeric polyhydroxyalkanoate in Escherichia coli froma single carbon source. Int. J. Biol. Macromol. 19, 121–130.

Fukui, T., Shiomi, N., Doi, Y., 1998. Expression and characterizationof (R)-specific enoyl coenzyme A hydratase involved in polyhydrox-yalkanoate biosynthesis by Aeromonas caviae. J. Bacteriol. 180,667–673.

Hall, B., Baldwin, J., Rhie, H.G., Dennis, D., 1998. Cloning of theNocardia corallina polyhydroxyalkanoate synthase gene and produc-tion of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate). Can. J. Microbiol. 44,687–691.

Hein, S., Sohling, B., Gottschalk, G., Steinbuchel, A., 1997. Biosynthesisof poly(4-hydroxybutyric acid) by recombinant strains of Escherichia

coli. FEMS Microbiol. Lett. 153, 411–418.Hein, S., Tran, H., Steinbuchel, A., 1998. Synechocystis sp. PCC6803

possesses a two-component polyhydroxyalkanoic acid synthase similarto that of anoxygenic purple sulfur bacteria. Arch. Microbiol. 170,162–170.

Holmes, P.A., Lim, G.B., 1990. Separation process. US Patent 4,910,914.Jung, Y.M., Lee, J.N., Shin, H.D., Lee, Y.H., 2004. Role of tktA gene in

pentose phosphate pathway on odd-ball biosynthesis of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCABoperon. J. Biosci. Bioeng. 98, 224–227.

Jung, I.L., Phyo, K.H., Kim, K.C., Park, H.K., Kim, I.G., 2005.Spontaneous liberation of intracellular polyhydroxybutyrate granulesin Escherichia coli. Res. Microbiol. 156, 865–873.

Kabir, M.M., Shimizu, K., 2003. Fermentation characteristics and proteinexpression patterns in a recombinant Escherichia coli mutant lackingphosphoglucose isomerase for poly(3-hydroxybutyrate) production.Appl. Microbiol. Biotechnol. 62, 244–255.

Kessler, B., Witholt, B., 2001. Factors involved in the regulatorynetwork of polyhydroxyalkanoate metabolism. J. Biotechnol. 86,97–104.

Kim, B.S., 2000. Production of poly(3-hydroxybutyrate) from inexpensivesubstrates. Enzyme Microb. Technol. 27, 774–777.

Klinke, S., Ren, Q., Witholt, B., Kessler, B., 1999. Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombi-nant Escherichia coli. Appl. Environ. Microbiol. 65, 540–548.

Langenbach, S., Rehm, B.H., Steinbuchel, A., 1997. Functional expressionof the PHA synthase gene phaC1 from Pseudomonas aeruginosa inEscherichia coli results in poly(3-hydroxyalkanoate) synthesis. FEMSMicrobiol. Lett. 150, 303–309.

Law, K.H. et al., 2004. Construction of recombinant Escherichia coli

strains for production of poly-(3-hydroxybutyrate-co-3-hydroxyvaler-ate). Appl. Biochem. Biotechnol. 113–116, 361–372.

Lee, S.Y., Chang, H.N., 1995. Production of poly(hydroxyalkanoic acid).Adv. Biochem. Eng. Biotechnol. 52, 27–58.

Lee, S.Y., Yim, K.S., Chang, H.N., Chang, Y.K., 1994. Construction ofplasmids, estimation of plasmid stability, and use of stable plasmidsfor the production of poly(3-hydroxybutyric acid) by recombinantEscherichia coli. J. Biotechnol. 32, 203–211.

Lee, S.Y., Choi, J., Han, K., Song, J.Y., 1999. Removal of endotoxinduring purification of poly(3-hydroxybutyrate) from gram-negativebacteria. Appl. Environ. Microbiol. 65, 2762–2764.

Lim, S.J., Jung, Y.M., Shin, H.D., Lee, Y.H., 2002. Amplification of theNADPH-related genes zwf and gnd for the oddball biosynthesis ofPHB in an E. coli transformant harboring a cloned phbCAB operon.J. Biosci. Bioeng. 93, 543–549.

Ling, Y., Williams, D.R.G., Thomas, C.J., Middelberg, A.P.J., 1997a.Recovery of poly-3-hydroxybutyrate from recombinant Escherichia

coli by homogenization and centrifugation. Biotechnol. Tech. 11,409–412.

Ling, Y., Wong, H.H., Thomas, C.J., Williams, D.R., Middelberg, A.P.,1997b. Pilot-scale extraction of PHB from recombinant E. coli byhomogenization and centrifugation. Bioseparation 7, 9–15.

Liu, F., Li, W., Ridgway, D., Gu, T., Shen, Z., 1998. Production of poly-b-hydroxybutyrate on molasses by recombinant Escherichia coli.Biotechnol. Lett. 20, 345–348.

Lu, X.Y., Wu, Q., Zhang, W.J., Zhang, G., Chen, G.Q., 2004. Molecularcloning of polyhydroxyalkanoate synthesis operon from Aeromonas

hydrophila and its expression in Escherichia coli. Biotechnol. Prog. 20,1332–1336.

Matsusaki, H., Manji, S., Taguchi, K., Kato, M., Fukui, T., Doi, Y., 1998.Cloning and molecular analysis of the poly(3-hydroxybutyrate) andpoly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes inPseudomonas sp. strain 61-3. J. Bacteriol. 180, 6459–6467.

Miyake, M., Miyamoto, C., Schnackenberg, J., Kurane, R., Asada, Y.,2000. Phosphotransacetylase as a key factor in biological produc-tion of polyhydroxybutyrate. Appl. Biochem. Biotechnol. 84–86,1039–1044.

Nikel, P.I., Pettinari, M.J., Mendez, B.S., Galvagno, M.A., 2005.Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strainusing agroindustrial byproducts. Int. Microbiol. 8, 243–250.

Nikel, P.I., Pettinari, M.J., Galvagno, M.A., Mendez, B.S., 2006. Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA

mutants in microaerobiosis. Appl. Environ. Microbiol. 72, 2614–2620.Nomura, C.T., Taguchi, K., Taguchi, S., Doi, Y., 2004a. Coexpression of

genetically engineered 3-ketoacyl-ACP synthase III (fabH) and poly-hydroxyalkanoate synthase (phaC) genes leads to short-chain-length–medium-chain-length polyhydroxyalkanoate copolymer production

R. Li et al. / Bioresource Technology 98 (2007) 2313–2320 2319

from glucose in Escherichia coli JM109. Appl. Environ. Microbiol.70, 999–1007.

Nomura, C.T. et al., 2004b. Effective enhancement of short-chain-length–medium-chain-length polyhydroxyalkanoate copolymer production bycoexpression of genetically engineered 3-ketoacyl-acyl-carrier-proteinsynthase III (fabH) and polyhydroxyalkanoate synthesis genes. Bio-macromolecules 5, 1457–1464.

Nomura, C.T. et al., 2005. Expression of 3-ketoacyl-acyl carrier proteinreductase (fabG) genes enhances production of polyhydroxyalkanoatecopolymer from glucose in recombinant Escherichia coli JM109. Appl.Environ. Microbiol. 71, 4297–4306.

Park, S.J., Lee, S.Y., 2003. Identification and characterization of a newenoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli.J. Bacteriol. 185, 5391–5397.

Park, S.J., Lee, S.Y., 2004. Biosynthesis of poly(3-hydroxybutyrate- co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli

strains. Appl. Biochem. Biotechnol. 113–116, 335–346.Park, S.J., Yup Lee, S., 2004. New FadB homologous enzymes and their

use in enhanced biosynthesis of medium-chain-length polyhydroxyalk-anoates in FadB mutant Escherichia coli. Biotechnol. Bioeng. 86,681–686.

Park, S.J., Ahn, W.S., Green, P.R., Lee, S.Y., 2001. Biosynthesis ofpoly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)by metabolically engineered Escherichia coli strains. Biotechnol.Bioeng. 74, 81–86.

Park, S.J., Park, J.P., Lee, S.Y., 2002a. Metabolic engineering ofEscherichia coli for the production of medium-chain-length polyhy-droxyalkanoates rich in specific monomers. FEMS Microbiol. Lett.214, 217–222.

Park, S.J., Park, J.P., Lee, S.Y., 2002b. Production of poly(3-hydroxybu-tyrate) from whey by fed-batch culture of recombinant Escherichia coli

in a pilot-scale fermenter. Biotechnol. Lett. 24, 185–189.Peoples, O.P., Sinskey, A.J., 1989. Poly-beta-hydroxybutyrate (PHB)

biosynthesis in Alcaligenes eutrophus H16. Identification and charac-terization of the PHB polymerase gene (phbC). J. Biol. Chem. 264,15298–15303.

Prieto, M.A., Kellerhals, M.B., Bozzato, G.B., Radnovic, D., Witholt, B.,Kessler, B., 1999. Engineering of stable recombinant bacteria forproduction of chiral medium-chain-length poly-3-hydroxyalkanoates.Appl. Environ. Microbiol. 65, 3265–3271.

Qi, Q., Rehm, B.H., 2001. Polyhydroxybutyrate biosynthesis in Caulob-

acter crescentus: molecular characterization of the polyhydroxybuty-rate synthase. Microbiology 147, 3353–3358.

Qi, Q., Rehm, B.H., Steinbuchel, A., 1997. Synthesis of poly(3-hydroxy-alkanoates) in Escherichia coli expressing the PHA synthase genephaC2 from Pseudomonas aeruginosa: comparison of PhaC1 andPhaC2. FEMS Microbiol. Lett. 157, 155–162.

Qi, Q., Steinbuchel, A., Rehm, B.H., 1998. Metabolic routing towardspolyhydroxyalkanoic acid synthesis in recombinant Escherichia coli

(fadR): inhibition of fatty acid beta-oxidation by acrylic acid. FEMSMicrobiol. Lett. 167, 89–94.

Ramachander, T.V., Rawal, S.K., 2005. PHB synthase from Strepto-

myces aureofaciens NRRL 2209. FEMS Microbiol. Lett. 242,13–18.

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

Rehm, B.H., Steinbuchel, A., 2001. Heterologous expression of the acyl-acyl carrier protein thioesterase gene from the plant Umbellularia

californica mediates polyhydroxyalkanoate biosynthesis in recombi-nant Escherichia coli. Appl. Microbiol. Biotechnol. 55, 205–209.

Ren, Q., Sierro, N., Kellerhals, M., Kessler, B., Witholt, B., 2000a.Properties of engineered poly-3-hydroxyalkanoates produced inrecombinant Escherichia coli strains. Appl. Environ. Microbiol. 66,1311–1320.

Ren, Q., Sierro, N., Witholt, B., Kessler, B., 2000b. FabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides

precursors for medium-chain-length poly-3-hydroxyalkanoate biosyn-thesis in Escherichia coli. J. Bacteriol. 182, 2978–2981.

Resch, S., Gruber, K., Wanner, G., Slater, S., Dennis, D., Lubitz, W.,1998. Aqueous release and purification of poly(beta-hydroxybutyrate)from Escherichia coli. J. Biotechnol. 65, 173–182.

Rhie, H.G., Dennis, D., 1995. Role of fadR and atoC(Con) mutations inpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombi-nant pha + Escherichia coli. Appl. Environ. Microbiol. 61, 2487–2492.

Salehizadeh, H., Van Loosdrecht, M.C., 2004. Production of polyhydrox-yalkanoates by mixed culture: recent trends and biotechnologicalimportance. Biotechnol. Adv. 22, 261–279.

Schubert, P., Steinbuchel, A., Schlegel, H.G., 1988. Cloning of theAlcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyricacid (PHB) and synthesis of PHB in Escherichia coli. J. Bacteriol. 170,5837–5847.

Sharma, L., Kumar Singh, A., Panda, B., Mallick, N., in press. Processoptimization for poly-beta-hydroxybutyrate production in a nitrogenfixing cyanobacterium, Nostoc muscorum using response surfacemethodology. Bioresour. Technol.

Slater, S., Gallaher, T., Dennis, D., 1992. Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia

coli strain. Appl. Environ. Microbiol. 58, 1089–1094.Snell, K.D., Feng, F., Zhong, L., Martin, D., Madison, L.L., 2002. YfcX

enables medium-chain-length poly(3-hydroxyalkanoate) formationfrom fatty acids in recombinant Escherichia coli fadB strains. J.Bacteriol. 184, 5696–5705.

Solaiman, D.K., Ashby, R.D., Foglia, T.A., Marmer, W.N., 2006.Conversion of agricultural feedstock and coproducts into poly(hydrox-yalkanoates). Appl. Microbiol. Biotechnol. 71, 783–789.

Song, S., Hein, S., Steinbuchel, A., 1999. Production of poly(4-hydroxy-butyric acid) by fed-batch cultures of recombinant strains of Esche-

richia coli. Biotechnol. Lett. 21, 193–197.Steinbuchel, A., Hein, S., 2001. Biochemical and molecular basis of

microbial synthesis of polyhydroxyalkanoates in microorganisms.Adv. Biochem. Eng. Biotechnol. 71, 81–123.

Steinbuchel, A., Lutke-Eversloh, T., 2003. Metabolic engineering andpathway construction for biotechnical production of relevant poly-hydroxyalkanotes in microoganisms. Biochem. Eng. J. 15, 81–96.

Steinbuchel, A., Hustede, E., Liebergesell, M., Pieper, U., Timm, A.,Valentin, H., 1992. Molecular basis for biosynthesis and accumulationof polyhydroxyalkanoic acids in bacteria. FEMS Microbiol. Rev. 9,217–230.

Steinbuchel, A., Aerts, K., Babel, W., Follner, C., Liebergesell, M.,Madkour, M.H., Mayer, F., Pieper-Furst, U., Pries, A., Valentin,H.E., et al., 1995. Considerations on the structure and biochemistry ofbacterial polyhydroxyalkanoic acid inclusions. Can. J. Microbiol. 41(Suppl. 1), 94–105.

Stubbe, J., Tian, J., He, A., Sinskey, A.J., Lawrence, A.G., Liu, P., 2005.Nontemplate-dependent polymerization processes: polyhydroxyalk-anoate synthases as a paradigm. Annu. Rev. Biochem. 74, 433–480.

Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., Doi, Y., 1999a. Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoatesynthase genes induces PHA production in Escherichia coli HB101strain. FEMS Microbiol. Lett. 176, 183–190.

Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., Doi, Y., 1999b. Over-expression of 3-ketoacyl-ACP synthase III or malonyl-CoA-ACPtransacylase gene induces monomer supply for polyhydroxybutyrateproduction in Escherichia coli HB101. Biotechnol. Lett. 21, 579–584.

Thakor, N., Trivedi, U., Patel, K.C., 2005. Biosynthesis of medium chainlength poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testos-

teroni during cultivation on vegetable oils. Bioresour. Technol. 96,1843–1850.

Timm, A., Steinbuchel, A., 1992. Cloning and molecular analysis of thepoly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa

PAO1. Eur. J. Biochem. 209, 15–30.Tombolini, R., Povolo, S., Buson, A., Squartini, A., Nuti, M.P., 1995.

Poly-beta-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium

meliloti 41. Microbiology 141 (Pt 10), 2553–2559.

2320 R. Li et al. / Bioresource Technology 98 (2007) 2313–2320

Tsuge, T. et al., 2000. Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their usefor polyhydroxyalkanoate synthesis. FEMS Microbiol. Lett. 184,193–198.

Tsuge, T., Taguchi, K., Seiichi, T., Doi, Y., 2003. Molecular character-ization and properties of (R)-specific enoyl-CoA hydratases fromPseudomonas aeruginosa: metabolic tools for synthesis of polyhy-droxyalkanoates via fatty acid beta-oxidation. Int. J. Biol. Macromol.31, 195–205.

Valentin, H.E., Dennis, D., 1997. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown onglucose. J. Biotechnol. 58, 33–38.

Valentin, H.E., Steinbuchel, A., 1993. Cloning and characterization ofthe Methylobacterium extorquens polyhydroxyalkanoic-acid-synthasestructural gene. Appl. Microbiol. Biotechnol. 39, 309–317.

Valentin, H.E., Mitsky, T.A., Mahadeo, D.A., Tran, M., Gruys, K.J.,2000. Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombi-nant Escherichia coli. Appl. Environ. Microbiol. 66, 5253–5258.

Wang, F., Lee, S.Y., 1997. Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia

coli. Appl. Environ. Microbiol. 63, 4765–4769.Wong, H.H., Lee, S.Y., 1998. Poly-(3-hydroxybutyrate) production from

whey by high-density cultivation of recombinant Escherichia coli.Appl. Microbiol. Biotechnol. 50, 30–33.

Wu, G.F., Wu, Q.Y., Shen, Z.Y., 2001. Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803.Bioresour. Technol. 76, 85–90.

Yu, J., Chen, L.X., 2006. Cost-effective recovery and purification ofpolyhydroxyalkanoates by selective dissolution of cell mass. Bio-technol. Prog. 22, 547–553.

Yu, H., Yin, J., Li, H., Yang, S., Shen, Z., 2000. Construction andselection of the novel recombinant Escherichia coli strain for poly(beta-hydroxybutyrate) production. J. Biosci. Bioeng. 89, 307–311.

Yu, H., Shi, Y., Zhang, Y., Yang, S., Shen, Z., 2002. Effect of Vitreoscilla

hemoglobin biosynthesis in Escherichia coli on production of poly(beta-hydroxybutyrate) and fermentative parameters. FEMS Micro-biol. Lett. 214, 223–227.