-
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:
[email protected];Fax: 0086-10-62794217; Tel:
0086-10-62783844
bMultidisciplinary Research Center, Shantou University,Shantou
515063, China. E-mail: [email protected];Fax: 0086-754-82901175;
Tel: 0086-754-82901186
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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).
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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
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ished
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