A microbial polyhydroxyalkanoates (PHA) based bio- andmaterials industry

Guo-Qiang Chen*ab

Received 5th January 2009

First published as an Advance Article on the web 8th May 2009

DOI: 10.1039/b812677c

Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated

by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer

experts and medical researchers. PHA applications as bioplastics, ne chemicals, implant

biomaterials, medicines and biofuels have been developed and are covered in this critical review.

Companies have been established or involved in PHA related R&D as well as large scale

production. Recently, bacterial PHA synthesis has been found to be useful for improving

robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield

improvement on some fermentation products. In addition, amphiphilic proteins related to PHA

synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein

purication and even specic drug targeting. It has become clear that PHA and its related

technologies are forming an industrial value chain ranging from fermentation, materials, energy

to medical elds (142 references).

1. Introduction

Polyhydroxyalkanoates (PHA), as a family of diverse

biopolyesters,1 have gone through many years of eorts

towards commercialization, with limited successes.

Beginning in the 1980s, many companies have tried to

produce various PHA on pilot or industrial scales (Table 1)

based on the expectation that petroleum prices would increase

due to its exhaustion and people might be willing to use

environmentally friendly non-petrochemical based plastics,

termed biodegradable plastics, green plastics, bioplastics or

ecoplastics.2 Scientic breakthroughs led to the successful

large scale production of poly-(R)-3-hydroxybutyrate

(PHB) by Chemie Linz AG Austria, copolymer PHBV of

(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate

(3HV) by ICI UK and TianAn China, and copolymer

PHBHHx of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxy-

hexanoate (3HHx) by the joint eorts of Tsinghua University,

KAIST and P&G (Table 1). Many applications have also been

developed based on the availability of the above PHA

(Table 4). New molecular biology technology contributes

more and more to these industrial breakthroughs. Beginning

in 2009, Metabolix (USA) and Tianjin Green Bioscience

(China) will produce 50 000 and 10 000 tons/year of PHA,

respectively (Table 1). By then, global polymer companies

should have sucient PHA materials to research with. A

new wave of PHA development with a focus on new applications

is expected soon.

However, the petroleum price did not increase signicantly,

resulting in the closure of many PHA related projects in some

companies (Table 1). Right after 2001, the petroleum price

began a sharp increase. In mid-2008, $140 per barrel was

reached. This has promoted the development of plastics that

may be independent of petroleum. Two polyesters, namely

polylactic acid (PLA) and PHA, come into play, each has its

advantages and disadvantages (Table 2). Typically, PLA is

cheaper and PHA more expensive yet application properties

can be varied, depending on the co-monomer structures and

content in the copolyesters. Since PLA has been available in

large quantity, its application research is ahead of PHAs.

The recent energy crisis has prompted a renewed interest in

producing PHA with a competitive edge compared with other

bulk plastics including the already mass produced PLA, which

possesses similar biodegradability and sustainability (Table 2).

Although the cost of petroleum has been drawn down by the

Professor Guo-Qiang CHEN

obtained his BSc from South

China University of Technology

in 1985 and PhD from Graz

University of Technology in

Austria in 1989. He has been

focusing his research on micro-

bial polyhydroxyalkanoates

(PHA) since 1986. With 20+

years of basic research and

R&D experience on PHA

production and applications,

he is currently heading the

Laboratory of Microbiology at

Tsinghua University and the

Multidisciplinary Research Center at Shantou University, and

has contributed to the founding of several PHA based biotech

companies in China.

Guo-Qiang Chen

aDept Biological Sciences and Biotechnology, Tsinghua University,Beijing 100084, China. E-mail: chengq@mail.tsinghua.edu.cn;Fax: 0086-10-62794217; Tel: 0086-10-62783844

bMultidisciplinary Research Center, Shantou University,Shantou 515063, China. E-mail: chengq@stu.edu.cn;Fax: 0086-754-82901175; Tel: 0086-754-82901186

2434 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

. View Article Online / Journal Homepage / Table of Contents for this issue

nancial tsunami in late 2008, PHA as a bioplastic has been

considered as useful for reducing CO2 emissions. To achieve

the mass production and application of PHA, eorts should be

made on eectively lowering the PHA production cost.3

In addition, more PHA applications should be developed,

including the high value added ones.

Over the past years, PHA as polymeric materials have been

developed into applications in plastics, medical implants,

drug delivery carriers, printing and photographic materials,

nutritional supplements, drugs and ne chemicals.49

Recently, PHA has been found useful as a new type of

biofuel.10 In addition, PHA related proteins and genes have

been used to regulate metabolisms and to enhance the

robustness of industrial microorganisms,11,12 even for specic

drug targeting13 and protein purications.1416 Its applications

are rapidly expanding.

This paper reviews what has been achieved and what should

be done to make PHA competitive as bulk plastics, chemicals,

biofuels or other relative applications. An outlook for PHA

development is also provided (Table 5).

2. Fermentation industry: PHA production

Since PHA are produced by large scale microbial fermentation, it

contributes to the fermentation industry, much like the

antibiotic industry started in the early 1920s. So far, PHB,

PHBV, P3HB4HB, PHBHHx and mcl PHA are produced in

large scale (Table 3).

PHA production involves strain development, shake ask

optimization, lab and pilot fermentor studies and then industrial

scale up (Fig. 1). Eective microbial production of PHA

depends on several factors, including the nal cell density,

bacterial growth rate, percentage of PHA in cell dry weight,

time taken to reach high nal cell density, substrate to product

transformation eciency, price of substrates and a convenient

and cheap method to extract and purify the PHA (Fig. 1).

Table 1 Worldwide PHA producing and researching companies

Company Types of PHA Production scale (t/a) Period Applications

ICI, UK PHBV 300 1980s to 1990s PackagingChemie Linz, Austria PHB 20100 1980s Packaging & drug deliverybtF, Austria PHB 20100 1990s Packaging & drug deliveryBiomers, Germany PHB Unknown 1990s to present Packaging & drug deliveryBASF, Germany PHB, PHBV Pilot scale 1980s to 2005 Blending with EcoexMetabolix, USA Several PHA Unknown 1980s to present PackagingTepha, USA Several PHA PHA medical implants 1990s to present Medical bio-implantsADM, USA (with Metabolix) Several PHA 50 000 2005 to present Raw materialsP&G, USA Several PHA Contract manufacture 1980s to 2005 PackagingMonsanto, USA PHB, PHBV Plant PHA production 1990s Raw materialsMeredian, USA Several PHA 10 000 2007 to present Raw materialsKaneka, Japan (with P&G) Several PHA Unknown 1990s to present PackagingMitsubishi, Japan PHB 10 1990s PackagingBiocycles, Brazil PHB 100 1990s to present Raw materialsBio-On, Italy PHA (unclear) 10 000 2008 to present Raw materialsZhejiang Tian An, China PHBV 2000 1990s to present Raw materialsJiangmen Biotech Ctr, China PHBHHx Unknown 1990s Raw materialsYikeman, Shandong, China PHA (unclear) 3000 2008 to present Raw materialsTianjin Northern Food, China PHB Pilot scale 1990s Raw materialsShantou Lianyi Biotech, China Several PHA Pilot scale 1990s to 2005 Packaging and medicalJiang Su Nan Tian, China PHB Pilot scale 1990s to present Raw materialsShenzhen OBioer, China Several PHA Unknown 2004 to present UnclearTianjin Green Bio-Science (+DSM) P3HB4HB 10 000 2004 to present Raw materials & packagingShandong Lukang, China Several PHA Pilot scale 2005 to present Raw materials & medical

Table 2 Comparison between polylactic acid (PLA) and polyhydroxyalkanoates (PHA)

PLA vs. PHA PLA PHA

Monomer structures Only D- and L-lactic acids (LA) At least 150 monomersProduction methods Bio-production of LA and chemical synthesis of PLA Totally biosynthesized as intracellular polyestersProduction cost Comparable with conventional plastics like PET At least twice that of PLAMaterial properties Poor, could be adjusted by regulating D- and L-LA

ratiosFrom brittle, exible to elastic, fully controllable

Technology maturity LA production well established, yet LA polymerizationto PLA is complicated. Only one company,NatureWork, produces PLA on a large scale

At least 10 companies worldwide produced or areproducing PHA up to 2000 t per year scale viamicrobial fermentation

Investment Large xed capital investment: NatureWork hasinvested 1 billion US$ over the past several years to runa 140 000 ton PLA plant

Small investment: existing aerobic microbial fer-mentation plants with process modications canbe used for PHA production

Intellectual properties Cover almost all areas of production and applications Still a lot of spaces for exploitationApplication areas Packaging, medical implants, printing, coating et al., yet

limited by Tg of 6575 1C for cheaper P(L-LA)Almost all areas of conventional plastic industry,limited only by current higher cost

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2435

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

Various factors must be considered in dierent stages of

development. Both wild type and recombinant bacteria were

used for large scale production of various PHA (Table 3).

For large scale application, PHA production costs should be

as low as possible. Therefore, energy saving micro-aerobic

processes, and, increasingly, the use of waste water or activated

sludges for PHA production should be paid attention to. This

requires the development of industrial strains or even mixed

cultures that are capable of growing in low intensity aeration and

producing high content PHA within a reasonable period of time.

Microbial production of PHA by wild type strains

Ralstonia eutropha (formerly called Alcaligenes eutrophus,

Wautersia eutropha, or Cupriavidus necator) has been the most

commonly used wild type strain for the industrial production

of poly-(R)-3-hydroxybutyrate (PHB),17 poly((R)-3-hydroxy-

butyrate-co-4-hydroxybutyrate) (P3HB4HB)18 and poly((R)-

3-hydroxybutyrate-co-(R)-3-hydroxyvalerate) (PHBV)19 (Table 3).

The strain was able to grow to a density of over 100 g l1 after72 h (ICI, UK). The highest cell density of over 200 g l1

containing over 80% PHB was observed in a one cubic metre

fermentor after 60 h of fermentation (Tianjin Northern Food

Co. Ltd). When glucose was fed together with propionate,

160 g l1 cell dry weight containing over 75% PHBV wasproduced over 48 h of growth in a ve cubic metre fermentor

(Zhejiang Tian An Co. Ltd, China) (Table 3). R. eutropha and

recombinant E. coli could grow to over 100 g l1 containingover 75% P3HB4HB after 100 h of fermentation in a one cubic

metre fermentor vessel (Tianjin Green Bioscience Co. Ltd).

All these results indicated that R. eutropha and recombinant

Table 3 Wild type and industrial bacterial strains commonly used for pilot and large scale production of PHAa

StrainDNAmanipulation

PHA type andscale (t/a) C-Source

Final CDW(g l1)

Final PHA(% CDW) Company (Table 1)

Ralstonia eutropha No PHB (10) Glucose 4200 480% Tianjin North. Food, ChinaAlcaligenes latus No PHB (10300) Glucose or

sucrose460 475% Chemie Linz, btF, Austria

Biomers, GermanyEscherichia coli phbCAB + vgb PHB (10) Glucose 4150 480% Jiang Su Nan Tian, ChinaRalstonia eutropha No PHBV

(3002000)Glucose +propionate

4160 475% ICI, UKZhejiang Tian An, China

Ralstonia eutropha No P3HB4HB(410 000)

Glucose +1,4-BD

4100 475% Metabolix, USAEscherichia coli phbCAB Tianjin Green Biosci. ChinaRalstonia eutropha phaCAc PHBHHx (1) Fatty acids 4100 480% P&G, Kaneka, JapanAeromonas hydrophila No PHBHHx (1) Lauric acid o50 o50% P&G, Jiangmen Biotech Ctr,

ChinaAeromonas hydrophila phbAB + vgb PHBHHx (0.1) Lauric acid B50 450% Shandong Lukang,Pseudomonas putida No mcl PHA (0.1) Fatty acids B45 460% ETH, SwitzerlandP. oleovoransBacillus spp. No PHB (5) Sucrose 490 450% Biocycles, Brazila CDW: cell dry weight; vgb: gene encoding Vitreoscilla hemoglobin; phbCAB: PHB synthesis genes encoding b-ketothiolase, acetoacetyl-CoAreductase and PHB synthase; A. caviae: Aeromonas caviae; 1,4-BD: 1,4-butanediol; phaCAc: PHA synthase gene phaC from Aeromonas caviae;

phbAB encodes b-ketothiolase and acetoacetyl-CoA reductase.

Fig. 1 Strain and process development for industrial production of PHA.

2436 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

E. coli were and will be important industrial strains for the

production of at least the three PHAmentioned above due to its

simplicity of growth requirements. Recently, the whole

genome of R. eutropha was sequenced,20 allowing more genetic

manipulations on the PHA production related pathways.

Alcaligenes latus, similar to R. eutropha in terms of PHA

production, was used for PHB and PHBV production

by Chemie Linz AG, Austria and later Biotechnologische

Forschungsgesellschaft (btF, Austria), then Biomers

(Germany).17,21 A. latus in a lab scale 1 L fermentor was

reported to reach a cell density of 142 g l1 containing 50%PHB after just 18 h of growth.22

Aeromonas hydrophila was employed for large scale

production of random copolymer poly((R)-3-hydroxybutyrate-

co-(R)-3-hydroxyhexanoate) (PHBHHx) in a 20 000 L fermentor.

A nal cell concentration, PHBHHx concentration and PHA

content of 50 g l1, 25 g l1, and 50 wt%, respectively, wereobtained in 46 h.23 Wild type A. hydrophila is not an eective

PHBHHx producer.

Pseudomonas oleovorans grown on n-alkanes was reported

to produce 63 wt% PHA containing medium-chain-length

(mcl) monomers in its cell dry weight with a volumetric

PHA productivity of 1.06 g l1 h1.24 The wide choice ofsubstrates and the low substrate specicity of PHA synthase

PhaC enable P. oleovorans and P. putida to synthesize over

100 PHA with various monomer structures.25,26

Microbial production of PHA by recombinant strains

Recombinant E. coli has been commonly employed for PHA

production due to its convenience for genetic manipulation,

fast growth, high nal cell density and ability to utilize

inexpensive carbon sources. Metabolix and Jiang Su Nan Tian

Co. Ltd. employed recombinant E. coli for their PHA

production (Table 3). A recombinant E. coli harboring the

PHA synthetic genes phbCAB from R. eutropha and bacterial

hemoglobin gene vgb produced a cell dry weight of 206 g l1

containing 73% PHB with a productivity of 3.4 g l1 h1.2729

A starch hydrolysis capable recombinant E. coli VG1

harboring phbCAB, vgb and lytic genes of phage lambda

grown on starch produced 167 g l1 PHB with a productivityof 3.05 g l1 h1, a simple temperature-inducing treatment ledto 95% PHB purity recovered.29

Recombinant E. coli can also be used to produce other PHA

including poly-4-hydroxybutyrate (P4HB) homopolymer,30

copolymer P3HB4HB,31 and PHBVHHx terpolymer of

(R)-3-hydroxybutyrate (HB), (R)-3-hydroxyvalerate (HV)

and (R)-3-hydroxyhexanoate (HHx).32,33

Recombinant E. coli containing the phaC1 gene from

P. aeruginosa was able to produce medium-chain-length

PHA from fatty acids when the beta-oxidation gene fadB

was deleted.33

R. eutropha PHB-4 unable to accumulate PHA is the PhaC

mutant of wild type strain R. eutropha. Recombinant

R. eutropha PHB-4 containing PHA synthase gene phaC

from Aeromonas caviae produced PHBHHx from fatty acids

including palm olein, crude palm oil and palm acid oil,34,35

approximately 87 wt% PHBHHx containing 5 mol% HHx

was obtained.36

Recombinant Aeromonas hydrophila 4AK4 harboring

phbAB or phaPCJ was able to accumulate terpolyesters of

PHBVHHx or P3HB4HB3HHx of HB, 4-hydroxybutyrate

(4HB) and HHx.37,38 A. hydrophila 4AK4, harboring a

truncated tesA gene encoding cytosolic thioesterase I of E. coli

which catalyzes the conversion of acyl-ACP into free fatty

acids, was able to synthesize PHBHHx containing 1014 mol%

HHx using gluconate and glucose rather than fatty acids.39 To

enhance PHBHHx production by A. hydrophila, vgb gene

encoding Vitreoscilla haemoglobin or fadD gene encoding

E. coli acyl-CoA synthase was co-expressed with PHA

synthesis related genes including phbAB from R. eutropha

and phaPCJ from A. hydrophila, leading to at least 20%

increases in PHBHHx and HHx content.40

P. putida KT2442 is a well-known mcl PHA producer

with its whole genomic DNA fully sequenced.41 Using

suicide plasmid pK18mobsacB, beta-oxidation genes fadA

and fadB knock-out mutants of P. putida KT2442

produced 84% intracellular mcl PHA consisting of 41 mol%

3-hydroxydodecanoate (HDD), signicantly higher than its

wild type strain KT2442, which produces around 50% mcl

PHA containing only 8.5 mol% HDD. A higher HDD

fraction surprisingly increased crystallization degree and

tensile strength of the mcl PHA.42 When tetradecanoate was

fed, P. putida KTOY06 produced about 80% mcl PHA

containing over 30% 3-hydroxytetradecanoate (HTD).43

Anaerobic PHA production

Recombinant E. coli anaerobically accumulated PHB to more

than 50% of its cell dry weight during cultivation in either

growth or nongrowth medium.44 The maximum theoretical

carbon yield for anaerobic PHB synthesis in E. coli is 0.8,

much higher than the aerobic one of 0.48. Anaerobic PHA

production is perhaps one of the most important ways to

reduce PHA production cost. However, the slow growth of

bacteria under anaerobic conditions must be considered.

Vijayasankaran et al.45 constructed a tandem gene expression

cassette containing three R. eutropha PHB synthesis genes

under the control of the Pichia pastoris glyceraldehyde-3-

phosphate promoter and the green uorescent protein (GFP)

under the control of the P. pastoriss alcohol oxidase promoter.

Oxygen stress led to increased PHB production. PHB exceeding

30% of CDW of recombinant P. pastoris appeared to be the

consequence of the decreased biomass growth rate under

severe oxygen limitation.45 Therefore, micro-aerobic process

appears to be more attractive as it combines the advantages of

low energy cost and fast growth.46

PHA production from waste materials

Waste materials or waste water can be used to produce

PHA, which provides a cost reduction. Khardenavis et al.47

evaluated waste activated sludge generated from a combined

dairy and food processing industry waste water treatment

plant for PHB production. Deproteinized jowar grain-based

distillery spentwash yielded 42.3 wt% PHB, followed by 40

wt% PHB from ltered rice grain-based distillery spentwash.

Addition of diammonium hydrogen phosphate (DAHP)

resulted in an increase in PHB production to 67% when raw

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2437

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

rice grain-based spentwash was used. The same waste water

after removal of suspended solids by ltration and with DAHP

supplementation resulted in lower PHB production (57.9%).

However, supplementing other wastes with DAHP led to a

substantial decrease in PHB content in comparison to what

was observed in the absence of DAHP.47 Such study indicates

the feasibility of using waste water for PHA production.

Mixed culture production of PHA from waste water was

nancially attractive in comparison to pure culture PHA

production.48 Both PHA production processes had similar

environmental impacts that were signicantly lower than high

density polyethylene (HDPE) production. A large potential

for optimisation exists for the PHA process as nancial and

environmental costs were primarily due to energy use for

downstream processing.49 Therefore, mixed culture bio-

technology could become an attractive addition or alternative

to traditional pure culture based biotechnology for the

production of PHA, other chemicals and/or bioenergy,47

especially, mixed culture could become stable and continuous.

3. Energy industry: biofuels based on PHA

PHA including poly-(R)-3-hydroxybutyrate (PHB) and medium-

chain-length PHA (mcl PHA) were respectively converted to

(R)-3-hydroxybutyrate methyl ester (3HBME) and medium

chain length hydroxyalkanoate methyl ester (3HAME) by acid

catalyzed hydrolysis.10 It was found that 3HBME and 3HAME

had combustion heat values of 20 kJ g1 and 30 kJ g1,respectively. Ethanol has a combustion heat of 27 kJ g1 whileaddition of 10% 3HBME or 3HAME enhanced the combustion

heat of ethanol to 30 kJ g1 and 35 kJ g1, respectively. Theaddition of 3HBME or 3HAME into n-propanol and n-butanol

led to slight reductions in their combustion heats. Combustion

heats of blended fuels 3HBMEdiesel or 3HBMEgasoline and

of 3HAMEdiesel or 3HAMEgasoline were lower than that of

pure diesel or gasoline but were reasonable as fuels. It was

roughly estimated that the production costs of PHA based

biofuels from waste resources including waste water and

activated sludges should be around US$1200 per ton.10

Since biofuels including ethanol and biodiesel have always

had controversy regarding food vs. fuel and fuel vs.

arable land, PHA based biofuel production from waste water

or from activated sludge enjoys the advantages of waste water

treatment accompanied by energy generation. This result

opens a new area for PHA application in the energy sector.

4. Material industry: PHA as polymeric materials

Owing to its special polymer features, PHA with diverse

structures and properties have been researched as bioplastics,

bers, biomedical implants and drug delivery carriers et al.49

(Table 4). Similar to the rapid development of polylactic acid

(PLA) promoted by NatureWork as a bulk bioplastic, the

large scale supply of PHA will speed up its development as

new plastics with sustainable properties.

PHA as biodegradable plastics and ber materials

PHA were initially used to make everyday articles such

as shampoo bottles and packaging materials by Wella AG

Germany.50 PHA were also developed as packaging lms

mainly for use as shopping bags, containers and paper

coatings, disposable items such as razors, utensils, diapers,

feminine hygiene products, cosmetic containers and cups, as

well as medical surgical garments, upholstery, carpets, packaging,

compostable bags and lids or tubs for thermoformed

articles by P&G, Biomers, Metabolix and several other

companies.2,51,52

PHB bers with high tensile strength were prepared by

stretching the bers after isothermal crystallization near

the glass transition temperature.53 Increasing the time for

isothermal crystallization of PHB bers resulted in a decrease

in the maximum draw ratio. Yet the tensile strength of PHA

bers increased remarkably when the isothermal crystallization

time was prolonged to more than 24 h. The tensile strength of

low-molecular-weight drawn bers was higher than that of

high-molecular-weight bers.53 PHB bers stretched after

isothermal crystallization had the oriented alpha-form crystal

with a 2(1) helix conformation and the beta-form with a planar

zigzag conformation.53

Vogel et al.54 attempted to use reactive extrusion

with peroxide as a comfortable pathway for improving the

crystallization of PHB in a melt spinning process. They

succeeded in improving the crystallization in the spinline and

Table 4 Applications of PHA in various elds

Applications Examples

Packagingindustry

All packaging materials that are used for a shortperiod of time, including food utensils, lms,daily consumables, electronic appliances et al.

Printing &photographicindustry

PHA are polyesters that can be easily stained111

Other bulkchemicals

Heat sensitive adhesives, latex, smart gels. PHAnonwoven matrices can be used to remove facialoils

Blockcopolymerization

PHA can be changed into PHA diols for blockcopolymerization with other polymers

Plastic processing PHA can be used as processing aids for plasticprocessing

Textile industry Like nylons, PHA can be processed into bersFine chemicalindustry

PHA monomers are all chiral R-forms, and canbe used as chiral starting materials for thesynthesis of antibiotics and other ne chemicals8

Medical implantbiomaterials

PHA have biodegradability and biocompatibility,and can be developed into medical implantmaterials.9 PHA can also be turned into drugcontrolled release matrices

Medical PHA monomers, especially R3HB, havetherapeutic eects on Alzheimers andParkinsons diseases, osteoporosis and evenmemory improvement et al.8991

Healthy foodadditives

PHA oligomers can be used as food supplementsfor obtaining ketone bodies58

Industrialmicrobiology

The PHA synthesis operon can be used as ametabolic regulator or resistance enhancer toimprove the performances of industrial microbialstrains95101

Biofuels or fueladditives

PHA can be hydrolyzed to form hydroxy-alkanoate methyl esters that are combustible10

Proteinpurication

PHA granule binding proteins phasin or PhaP areused to purify recombinant proteins14,102109

Specic drugdelivery

Coexpression of PhaP and specic ligands canhelp achieve specic targeting to diseasedtissues13

2438 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

the inhibition of the secondary crystallization in the bers.54

These processes overcame the brittleness of PHA and created

very strong bers with promising applications.

PHA as medical implant materials

PHA including PHB, PHBV, P3HB4HB, P4HB, P3HO

(poly-(R)-3-hydroxyoctanoate) and PHBHHx are frequently

investigated for use as sutures, repair devices, repair patches,

slings, cardiovascular patches, orthopedic pins, adhesion

barriers, stents, guided tissue repair/regeneration devices,

articular cartilage repair devices, nerve guides, tendon repair

devices, bone marrow scaolds, articial oesophagus and

wound dressings.9 Boston based company Tepha specializes

in manufacturing pericardial patches, artery augments,

cardiological stents, vascular grafts, heart valves, implants

and tablets, sutures, dressings, dusting powders and prodrugs;

it markets P4HB for medical application under the name of

PHA4400.55 Recently, PHBHHx was successfully used as an

osteosynthetic material for stimulating bone growth owing to

its piezoelectric properties,56 and for eectively repairing

damaged nerves.57 Oligomers of PHA were found to have

nutritional and therapeutic uses.58

Shishatskaya et al. found that the monolament sutures

made of PHB and PHBV did not cause any in vivo acute

vascular reaction at the site of implantation or any adverse

event for more than one year.59 Similar phenomena have been

discovered with PHBHHx.60 One of the most important issues

for PHBHHx as an implant biomaterial is the non-toxicity and

lack of immuno-stimulation properties of its degradation

products including monomers and oligomers,6163 they

even stimulate the Ca2+-channel activation and promote

regeneration of damaged tissues.

With successful approval of P4HB as an implant bio-

material, more PHA based biomaterials are expected to go

into clinical trials soon. With the diversity of PHA materials,

one can expect the PHA to become a family of bioimplant

materials with rich applications.

PHA as drug delivery carrier

Homo- and co-polymers of lactate and glycolate are widely

used in commercially available sustained release products for

drug delivery. However, lactate and glycolate co-polymers are

degraded by bulk hydrolysis. Hence drug release can not be

fully controlled.64 In the early 1990s, PHA became candidates

for use as drug carriers due to their biodegradability,

biocompatibility and their degradation by surface erosion.65

PHA used as a drug carrier was reviewed in 1989 by Koosha

et al.66 The potential of matrices produced by direct compression

of PHBV for oral administration has been proven with the

benets of simplied processing over alternative sustained

release technologies.67 Increasing the polymer molecular mass

caused an increased rate of sulfamethizole release from

irregularly shaped PHB microparticles.68 When comparing the

in vitro and in vivo release of the anti-cancer agent lomustine

from PHB and PLA microspheres as potential carriers for drug

targeting, it was found that the drug was released from the PHB

microspheres faster.69 Incorporation of ethyl- or butyl esters of

fatty acids into the PHB microspheres increased the rate of the

drug release.70

So far only PHB and PHBV have been studied for drug

controlled release, it is expected that other PHA family

members with diverse properties will bring more control

release properties for the drug release eld. This is still an

area that remains to be exploited.

5. Fine chemical industry: PHA chiral monomers

More than 120 dierent structures of carboxylic acids

hydroxylated at the 3-, 4-, 5-, or 6-position, all in the

(R)-conguration if they possess a chiral center at the position

of the hydroxyl group, have been reported in PHA with an

increasing number of new monomers being discovered.71,72 In

addition, if the cells are under carbon limitation, the accumu-

lated PHA can be degraded to the monomers and can be

reutilized by the bacteria as a carbon and energy source which

can also serve to produce PHA monomers.73 Due to the chiral

purity, modiable OH and COOH groups and some other

special characteristics, PHA monomers have attracted much

attention in industry and academic areas. Technology for

production of PHA monomers by chemical synthesis,

biotransformation, chemical degradation and enzymatic

degradation has been developed.8

Production of PHA monomers by microorganisms

Various enantiomerically pure (R)-3-hydroxyalkanoic acids

(RHA) can be conveniently prepared by depolymerizing the

biosynthesized PHA. A method for producing (R)-3-hydroxy-

butyric acid (R3HB) and (R)-3-hydroxyvaleric acid (R3HV)

from PHB and PHBV by chemical degradation has been

reported.74 de Roo et al. produced the chiral (R)-3-hydroxy-

alkanoic acid and (R)-3-hydroxyalkanoic acid methyl esters

via hydrolytic degradation of polyhydroxyalkanoates synthe-

sized by Pseudomonads.75 They rst hydrolyzed the recovered

PHA by acid methanolysis and then distilled the 3-hydroxy-

alkanoic acid methyl ester mixture into several fractions.

Subsequently, the (R)-3-hydroxyalkanoic acid methyl esters

were saponied to yield the corresponding 3-hydroxyalkanoic

acid with nal yields of the RHA up to 92.8% (w/w).

Lee and co-workers demonstrated that R3HB could be

eciently produced via in vivo depolymerization by providing

the appropriate environmental conditions.76 In their study

with the strain Alcaligenes latus, they found that lowering

the pH to 34 induced the highest activity of intracellular PHB

depolymerase and blocked the reutilization of R3HB by the

cells.76 Ren et al. suspended the PHA containing Pseudomonas

putida cells in phosphate buer at dierent pH. When the pH

was 11, the degradation and monomer release were at their

best.77 Under the conditions, 3-hydroxyoctanoic acid (R3HO)

and 3-hydroxyhexanoic acid (R3HHx) were degraded with an

eciency of over 90% (w/w) in 9 h and the yields of the

corresponding monomers were also over 90%. Under the same

conditions, unsaturated monomers 3-hydroxy-6-heptenoic

acid, 3-hydroxy-8-nonenoic acid and 3-hydroxy-10-undecenoic

acid were also produced though in a lower yield compared

with the saturated monomer.77

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2439

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

Wild type E. coli DH5a was engineered to produce extra-cellular R3HB by simultaneous expression of PHB precursor

genes of b-ketothiolase (phbA), acetoacetyl-CoA reductase(phbB), phosphor-transbutyrylase (ptb) and butyrate kinase

(buk). A batch process run in a 5 L fermentor produced

approximately 5 g l1 3HB after 24 h. A fed-batchprocess increased R3HB production to 12 g l1 after 48 h offermentation.78

Medium-chain-length (R)-3-hydroxyhexanoic acid (R3HHx)

and (R)-3-hydroxyoctanoic acid (R3HO) were produced by

over-expressing PHA depolymerase gene phaZ, together

with putative long chain fatty acid transport gene fadL of

Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD)

of E. coli MG1655 in P. putida KT2442. In a 48 h fed-batch

fermentation process conducted in a 6 L fermentor with 3 L

sodium octanoate mineral medium, 5.8 g l1 extracellularR3HHx and R3HO were obtained in the fermentation broth.79

Now that chiral PHA monomers can be produced using

dierent ways, the cost of the chiral hydroxyalkanoic acids is

no longer an issue hindering their applications. With proper

scale up, the chiral PHA monomers can become bulk

chemicals for various applications.

Applications of PHA monomers

The PHA-origin RHA monomers contain a chiral center and

two easily modied functional groups (OH and COOH).

Therefore, one of the important applications for RHA is to

be valuable synthons and to act as starting materials for

the synthesis of ne chemicals such as antibiotics, vitamins,

aromatics and pheromones.80

The dierent RHA will have dierent physiological impacts.

Therefore, the diversied types of RHA will sometimes

have dierent functions. It was reported that some RHA

exhibit specic important biological activities such as

antimicrobial or antiviral potential while other RHA do not.

For example, (R)-3-hydroxy-n-phenylalkanoic acid was used

to eectively attack Listeria monocytogenes, which is a

ubiquitous microorganism, able to multiply at refrigeration

temperatures and is resistant to both high temperature and

low pH.81,82

The most common PHA monomer, namely R3HB has been

used as the starting material for producing carbapenem anti-

biotics and macrolides.83 A novel class of polymers named

dendrimers was also synthesized using R3HB. Besides the

application as building blocks of chiral compounds, RHA

monomers can also be used to synthesize novel chiral poly-

esters. Two new 3-hydroxyalkanoates: 3-hydroxy-3-cyclo-

propylpropionate and 3-hydroxy-4-chlorobutyrate and their

CoA thioester derivatives have been successfully synthesized.84

Some peptides modied by the RHA exhibit novel features

and better application abilities.85,86 Novel b- and g-peptideswere produced by replacing amino acids by 3-hydroxybutyrate

residues in peptides and by replacing the chain bound oxygens

in 3HB or 4HB with NH. These novel peptides showed

better resistance to peptidases and environmental microbial

degradation and longevity in mammalian serum. Some

of these peptides even possessed useful antibacterial, anti-

proliferative and haemolytic properties.85,86

Ketone bodies, including R3HB, can correct defects in

mitochondrial energy generation in the heart. R3HB was

administered into rats in hemorrhagic hypotension and the

plasma concentration of substrates related to glucose metabolism

was evaluated.87 The results suggest that administered R3HB

may suppress glycolysis during hemorrhagic shock.

One of the biggest advantages for R3HB is that it has good

tolerance in humans and a short half-life in vivo. Therefore,

R3HB was directly used as an oral drug. Recently, R3HB has

been employed to treat traumatic injuries that benet from

elevated levels of ketone bodies such as hemorrhagic shock,

extensive burns, myocardial damage, and cerebral hypoxia,

anoxia, and ischemia.8890 Furthermore, R3HB has been

found to be able to reduce the death rate of the human

neuronal cell model culture for Alzheimers and Parkinsons

diseases and to ameliorate the appearance of corneal epithelial

erosion through suppression of apoptosis; R3HB methyl

ester was also found to dramatically improve the memory

of mice.8991

R3HB was found to have a stimulatory eect on cell cycle

progression of murine broblast L929 cells, human umbilical

vein endothelial cells, and rabbit articular cartilages that are

mediated by a signaling pathway dependent upon increases in

[Ca2+].92

R3HB was clearly demonstrated to have a positive eect on

the growth of osteoblasts in vitro and on anti-osteoporosis

in vivo.93 It was found that R3HB increased serum alkaline

phosphatase activity and calcium deposition, decreased

serum osteocalcin, prevented bone mineral density reduction

resulting from ovariectomization, leading to enhanced femur

maximal load and bone deformation resistance, as well as

improved trabecular bone volume.93

So far, most of the applications of PHA monomers are

based on R3HB due to its availability in sucient quantities.

With the availability of other chiral monomers, new PHA

monomer based polymers and new medical applications of

chiral R3HA will emerge rapidly. Again, this is an area waiting

for exploitation.

6. The application of the PHA synthesismechanism for improving industrial microorganisms

There have been many reports on the physiological functions

of PHA, mostly centered on increasing the survival ability

under adverse conditions such as starvation, desiccation, UV

radiation, high osmotic pressure and the presence of organic

solvents et al.94 It was also reported that the ability to

accumulate PHB makes the bacteria more responsive to

sudden increases in substrate concentrations, which explains

their ecological advantage.94,95

Feast and famine cycles are common in activated sludge

waste water treatment systems, and they select for bacteria

that accumulate storage compounds, such as PHB.94 Recently,

Zhao et al. compared the survival abilities of a PHBHHx

producing strain Aeromonas hydrophila 4AK4 and its

corresponding PhaC-disrupted mutant termed A. hydrophila

CQ4. It was found that PHBHHx synthesis in the wild type

strain A. hydrophila 4AK4 provided the strain with improved

resistance against environmental stress factors including heat

2440 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

and cold treatments, hydrogen peroxide, UV irradiation,

ethanol and high osmotic pressure.96 The real-time PCR study

revealed that the synthesis of PHBHHx enhanced the expression

levels of sigma factor ss encoded by groS.95,96 Therefore, itmay be possible to improve the robustness of some industrial

microorganisms for the benet of fermentation processes. In

addition, PHA synthesis that consumes a lot of acetyl-CoA

and NADH may be used to help regulate certain microbial

metabolisms for enhanced product formation.

For example, Streptococcus zooepidemicus is an important

strain for the industrial production of hyaluronic acid for

cosmetic applications. In the hyaluronic acid fermentation

process, S. zooepidemicus produces a large amount of lactic

acid, which consumes a great deal of carbon source and is

harmful to the microbial cells. When PHB synthesis genes

phbCAB of R. eutrophawere transformed into S. zooepidemicus,

the recombinant produced only 40 g l1 lactic acid and 7.5 g l1

hyaluronic acid, whereas the wild type produced 65 g l1

lactic acid and 5.5 g l1 hyaluronic acid.97 The enhancedproduction of hyaluronic acid via introducing the phbCAB

genes was explained by the regulatory eects of PHB synthesis

pathway on the cellular oxidation/reduction potential. This

study successfully demonstrated that the PHA synthesis

related to energy and carbon metabolism could be employed

as a pathway to regulate other cellular metabolism and possibly

to regulate the production of other metabolic products.97

Another example, Corynebacterium glutamicum, a

gram-positive soil bacterium, has been used extensively for

the industrial production of L-glutamate and other amino

acids. C. glutamicum harboring phbCAB from R. eutropha

accumulated 3B13% PHB when grown on glucose.L-Glutamate production increased to 39B68% in twoC. glutamicum strains harboring PHB synthesis genes

compared with their wild type parent strains in shake ask

experiments. In fermentor studies, the recombinant produced

approximately 23% more L-glutamate compared with that of

the wild type, and yielded less intermediate metabolites or

by-products including a-ketoglutarate, L-glutamine andlactate. These results suggested that the expression of phbCAB

genes in C. glutamicum helped regulate glutamate production

metabolism. This demonstrated that the expression of PHB

synthesis genes has a positive eect on L-glutamate production

in C. glutamicum.98

In addition, methanol production from carbon dioxide

was successfully achieved using resting cells of Methylosinus

trichosporium IMV 3011 as biocatalysts. It was found that the

catabolism of stored PHB can provide intracellular reducing

equivalents to improve the intrinsic methanol production

capacity. It appeared that the total methanol production

capacity was increased with increasing PHB content in cells.

Resting cells containing 38.6% PHB exhibited the highest total

methanol production capacity. The eects of the methanol

production process on the survival and recovery of

M. trichosporium IMV 3011 showed that the methanol

production from carbon dioxide reduction was not detrimental

to the viability of methanotrophs.99

As molecular evidence for PHA synthesis related to

enhanced stress resistance, Han et al. analyzed and compared

the proteomes of a metabolically engineered E. coli under

PHB-production and non-PHB-production conditions.100 The

proteome expression patterns of the recombinant strain were

resolved on 2D gels. It was found that three heat shock

proteins, GroEL, GroES, and DanK were signicantly

up-regulated in the PHB-accumulating cells. The expression

of the yD gene encoding a 14.3 kDa protein, which is known

to be produced at low pH, was greatly induced with the

accumulation of PHB. This may be a good indication for

enhancing the E. coli stress resistance by introducing the PHA

biosynthesis pathways. Generally, results in this research

indicated that accumulation of PHB in E. coli acted as a stress

on the cells, which reduced the cells ability to synthesize

proteins and induced the expression of various protective

proteins,100 these proteins help improve the robustness of the

strains.

When PHB biosynthesis operon phbCAB was fused with the

promoter of pyruvate decarboxylase of Zymomonas mobilis,

and then the pBBR1MCS-1 plasmid containing the fused

genes was introduced into Z. mobilis, the expression of PHB

in Z. mobilis was achieved. The shake-ask fermentation

results showed that the recombinant strains accumulated

10% more ethanol than the wild type strain after culturing

the strains for 48 h.101

It has become clear that the PHA synthesis mechanism that

improves the anti-stress ability of non-PHA producers, can

be borrowed to improve the yield of non-PHA producing

industrial strains that are constantly under environmental

stresses including changing temperature, pH, substrate types

and concentrations et al.

7. Application of PHA granule surface proteins

Several kinds of proteins are found to locate on the surface of

in vivo PHA granules.102,103 Among these proteins, PHA

synthase has been employed to covalently immobilize

b-galactosidase on the in vivo PHA granule surface by fusingb-galactosidase to the N-terminus of PHA synthase fromPseudomonas aeruginosa.104 Similarly, both the substrate bind-

ing domain of PHA depolymerase and the N-terminal domain

of PhaF phasin or PhaP (PHA granule-associated protein)

have been used to anchor fusion proteins to PHA micro-

beads.105 Auto-regulator protein PhaR has been conrmed

to have two separate domains that bind to DNA and PHB,

respectively,106 and PhaR can be adsorbed to various types of

hydrophobic polymers, such as PHB, poly(L-lactide), poly-

ethylene and polystyrene, mainly by nonspecic hydrophobic

interactions.107 Banki et al. developed a novel purication

system for recombinant proteins based on a self-cleaving intein

fused between phasin and the target protein, and the fusion

protein was anity bound to in vivo PHB particles produced

by the cell itself.108 Following the recovery of native PHB

particles, self-cleavage of the intein resulted in the release of

puried recombinant proteins. This PHB system pushed

bio-separation technology several steps forward in the direction

towards convenience and cheapness.

It appears that proteins locating on the in vivo PHA granule

surface may be potential anity tags for protein purication.

Among them, the nonspecic PHA granule binding protein

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2441

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

phasin as a hydrophobic anity tag appears to be the most

attractive due to its richness compared with others.

Backstrom et al. constructed hybrid genes encoding either the

mouse interleukin-2 (IL2) or the myelin oligodendrocyte glyco-

protein (MOG) fused via an enterokinase site providing linker

region to the C-terminus of PhaP, respectively.109 The hybrid

genes were expressed in PHA-accumulating recombinant

E. coli. MOG and IL2 fusion proteins were found abundantly

attached to PHA granules. A more abundant second fusion

protein of either MOG or IL2 resulted from an additional

N-terminal fusion, which surprisingly did not interfere with

attachment to the PHA granule. PHA granules displaying

either IL2 or MOG were used for FACS using monoclonal

anti-IL2 or anti-MOG antibodies conjugated to a uorescent

dye. FACS analysis showed signicant and specic binding of

respective antibodies. Enterokinase treatment of IL2 displaying

PHA granules enabled removal of IL2 as monitored by FACS

analysis. Mice were immunized with either MOG or OVA

(ovalbumin) and the respective sera were analysed using

MOG-displaying PHA granules and FACS analysis showed a

specic and sensitive detection of antigen-specic antibodies

within a wide dynamic range.109

Wang et al. developed a novel protein purication method

based on phasin, a pH-inducible self-cleaving intein and PHA

nanoparticles.14 Genes for the target proteins to be produced

and puried were fused to genes of the intein and phasin, and

the genes were jointly over-expressed in vivo, such as in E. coli.

The fused proteins containing target protein, intein and phasin

produced by the recombinant E. coli were released together

with all other E. coli proteins via a bacterial lysis process, they

were then adsorbed in vitro to the surfaces of the hydrophobic

polymer nanoparticles incubated with the cell lysates. The

nanoparticles attached to the fused proteins were concentrated

via centrifugation. Then, the reasonably puried target protein

was released by self-cleavage of the intein and separated with

nanoparticles by a simple centrifugation process. This system

was successfully used to produce and purify the enhanced

green uorescent protein (EGFP), maltase binding protein

(MBP) and b-galactosidase. This method allows the productionand purication of high value added proteins in a continuous

way with low cost.14

A receptor mediated drug delivery system was developed

based on PhaP.13 The system consists of PHA nanoparticles,

PhaP and polypeptide or protein ligands fused to PhaP. The

PHA nanoparticles were used to package mostly hydrophobic

drugs; PhaP fused with ligands produced by over-expression

of their corresponding genes in Pichia pastoris or E. coli was

able to attach to hydrophobic PHA nanoparticles. In the end,

the ligands were able to pull the PhaPPHA nanoparticles to

the targeted cells with receptors recognized by the ligands. It

was found that the receptor mediated drug specic delivery

system ligandPhaPPHA nanoparticles was taken up by

macrophages, hepatocellular carcinoma cell BEL7402 in vitro

and liver, hepatocellular carcinoma cells in vivo, respectively,

when the ligands were mannosylated human a1-acid glyco-protein (hAGP) and human epidermal growth factor (hEGF),

respectively, which were able to bind to receptors of

macrophages or hepatocellular carcinoma cells. The delivery

system of hEGFPhaPnanoparticles carrying rhodamine B

isothiocyanate (RBITC) was found to be endocytosed by

the tumor cells in tumorous model mice. Thus, the ligand

PhaPPHA specic drug delivery system was proven eective

both in vitro and in vivo.13

More applications of PHA granule surface proteins should

be exploited since similar ideas could be easily developed.

8. Future development of PHA based industry

Two aspects should be taken into consideration to help the

commercial applications of PHA. One is to lower the production

costs of PHA, and the other is to nd high value added

applications of PHA. Besides the basic research, many eorts

have been directed to these two aspects (Table 5).

The development of low cost PHA production technology

To lower PHA production costs, genetic engineering technology,

pathway modication or even synthetic biology approaches

should be taken to develop super PHA production strains that

are able to grow to high cell density within a short period of

time on lower cost substrates under less demanding fermentation

conditions, such as micro-aerobic conditions.110115 A synthetic

strain containing the minimum genome could help increase the

substrate to PHA yield. Simple purication and extraction

technology employing controllable lysis of high PHA content

containing bacteria accumulated large PHA granules should

be developed,116122 such a process can dramatically reduce

the cost of centrifugation, ltration and extraction as it can be

coupled with inorganic aqueous treatment.116,117,125 On the

other hand, the use of simple substrates such as glucose only

for production of copolymers including PHBV, PHBHHx,

copolymers of scl- and mcl 3-hydroxyalkanoates, and

P3HB4HB will reduce the PHA cost attributed to co-substrates

including propionate, fatty acids or 1,4-butanediol.123,124,130

PHA produced should have a controllable molecular weight to

meet various applications.127 Particularly, the use of continuous

mixed culture fermentation without sterilization is a newly

developed technology to signicantly lower the PHA production

cost (K. Johnson et al., ISBP 2008 Lecture). Such a continuous

process without sterilization avoids the cost of energy

consumption for sterilization, however, robust PHA production

strains are needed for this purpose (Table 5). Johnson et al.

reported the development of a PHB continuous fermentation

process without sterilization; the process produced 90% PHB

in the cell dry weight and it lasted for three years without

troubles. This should be considered as a breakthrough in low

cost production of PHA.

Low cost PHA will not only benet the PHA material

application as bioplastics, but also promote the application

of PHA as biofuels. This is an area full of promise, since low

cost PHA could also be obtained from activated sludge

and waste water fermentation, so it will not run into the

controversy of food vs. fuel or fuel vs. arable land.10 In a

not so distant future, plant production of PHA could become

a reality, as indicated by some promising results.128

Unusual PHA with special properties

Unusual PHA containing various functional side groups such

as double bonds, hydroxyl- and/or carboxyl-groups should be

2442 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

produced since these PHA have not only intrinsic novel

properties but also easily-modied side groups which greatly

expand their applications.

It is now possible to use low specicity PHA synthases for

the production of scl- and mcl PHA copolymers through

screening or molecular evolution,120,122,129,130 and it has

become a reality to design and produce PHA with the expected

structures. PHA containing designed functional side groups

allow chemical modication to change the polymer properties,

leading to expansion of PHA applications.

It is now also possible to produce PHA containing various

blocks like PHB-b-PHV, or PHB-b-PHBV, PHB-b-PHA and

the similar.131,132 Such block PHA have been found to show

new properties. More PHB block copolymers are under

development and they could generate some more unique

applications.

PHA with ultra-high molecular weights of over several

million were reported.127,133 Molecular weights of PHA

were found to depend on PHA production strains and the

N-terminus of PHA synthase. Ultra-high molecular weight

Table 5 Future development for PHA production technology and applications

Development Technology employed and other issues Ref.

To lower PHA production costsHigh cell density growth withinshort period of time

Manipulating quorum sensing pathways. Process control,pathway manipulation

This lab, 112115

Controllable lysis of PHAcontaining cells

Genes related to cell lysis, e.g., lambda lysis factor and lysozyme 116, 117

Large PHA granules for easyseparation

Delete phasin led to large in vivo PHA granules 118, 119

Super high PHA content in cell dryweight

Delete PhaZ or over-expression of PHA synthesis genesincluding PhaF et al.

120122

Micro-aerobic PHA production Employing anaerobic promoter, and/or facultative anaerobicbacteria or other technology including synthetic biology thatturns aerobic processes into micro-aerobic processes

This lab

Simple carbon sources for scl- andmcl copolymers

Application of low specicity PHA synthase, and construction ofpathways to supply mcl PHA monomers from non-fatty acidoxidation pathways

123, 124, 130

Enhanced substrates for PHAtransformation eciency

Delete pathways that aect PHA synthesis, or employ aminimum genome containing cells with an inserted PHA pathway

This lab

Inorganic extraction & purication Apply to over 90% PHA containing cells 116, 117, 125Mixed cultures withoutsterilization

Employ feast and famine selection process to nd robust PHAproduction strain

Johnson K. ISBP2008 lecture

Continuous fermentation The use of robust PHA production strains, better undernon-sterilization conditions

126, Johnson K.ISBP 2008 lecture

Controllable PHA molecularweight

Manipulation of the N-terminus of PHA synthase 127

The use of PHA monomer methylesters as a biofuel or fuel additives

The development of low cost technology for production of lowcost PHA biofuels or fuel additives

This lab

Plants as PHA productionmachines

Plant molecular biology 128

PHA with special properties:Novel PHA with unique properties The use of low specicity PHA synthases for production of PHA

with functional groups for chemical modications, and PHAwith controllable compositions including block PHA copolymers

120, 122, 129, 130Controllable PHA compositions 131, 132

Ultra-high PHA molecular weights The use of special strain and mutated PHA synthases 127, 133Block copolymerization of PHA The making of PHA-diols and the block copolymerization with

other polymers134, 135

PHA monomers as building blocksfor new polymers

Copolymerization of PHA monomers with other monomersincluding lactides for the formation of novel copolymers withnew properties

136, 137 andthis lab

To develop low cost PHA applications:New PHA processing technology Cost eective processing of PHA as plastic packaging materials 138140PHA blending with low costmaterials

PHA blending with starch, cellulose et al.

High value added applicationsPHA as bio-implant materials Further improve the in vivo controllable degradation of PHA

implants, seek FDA approval for clinical applications9, Tepha Co. Ltd.

PHA as tissue engineering materials Develop 3-D scaolds as tissue engineering matrices 141PHA monomers and oligomers asnutritional and energy supplements

Understand the mechanism behind the Ca2+ stimulation eect ofoligomers and monomers of PHA

6062

PHA monomers as drugs Study of other non-R3HB monomers as drug candidates 89PHA monomers as ne chemicals Chiral monomers should be exploited as intermediates for chiral

synthesis8

PHA as smart materials Tailor-made PHA as shape memory or temperature sensitive gels 142

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2443

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

PHA can be turned into high strength bers for shing nets or

shing string et al.

Many studies showed that PHA-diols can be used to

prepare PHA based block copolymers. These block copoly-

mers exhibited biocompatibility and temperature sensitive

behavior,134,135 and they are candidates for applications as

implant biomaterials and drug delivery matrices.

Finally, the diverse PHA monomers are a rich pool for

novel polymer synthesis.136,137 Copolymerization of PHA

monomers with commercially available polymer monomers

will generate limitless new copolymers. This is an area that has

not yet started to attract attention, possibly due to the high

cost of PHA monomer production. However, a copolymer of

lactide and 3HB has been recently reported,136,137 signifying

the start of a PHA monomer based new polymer era.

The development of low cost PHA applications

With cost competitive PHA developed, low cost applications

should also be developed. These include the new processing

technology that can exploit the existing extruders and other

molding machines used to make products from common

plastics like polyethylene and polypropylene.138140 In

addition, the blending of PHA with cheaper materials like

starch and cellulose will further reduce the cost without

the loss of degradability and sustainability (Table 5). In

addition, PHA can also be used to blend with other low cost

plastics to form bio-based plastics for improving the cost

competitiveness.

To our knowledge, PHA have been successfully turned into

heat sensitive adhesives, latex and smart gels in the labs.

Technology should be further developed to produce such

PHA based bulk materials into commercial products

(Table 4).

As indicated (Table 4), many large volume and low value

applications of PHA have been intensively investigated by the

materials industries worldwide, and they are approaching step

by step the reality when large amounts of low cost PHA

become available in the years ahead.

High value added applications

Among the wide variety of applications of PHA, medical

applications of PHA seemed to be the most economically

practical area. It is crucial to exploit and develop the

application of PHA in the medical eld. Almost all PHA

available in sucient quantities, including PHB, PHBV,

PHBHHx, P4HB, P3HB4HB and PHO, have been studied

for bio-implant applications. All of them showed good

biocompatibility and some biodegradability. Among them,

P4HB has been approved by the FDA for suture application

with a trade name TephaFLEX marketed by Tepha Inc., of

Cambridge, Mass., USA. Future eorts have been directed to

develop more medical applications for PHA,9 mostly, three

dimensional scaolds for implant purposes.141

PHA monomers and oligomers have been found to

stimulate Ca2+ channels in mammalian cells.60,62,89 They have

been proposed to be used as drug candidates or nutritional

and energy supplements based mainly on lab studies. More

eorts should be directed to understand the mechanisms

behind this, and more animal studies should also be conducted

to see the real benets.

The use of PHA chiral monomers, especially R3HB, as

chiral intermediates leading to chiral compounds have been

well documented.8 The large chiral R3HA pool has mostly

remained untouched. The greater involvement of organic

chemists could accelerate the expansion of the novel chiral

compound pool based on PHA chiral monomers.

In addition, PHA or PHA oligomers block copolymerized

with other polymers have been found to show some smart

properties with the possibility for medical applications. This

should also be taken into consideration for high value added

uses.142

Other future applications

The use of PHA operons expressed in prokaryotes or

eukaryotes can help enhance the cellular robustness.97100 This

mechanism should be tested in more industrial microbial

strains with an aim to select strains with better resistance to

the stressed conditions and thus enhanced yields of the

bio-products, including antibiotics, vitamins and amino acids

et al.

The amphiphilic proteins on PHA granule surfaces should

be exploited for more applications in specic drug targeting,

cell sorting, protein purication and many other applications.

Conclusions

Research on the production and application of PHA requires

interdisciplinary knowledge. Joint eorts by microbiologists,

geneticists, botanists, chemists, polymer scientists, chemical

engineers, biotechnologists, medical scientists, government

agencies and venture capitalists have strongly promoted the

PHA eld to become an industrial value chain ranging from

agriculture, fermentation, plastics, packaging, biofuels, ne

chemicals, and medicine to nutrition. With the availability of

large amounts of PHA in late 2009, more polymer specialized

companies will get involved, leading to more applications of

PHA, and we will see the formation of such a value chain

accelerate more quickly.

Acknowledgements

Over the past many years, I have been supported by the

Natural Sciences Foundation of China (Grants No.

30570024/C010103, No. 20334020). In addition, the NSFC

funding for Distinguished Young Scholar (No. 30225001) also

contributes to this research. Since 2003, the Li K-Shing

Foundation has begun to support the systematic PHA

development. The National High Tech 863 Grants (Project

No. 2006AA02Z242 and 2006AA020104) and Guangdong

Provincial Grant for collaboration among industry, university

and research organization have supported the application

research on PHA. From 19962002, I was supported by

P&G based in Cincinnati for the development of PHBHHx

industrial production technology. Beginning from 2007,

my lab has been supported by 973 Basic Research Fund

(Grant No. 2007CB707804).

2444 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

References

1 B. Hazer and A. Steinbuchel, Appl. Microbiol. Biotechnol., 2007,74, 1.

2 S. Philip, T. Keshavarz and I. Roy, J. Chem. Technol. Biotechnol.,2007, 82, 233.

3 R. A. J. Verlinden, D. J. Hill, M. A. Kenward, C. D. Williams andI. Radecka, J. Appl. Microbiol., 2007, 102, 1437.

4 W. J. Orts, G. A. R. Nobes, J. Kawada, S. Nguyen, G. E. Yu andF. Raveneile, Can. J. Chem., 2008, 86, 628.

5 Y. Tokiwa and B. P. Caiabia, Can. J. Chem., 2008, 86, 548.6 K. Sudesh and T. Iwata, Clean: Soil, Air, Water, 2008,36, 433.

7 T. Yano, T. Kenmoku, C. Mihara, T. Fukui, A. Kusakari,N. Fujimoto and I. Fukui, US Pat., 2006194071-A1, 2006.

8 G. Q. Chen and Q. Wu, Appl. Microbiol. Biotechnol., 2005, 67,592.

9 G. Q. Chen and Q. Wu, Biomaterials, 2005, 26, 6565.10 X. J. Zhang, R. C. Luo, Z. Wang, Y. Deng and G. Q. Chen,

Biomacromolecules, 2009, 10, 707.11 Q. Liu, S. P. Ouyang, J. Kim and G. Q. Chen, J. Biotechnol.,

2007, 132, 273.12 J. Y. Zhang, N. Hao and G. Q. Chen, Appl. Microbiol. Biotechnol.,

2006, 71, 222.13 Y. C. Yao, X. Y. Zhan, X. H. Zou, Z. H. Wang, Y. C. Xiong,

J. Zhang, J. Chen and G. Q. Chen, Biomaterials, 2008, 29, 4823.14 Z. H. Wang, H. N. Wu, J. Chen, J. Zhang and G. Q. Chen, Lab

Chip, 2008, 8, 1957.15 M. R. Banki, T. U. Gerngross and D. W. Wood, Protein Sci.,

2005, 14, 1387.16 V. Peters and B. H. A. Rehm, Appl. Environ. Microbiol., 2006, 72,

1777.17 O. Hrabak, FEMS Microbiol. Rev., 1992, 103, 251.18 M. Kunioka, Y. Nakamura and Y. Doi, Polym. Commun., 1988,

29, 174.19 D. Byrom, FEMS Microbiol. Rev., 1992, 103, 247.20 A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian,

H. Liesegang, R. Cramm, T. Eitinger, C. Ewering, M. Potter,E. Schwartz, A. Strittmatter, I. Voss, G. Gottschalk,A. Steinbuchel, B. Friedrich and B. Bowien, Nat. Biotechnol.,2006, 24, 1257.

21 G. Q. Chen, K. H. Koenig and R. M. Laerty, Antonie vanLeeuwenhoek, 1991, 60, 61.

22 T. Yamane, M. Fukunaga and Y. W. Lee, Biotechnol. Bioeng.,1996, 50, 197.

23 G. Q. Chen, G. Zhang, S. J. Park and S. Y. Lee, Appl. Microbiol.Biotechnol., 2001, 57, 50.

24 K. Jung, W. Hazenberg, M. Prieto and B. Witholt, Biotechnol.Bioeng., 2001, 72, 19.

25 M. J. de Smet, G. Eggink, B. Witholt, J. Kingma andH. Wynberg, J. Bacteriol., 1983, 154, 870.

26 A. Steinbuchel and H. E. Valentin, FEMS Microbiol. Lett., 1995,128, 219.

27 S. Y. Lee, E. coli moves into the plastic age, Nat. Biotechnol.,1997, 15, 17.

28 F. Wang and S. Y. Lee, Biotechnol. Bioeng., 1998, 58, 325.29 H. M. Yu, Y. Shi, J. Yin, Z. Y. Shen and S. L. Yang, J. Chem.

Technol. Biotechnol., 2003, 78, 283.30 S. Song, S. Hein and A. Steinbuchel, Biotechnol. Lett., 1999, 21,

193.31 H. E. Valentin and D. Dennis, J. Biotechnol., 1997, 58, 33.32 S. J. Park, W. S. Ahn, P. R. Green and S. Y. Lee, Biotechnol.

Bioeng., 2001, 74, 82.33 S. Langenbach, B. H. A. Rehm and A. Steinbuchel, FEMS

Microbiol. Lett., 1997, 150, 303.34 T. Fukui and Y. Doi, J. Bacteriol., 1997, 179, 4821.35 T. Fukui and Y. Doi, Appl. Microbiol. Biotechnol., 1998, 49, 333.36 C. Y. Loo, W. H. Lee, T. Tsuge, Y. Doi and K. Sudesh,

Biotechnol. Lett., 2005, 27, 1405.37 W. P. Xie and G. Q. Chen, Biochem. Eng. J., 2008, 38, 384.38 W. Zhao and G. Q. Chen, Process Biochem., 2007, 42, 1342.39 Y. Z. Qiu, J. Han, J. J. Guo and G. Q. Chen, Biotechnol. Lett.,

2005, 27, 1381.

40 S. P. Ouyang, J. Han, Y. Z. Qiu, L. F. Qin, S. Chen, Q. Wu,M. L. Leski and G. Q. Chen, Macromol. Symp., 2005, 224, 21.

41 C. Weinel, K. E. Nelson and B. Tummler, Environ. Microbiol.,2002, 4, 809.

42 S. P. Ouyang, R. C. Luo, S. S. Chen, Q. Liu, A. Chung, Q. Wuand G. Q. Chen, Biomacromolecules, 2007, 8, 2504.

43 W. K. Liu and G. Q. Chen, Appl. Microbiol. Biotechnol., 2007, 76,1153.

44 R. Carlson, A. Wlaschin and F. Srienc, Appl. Environ. Microbiol.,2005, 71, 713.

45 N. Vijayasankaran, R. Carlson and F. Srienc, Biomacromolecules,2005, 6, 604.

46 X. X. Wei, Z. Y. Shi, M. Q. Yuan and G. Q. Chen, Appl.Microbiol. Biotechnol., 2009, 82, 703.

47 A. A. Khardenavis, M. S. Kumar, S. N. Mudliar andT. Chakrabart, Bioresour. Technol., 2007, 98, 35793584.

48 N. Gurie and P. Lant, Bioresour. Technol., 2007, 98, 3393.49 R. Kleerebezem and M. C. M. van Loosdrecht, Curr. Opin.

Biotechnol., 2007, 18, 207.50 R. M. Weiner, Tibtech, 1997, 15, 390.51 A. M. Clarinval and J. Halleux, in Biodegradable Polymers for

Industrial Applications, ed. R. Smith, CRC, Fl, USA, 2005,pp. 356.

52 G. Mikova and I. Chodak, Chem. Listy, 2006, 100, 1075.53 T. Tanaka, T. Yabe, S. Teramachi and T. Iwata, Polym. Degrad.

Stab., 2007, 92, 1016.54 R. Vogel, B. Tandler, D. Voigt, D. Jehnichen, L. Haussler,

L. Peitzsch and H. Brunig, Macromol. Biosci., 2007, 7, 820.55 D. P. Martin and S. F. Williams, Biochem. Eng. J., 2003, 16, 97.56 Y. Wang, Y. Z. Bian, Q. Wu and G. Q. Chen, Biomaterials, 2008,

29, 2858.57 Y. Z. Bian, Y. Wang, S. Guli, G. Q. Chen and Q. Wu, Biomaterials,

2009, 30, 217.58 D. P. Martin, O. P. Peoples, S. F. Williams and L. Zhong, US

Pat., 359 086, 1999.59 E. I. Shishatskaya, T. G. Volova, A. P. Puzyr, O. A. Mogilnaya

and S. N. Efremov, J. Mater. Sci.: Mater. Med., 2004, 15, 719.60 X. H. Qu, Q. Wu, K. Y. Zhang and G. Q. Chen, Biomaterials,

2006, 27, 3540.61 S. Cheng, F. Yang, M. Xu, Q. Wu, M. Leski and G. Q. Chen,

Biomacromolecules, 2005, 6, 593.62 S. Cheng, G. Q. Chen, M. Leski, B. Zou, Y. Wang and Q. Wu,

Biomaterials, 2006, 27, 3758.63 J. Sun, Z. W. Dai and G. Q. Chen, Biomaterials, 2007, 28, 3896.64 C. W. Pouton and S. Akhtar, Adv. Drug Delivery Rev., 1996, 18,

133.65 P. L. Gould, S. J. Holland and B. J. Tighe, Int. J. Pharm., 1987,

38, 231.66 F. Koosha, R. H. Muller and S. S. Davis, CRC Crit. Rev. Ther.

Drug Carrier Syst., 1989, 6, 117.67 P. L. Gould, S. Holland and B. J. Tighe, Int. J. Pharm., 1987, 38,

231.68 M. R. Brophy and P. B. Deasy, Int. J. Pharm., 1986, 29, 223.69 M. C. Bissery, F. Valeriote and C. Thies, Proc. Int. Symp.

Controlled Release Bioact. Mater., 1985, 12, 69.70 M. Kubota, M. Nakano and K. Juni, Chem. Pharm. Bull., 1988,

36, 333.71 B. Witholt and B. Kessler, Perspective of medium chain length

poly(hydroxyalkanoates), a versatile set of bacterial bioplastics,Curr. Opin. Biotechnol., 1999, 10, 279.

72 A. Steinbuchel and H. E. Valentin, Diversity of bacterial poly-hydroxyalkanoic acids, FEMS Microbiol. Lett., 1995, 128, 219.

73 H. M. Muller and D. Seebach, Poly(hydroxyalkanoates) a fthclass of physiologically important organic biopolymers?, Angew.Chem., Int. Ed. Engl., 1993, 32, 477.

74 D. Seebach, A. K. Beck, R. Breitschuh and K. Job, Org. Synth.,1992, 71, 39.

75 G. de Roo, M. B. Kellerhals, Q. Ren, B. Witholt and B. Kessler,Biotechnol. Bioeng., 2002, 77, 717.

76 S. Y. Lee, Y. Lee and F. L. Wang, Biotechnol. Bioeng., 1999, 65,363.

77 Q. Ren, A. Grubelnik, M. Hoerler, K. Ruth, R. Hartmann,H. Felber and M. Zinn, Biomacromolecules, 2005, 6, 2290.

78 H. J. Gao, Q. Wu and G. Q. Chen, FEMS Microbiol. Lett., 2002,213, 59.

79 M. Q. Yuan, Z. Y. Shi, X. X. Wei, Q. Wu, S. F. Chen andG. Q. Chen, FEMS Microbiol. Lett., 2008, 283, 167.

This journal is c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 24342446 | 2445

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online

80 K. Ruth, A. Grubelnik, R. Hartman, T. Egli, M. Zinn andQ. Ren, Biomacromolecules, 2007, 8, 279.

81 A. Sandoval, E. Arias-Barrau, F. Bermejo, L. Canedo,G. Naharro, E. Olivera and J. Luengo,Appl. Microbiol. Biotechnol.,2005, 67, 97.

82 T. R. Burke, M. Knight, B. Chandrasekhar and J. Ferretti,Tetrahedron Lett., 1989, 30, 519.

83 O. Tasaki, A. Hiraide, T. Shiozaki, H. Yamamura, N. Ninomiyaand H. Sugimoto, J. Parenter. Enteral Nutr., 1999, 23, 321.

84 M. Kamachi, S. M. Zhang, S. Goodwin and R. W. Lenz,Macromolecules, 2001, 34, 6889.

85 S. H. Park, S. H. Lee and S. Y. Lee, J. Chem. Res., Synop., 2001,11, 498.

86 D. Seebach, M. Albert, P. Arvidsson, M. Rueping andJ. V. Schreiber, Chimia, 2001, 55, 345.

87 M. Katayama, A. Hiraide, H. Sugimoto, T. Yoshioka andT. Sugimoto, J. Parenter. Enteral Nutr., 1994, 18, 442.

88 K. Tieu, C. Perier, C. Caspersen, P. Teismann, D. C. Wu,S. D. Yan, A. Naini, M. Vila, V. Jackson-Lewis, R. Ramasamyand S. Przedborski, J. Clin. Invest., 2003, 112, 892.

89 L. Massieu, M. L. Haces, T. Montiel and K. Hernandez-Fonseca,Neuroscience, 2003, 120, 365.

90 X. H. Zou, H. M. Li, S. Wang, M. Leski, Y. C. Yao, X. D. Yang,Q. J. Huang and G. Q. Chen, Biomaterials, 2009, 30, 1532.

91 Y. Kashiwaya, T. Takeshima, N. Mori, K. Nakashima, K. Clarkeand R. L. Veech, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5440.

92 S. Cheng, Q. Wu, F. Yang, M. Xu, M. Leski and G. Q. Chen,Biomacromolecules, 2005, 6, 593.

93 Y. Zhao, B. Zou, Z. Y. Shi, Q. Wu and G. Q. Chen, Biomaterials,2007, 28, 3063.

94 D. Kadouri, E. Jurkevitch, Y. Okon and S. Castro-Sowinski,Crit. Rev. Microbiol., 2005, 31, 55.

95 S. Muller, T. Bley and W. Babel, J. Biotechnol., 1999, 75, 81.96 Y. H. Zhao, H. M. Li, L. F. Qin, H. H. Wang and G. Q. Chen,

FEMS Microbiol. Lett., 2007, 276, 34.97 J. Y. Zhang, N. Hao and G. Q. Chen, Appl. Microbiol.

Biotechnol., 2006, 71, 222.98 Q. Liu, S. P. Ouyang, J. Kim and G. Q. Chen, J. Biotechnol.,

2007, 132, 273.99 J. Y. Xin, Y. X. Zhang, S. Zhang, C. G. Xia and S. B. Li, J. Basic

Microbiol., 2007, 47, 426.100 M. J. Han, S. S. Yoon and S. Y. Lee, J. Bacteriol., 2001, 183, 301.101 W. J. Lai and G. Q. Chen, Chin. J. Biotechnol., 2006, 26, 52.102 B. H. A. Rehm, Curr. Issues Mol. Biol., 2007, 9, 41.103 E. S. Stuart, A. Tehrani, H. E. Valentin, D. Dennis, R. W. Lenz

and R. C. Fuller, J. Biotechnol., 1998, 64, 137.104 V. Peters and B. H. A. Rehm, Appl. Environ. Microbiol., 2006, 72,

1777.105 S. J. Lee, J. P. Park, T. J. Park, S. Y. Lee, S. Lee and J. K. Park,

Anal. Chem., 2005, 77, 5755.106 M. Yamada, K. Yamashita, A. Wakuda, K. Ichimura,

A. Maehara, M. Maeda and S. Taguchi, J. Bacteriol., 2007,189, 1118.

107 K. Yamashita, M. Yamada, K. Mumata and S. Taguchi,Biomacromolecules, 2006, 7, 2449.

108 M. R. Banki, T. U. Gerngross and D. W. Wood, Protein Sci.,2005, 14, 1387.

109 B. T. Backstrom, J. A. Brockelbank and B. H. A. Rehm, BMCBiotechnol., 2007, 7, 3.

110 X. W. Liu, H. H. Wang and G. Q. Chen, Biochem. Eng. J., 2009,43, 72.

111 Z. J. Li, L. Cai, Q. Wu and G. Q. Chen, Appl. Microbiol.Biotechnol., 2009, DOI: 10.1007/s00253-009-1943-6.

112 B. S. Kim, S. C. Lee, S. Y. Lee, H. N. Chang, Y. K. Chang andS. I. Woo, Biotechnol. Bioeng., 1994, 43, 892.

113 Y. Lee and S. Y. Lee, J. Environ. Polym. Degrad., 1996, 4, 131.114 J. I. Choi and S. Y. Lee, Bioprocess Eng., 1997, 17, 335.115 F. L. Wang and S. Y. Lee, Appl. Environ. Microbiol., 1997, 63,

3703.116 J. Yin, Y. Xu, H. M. Yu, P. J. Zhou and Z. Y. Shen,

Bioseparation Eng., 2000, 16, 213.117 H. M. Yu, Y. Shi, J. Yin, Z. Y. Shen and S. L. Yang, J. Chem.

Technol. Biotechnol., 2003, 78, 283.118 G. M. York, J. Stubbe and A. J. Sinskey, J. Bacteriol., 2001, 183,

2394.119 S. J. Tian, W. J. Lai, Z. Zheng, H. X. Wang and G. Q. Chen,

FEMS Microbiol. Lett., 2005, 244, 19.120 T. Hiraishi, Y. Hirahara, Y. Doi, M. Maeda and S. Taguchi,

Appl. Environ. Microbiol., 2006, 72, 7331.121 L. Cai, M. Q. Yuan, J. Jian, F. Liu and G. Q. Chen, Bioresour.

Technol., 2009, 100, 2265.122 A. Sandoval, E. Arias-Barrau, M. Arcos, G. Naharro,

E. R. Olivera and J. M. Luengo, Environ. Microbiol., 2007, 9, 737.123 T. Tsuge, Y. Saito, M. Narike, K. Muneta, Y. M. Normi,

Y. Kikkawa, T. Hiraishi and Y. Doi, Macromol. Biosci., 2004,4, 963.

124 Y. Z. Qiu, J. Han, J. J. Guo and G. Q. Chen, Biotechnol. Lett.,2005, 27, 1381.

125 D. V. Suzuki, J. M. Carter, M. F. A. Rodrigues, E. S. da Silva andA. E. Maiorano, World J. Microbiol. Biotechnol., 2008, 24, 771.

126 G. Braunegg, G. Lefebvre, G. Renner, A. Zeiser, G. Haage andK. Loidllanthaler, Can. J. Microbiol., 1995, 41(Suppl. 1), 239.

127 Z. Zheng, M. Li, X. J. Xue, H. L. Tian, Z. Li and G. Q. Chen,Appl. Microbiol. Biotechnol., 2006, 72, 896.

128 J. B. van Beilen and Y. Poirier, Plant J., 2008, 54, 684.129 J. M. Luengo, B. Garcia, A. Sandoval, G. Naharro and

E. R. Olivera, Curr. Opin. Microbiol., 2003, 6, 251.130 T. Tsuge, K. Yano, S. Imazu, K. Numata, Y. Kikkawa, H. Abe,

S. Taguchi and Y. Doi, Macromol. Biosci., 2005, 5, 112.131 C. W. J. McChalicher and F. Srienc, J. Biotechnol., 2007, 132,

296.132 E. N. Pederson, C. W. J. McChalicher and F. Srienc, Biomacro-

molecules, 2006, 7, 1904.133 S. Kusaka, T. Iwata and Y. Doi, J. Macromol. Sci., Pure Appl.

Chem., 1998, A35, 319.134 K. L. Liu, S. H. Goh and J. Li, Macromolecules, 2008, 41, 6027.135 L. P. Wu, S. T. Chen, Z. B. Li, K. T. Xu and G. Q. Chen, Polym.

Int., 2008, 57, 939.136 S. Taguchi, M. Yamadaa, K. Matsumoto, K. Tajima, Y. Satoh,

M. Munekata, K. Ohno, K. Kohda, T. Shimamura, H. Kambeand S. Obata, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 17323.

137 L. R. Rieth, D. R. Moore, E. B. Lobkovsky and G. W. Coates,J. Am. Chem. Soc., 2002, 124, 15239.

138 L. Wang, W. F. Zhu, X. J. Wang, X. A. Chen, G. Q. Chen andK. T. Xu, J. Appl. Polym. Sci., 2008, 107, 166.

139 K. C. Reis, J. Pereira, A. C. Smith, C. W. P. Carvalho, N. Wellnerand I. Yakimets, J. Food Eng., 2008, 89, 361.

140 L. H. Innocentini-Mei, J. R. Bartoli and R. C. Ballieri,Macromol.Symp., 2003, 197, 77.

141 S. T. Cheng, Z. F. Chen and G. Q. Chen, Biomaterials, 2008, 29,4187.

142 X. J. Loh, S. H. Goh and J. Li, Biomacromolecules, 2007, 8, 585.

2446 | Chem. Soc. Rev., 2009, 38, 24342446 This journal is c The Royal Society of Chemistry 2009

Publ

ished

on

08 M

ay 2

009.

Dow

nloa

ded

by F

AC

DE

QUIM

ICA

on 23

/09/20

13 23

:13:51

.

View Article Online