Biochem. J. (2009) 418, 219–232 (Printed in Great Britain)
219
doi:10.1042/BJ20081769
REVIEW ARTICLE
The second green revolution? Production of plant-based biodegradable
plastics
Brian P. MOONEY1
University of Missouri, Interdisciplinary Plant Group, Division of Biochemistry, and Charles W. Gehrke Proteomics Center, 214 Christopher S. Bond Life Sciences Center,
1201 Rollins St, Columbia, MO 65211, U.S.A.
Biodegradable plastics are those that can be completely degraded
in landfills, composters or sewage treatment plants by the action
of naturally occurring micro-organisms. Truly biodegradable
plastics leave no toxic, visible or distinguishable residues
following degradation. Their biodegradability contrasts sharply
with most petroleum-based plastics, which are essentially indestructible in a biological context. Because of the ubiquitous use of
petroleum-based plastics, their persistence in the environment and
their fossil-fuel derivation, alternatives to these traditional plastics
are being explored. Issues surrounding waste management of
traditional and biodegradable polymers are discussed in the context of reducing environmental pressures and carbon footprints.
The main thrust of the present review addresses the development
of plant-based biodegradable polymers. Plants naturally produce
numerous polymers, including rubber, starch, cellulose and storage proteins, all of which have been exploited for biodegradable
plastic production. Bacterial bioreactors fed with renewable resources from plants – so-called ‘white biotechnology’ – have also
been successful in producing biodegradable polymers. In addition
to these methods of exploiting plant materials for biodegradable
polymer production, the present review also addresses the
advances in synthesizing novel polymers within transgenic plants,
especially those in the polyhydroxyalkanoate class. Although
there is a stigma associated with transgenic plants, especially
food crops, plant-based biodegradable polymers, produced as
value-added co-products, or, from marginal land (non-food), crops
such as switchgrass (Panicum virgatum L.), have the potential to
become viable alternatives to petroleum-based plastics and an
environmentally benign and carbon-neutral source of polymers.
INTRODUCTION
Australia, the City of San Francisco and an attempted ban in the
State of California.
The search for alternatives to traditional petroleum-based
plastics is progressing to the point that not just the source,
but also the downstream consequences, are being addressed in
the form of biodegradable plastics. Although photodegradable
plastics continue to be explored [2], these alternatives must be
constantly exposed to sunlight and so are not suitable for landfill
disposal. Biodegradability in composters or municipal landfills
is the goal. Even large chemical companies, like BASF with its
Ecoflex® product, are touting their biodegradable-plastics efforts
to address the downstream consequences on the environment of
conventional plastics.
In this context, production of biodegradable plastics in plants
is an enviable goal. Plants naturally produce many polymers,
such as starch or cellulose, and these have been exploited
for plastics production. Additionally, novel plastics, like the
PHAs (polyhydroxyalkanoates), are also being synthesized in
plants. Plants can be considered as solar-driven biofactories with
the potential for being renewable, sustainable, scaleable and
relatively environmentally benign sources of edible vaccines or
‘plantibodies’ [3,4], novel oils and fatty acids (reviewed in [5]
and [6]) and biodegradable plastic. The present review will detail
the efforts to produce biodegradable plastics in plants and will be
extended to include the broader topics of biodegradable polymers
derived from plant materials, including starch and cellulose.
The term ‘plastic’ is defined as any of numerous organic synthetic
or processed materials that are mostly thermoplastic or thermosetting polymers of high molecular mass and that can be made into
objects, films or filaments (Merriam–Webster Dictionary definition). The majority of plastics are synthetic, using petroleum both
as feedstock and as energy during manufacture. With the price
of oil rising and easily accessible reserves dwindling, alternative
sources of fuel and of oil-based commodities such as plastics are
being explored across the world. The environmental concerns of
the oil-based economy are also being widely voiced as companies
and individuals attempt to reduce their carbon footprints.
Plastics are truly a huge industry. Data for first-quarter
2008 U.S. production show that total output of all petroleumbased plastics exceeded 11.34 billion kg (25 billion lb; American
Chemistry Council press release April 2008). With this volume of
production, the economies of scale become evident [for example,
polypropylene or polyethylene cost approx. 40 U.S. cents/lb or $1
(or approx. €1.5/£1.3)/kg]. With a market value of $100 billion
per year, packaging accounts for almost one-third of plastic use
in the United States, followed by construction and consumer
products [1]. The simple ‘paper or plastic’ decision one makes in
the supermarket eventually has consequences for the environment.
These consequences have been recognized in the form of bans on
plastic bags in many of the EU (European Union) countries, China,
Key words: biodegradable plastic, natural polymer, polyhydroxyalkanoate, transgenic plant.
Abbreviations used: ADM, Archer Daniels Midland; ASTM, American Society for Testing and Materials; CaMV, cauliflower mosaic virus; CAP,
cellulose acetate phthalate; CEN, European Committee for Standardization; ER, endoplasmic reticulum; EU, European Union; GMO, genetically
modified organism; HB, hydroxybutyrate; HV, hydroxyvalerate; IPP, isopentenyl diphosphate; PA, protective antigen; PCLH, polycaprolactone
(polyhexanolactone)/hexamethylene di-isocyanate; PDC, pyruvate dehydrogenase complex; PLA, poly(lactic acid); PHA, polyhydroxyalkanoate; PHB,
polyhydroxybutyrate; PHBV, PHB-co-polyhydroxyvalerate; SPI, soy protein isolate; UTR, untranslated region.
1
email [email protected]
c The Authors Journal compilation c 2009 Biochemical Society
220
B. P. Mooney
WASTE MANAGEMENT
Studies on the ecological footprint of humanity have suggested
that sustainability of human pressures on the world’s ecological
resources shifted from 70 % of global regenerative capacity in
1961 to 120 % in 1999 [7]. This translates to a clear and simple
fact: that we consume more than can be regenerated by the
biosphere. A clear example is crude oil, which obviously cannot be
regenerated on a human timescale. Petroleum-based plastics add
to this ecological imbalance, both from a source point of view and
from a waste management standpoint. Traditional plastics have
been engineered to be stable in many types of environments and
to persist for many years. This central ‘purpose’ of plastics and
their cost-effective production (i.e. that they are cheap) has led to
their ubiquitous use and is also the main reason that their disposal
presents such problems. The ubiquitous use of plastics has led
to their comprising about 12 % of the 227 tonnes/metric tons
(250 million short tons) of municipal waste produced annually
in the United States. Recycling recovered about 30 % of this
plastic from the wastestream, but that still allows plastics to
accumulate in the environment at a rate in excess of 18.2 metric
tons (20 million short tons) per year (source: http://www.epa.gov/
epaoswer/non-hw/muncpl/msw99.htm). Similar values for
plastic waste apply in the EU, where the typical generation of
solid waste in 2001 was 520 kg/year per person, of which approx.
10–15 % was plastics. Most of the EU’s municipal waste is sent
to landfills (45 %), but almost 40 % is recycled or composted,
and a further 18 % is used for energy production by incineration (source: http://www.eea.europa.eu/themes/waste/aboutwaste-and-material-resources). Most of the common types of
petroleum-based plastic (e.g. polyethylene, PVC, polypropylene
and polystyrene) are non-degradable, although incorporating
starch into polyethylene allows breakdown of the polymer
state (more correctly called ‘disintegration’ rather than true
‘biodegradation’) in composting environments [8]. Additionally,
municipal solid waste (including plastics) is being tested for power
generation due to a relatively new EU landfills directive [9]. In
Asia, plastic waste is also being explored as a source of fuels for
power generation; for example, in Korea, polyethylene has been
tested as an additive to coal-burning blast furnaces [10].
Although efforts have been explored in source reduction (i.e.
replacing plastics at the manufacturing/packaging stage), in most
cases recycling and disposal are the main ways in which plastics
are dealt with post-consumer. A clear example of one of the more
recent increases in use of plastics is bottled-water sales. Annual
U.S. sales of bottled water increased almost threefold between
1997 and 2006, growing from a $4 billion to a $10.8 billion
industry. In the space of 20 years (1987–2006), U.S. per capita
consumption of bottled water increased from 21.6 to 104.5 litres
(5.7 to 27.6 U.S. gallons) [11]. Unfortunately, many water and
soda bottles are used ‘on the go’ and so are more likely to
end up in ordinary rubbish collections rather than separated for
recycling. Although there have been some ingenious strategies
for separation of different types of plastics using electrostatics
[12] and hydraulics [13], consumer separation of classes of
plastics and recycling them in bulk is typically the best course
of action. However, even with recycling, traditional plastics have
a finite re-usability and eventually are only suitable for bulk
applications such as engineered lumber, WPC (wood plastic
composite) [14] or additives to concrete [15].
Clearly a source of biodegradable plastics is an obvious
alternative. A number of countries have implemented certification
standards for biodegradability, with concomitant labelling based
on their degradability and with an emphasis on composting
environments (Figure 1). North America, Europe, and some
c The Authors Journal compilation c 2009 Biochemical Society
Figure 1
Labelling standards for biodegradable plastics
The United States, the European Union and countries in Asia have specific requirements for
biodegradable polymer testing and certification. Companies producing biodegradable polymers
can submit the products for independent testing and certification through the US Composting
Council or the Biodegradable Products Institute in New York, Vincotte in Brussels, DIN
(Deutsches Institut fur Normung) Certco in Berlin, the European Bioplastics Association and
the Biodegradable Plastic Society in Japan. Their labels are based on the ASTM 6400-99
and EN 13432 standards for biodegradable and compostable plastics. As examples, the Figure
shows logos issued by Vincotte in Brussels, the European Bioplastics Association and the
US Composting Council/the Biodegradable Products Institute, and are reproduced with their
permission.
countries in Asia have enforced standard product testing to achieve
certification for labelling based on the ASTM/CEN (American
Society for Testing and Materials/European Committee for
Standardization) system of classification. The ASTM 640099 designation covers ‘standard specification for compostable
plastics’ and EN 13432 covers ‘proof of compostability of plastic
products’. Two pertinent definitions from the ASTM standard are
as follows:
r biodegradable plastic: a degradable plastic in which the
degradation results from the action of naturally occurring
micro-organisms such as bacteria, fungi and algae
r compostable plastic: a plastic that undergoes degradation
by biological processes during composting to yield CO2 ,
water, inorganic compounds, and biomass at a rate consistent
with other compostable materials and leaves no visible,
distinguishable, or toxic residue [16]
The ‘residue’ clause in the second definition is an important
distinction that favours truly biodegradable plastics rather than
blended materials that disintegrate or partially biodegrade.
PLANT-BASED BIODEGRADABLE PLASTICS
Although plant-based biodegradable plastics are not new, the
current interest in green technologies has lead to a renewed
interest in using plants for a number of applications. Many of these
Plant-based biodegradable plastics
Figure 2
Natural polymers: rubber and proteins
(A) Natural rubber is a polymer of isoprene units arranged in cis -1,4 linkages. Natural rubber
(unlike synthetic) also has minor protein, lipid, carbohydrate and mineral components that
are responsible for its properties of resistance to abrasion and impact, elasticity, efficient heat
dispersal, and resilience and malleability at cold temperatures. (B) Gluten is a polymer of
glutenin that has inter- and intra-disulfide bonds that promote elasticity of the gluten polymer.
The gliadin proteins are thought to intercalate within the glutenin matrix and are held in place
by hydrophobic interactions and hydrogen bonding.
applications are traditionally based on crop plants, be it for ethanol
production, biomass production for power generation and sources
of novel compounds such as pharmaceuticals. Additionally, with
pressures on land use for farming, the concomitant use of fertilizers and pesticides, and various (perceived?) threats of GMOs
(genetically modified organisms) to the human food chain, nonfood crops are also being explored as biofactories.
For plastics production, plant materials can be harvested and
used directly (natural rubber is a clear example), or plant polymers
can be derivatized to produce plastics. Plants can be used
indirectly as a nutrient source in bioreactors for biodegradable
plastic production. Plants can also be genetically modified to
directly synthesize novel polymers.
NATURAL PLANT POLYMERS
Plants naturally produce a number of structural and carbonreserve polymers. Polysaccharides are estimated to make up
approx. 70 % of all organic matter. Cellulose accounts for about
40 % of total organic matter and is the most abundant macromolecule on earth. Lignin comprises 15–25 % of a typical woody
plant. Starch is also a major component of global biomass, and
the ability to digest starchy foods is thought to have played a role
in human evolution and its success over other species [17]. A
number of natural polymers have been exploited for commodity
manufacturing, and most of these products retain the inherent
biodegradability of their carbohydrate building blocks.
Rubber
Rubber, a polymer of isoprene (cis-1,4-polyisoprene; Figure 2A),
is the most widely used natural plant polymer. All commercial
production of rubber comes from the Brazilian rubber tree
(Hevea brasiliensis). Despite the plant’s common name, more
221
than 90 % of the world’s natural rubber supply now actually comes
from South-East Asia. Although natural rubber production only
accounts for 40 % of demand (the remainder is synthetic), natural
rubber is superior to synthetic, owing to its molecular structure
and high-molecular mass (> 1 MDa), which confers resistance
to abrasion and impact, elasticity, efficient heat dispersal and
resilience and malleability at low temperatures. These properties
have not been duplicated in synthetic rubber because of the
unique, somewhat undefined, secondary compounds (proteins,
lipids, carbohydrates and minerals) in natural rubber.
So although synthetic-rubber production has reached a plateau,
natural-rubber production continues to increase, particularly in
China and Vietnam [18]. The main reason production has
switched to Asia from South America is the South American
leaf blight fungal infection caused by Microcyclus ulei, which
originates in the Amazon region. All attempts at commercialscale rubber cultivation in South and Central America have been
thwarted by this, as yet, uncontrolled fungus. More recently,
molecular-breeding approaches have been explored to attempt to
confer resistance traits, and, with strict quarantine, plantations in
Asia have been unaffected by the blight. Rubber plantations are
composed of clonal trees; in fact, the commercial rubber tree
is one of the most genetically restricted crops grown, making the
Hevea rubber tree particularly susceptible to pathogen attack [19].
Natural rubber’s isoprene monomers are derived almost
exclusively from IPP (isopentenyl diphosphate). IPP is derived
from cytosolic acetyl-CoA through the mevalonate pathway. Polymerization of the isoprenyl units is catalysed by ‘rubber elongation
factor’ [20] or ‘particle-bound rubber transferase’. This enzyme
is a cis-prenyltransferase, which adds isoprenyl units from IPP
to form the polymer. Two transferase cDNAs were cloned from
H. brasiliensis, then expressed in, and purified from, Escherichia
coli. Although low-molecular-mass rubber (< 10 kDa) was made
in vitro using the purified enzymes, the addition of a fraction from
latex containing washed rubber particles permitted the formation
of a high-molecular-mass product [21]. Clearly, then, other factors
present in the natural-rubber particles are required for highmolecular-mass polymer production, but these components are
ill-defined [22], although magnesium cations seem to regulate the
activity of the prenyltransferase [23].
Other natural sources of rubber are being explored owing to
H. brasiliensis’ disease susceptibility and also because of the prevalence of allergy to latex. Type I latex allergy is based on the
reaction to natural-rubber latex proteins, and type IV is based on
chemical additives, in gloves, for example [24]. Latex allergies
occur at a rate of about 1 % of the general population, but at
a 10–20-fold higher rate in healthcare workers [25]. Although a
number of plant species (about 2500) synthesize rubber, many of
these are not suitable for commercialization because they do not
produce the high-molecular-mass form of the polymer with its
concomitant useful properties.
Two promising alternatives to Hevea that do produce highmolecular-mass rubber are guayule (Parthenium argentatum
Gray) and Russian dandelion (Taraxacum kok-saghyz) [26].
Guayule is a non-tropical shrub native to Mexico and parts of the
U.S. South-West. Guayule rubber is considered hypoallergenic
compared with that from Hevea because it is less proteinaceous
and the proteins that are present do not cross-react with Hevea
immunoglobulins [27]. However, unlike Hevea, the guayule shrub
cannot be ‘tapped’ for latex extraction, and the kilogram yield per
acre of rubber is about one- to two- thirds that of Hevea. This
obviously increases the cost of production, and without significant
improvements in cultivation, harvesting and processing, guayule
rubber will likely remain as a specialized-use product (reviewed
in [26]). The Yulex Corporation in Arizona has built a pilot plant
c The Authors Journal compilation c 2009 Biochemical Society
222
B. P. Mooney
for its Yulex Natural Rubber product based on guayule latex and
they plan to couple latex production with cellulosic biomass for
biofuels and power generation.
Russian dandelion produces a higher-molecular-mass polymer
than either Hevea or guayule (about 2 MDa), which accumulates
in lactifers in the roots. It was discovered in Kazakhstan in the
1930s and was used as a source of motor-tyre rubber during World
War II by a number of countries, including the United States and
the U.K. Although production yields are poor and large-scale
cultivation is hampered by cross-breeding with weedy species
and labour-intensive crop maintenance, it can be useful as a model
species for rubber biosynthesis because, in part, of its relatively
short life cycle (altered rubber phenotypes can be screened within
6 months) compared with guayule and Hevea. A further advantage
is the ‘double-cropping’ potential of Russian dandelion, both as
a source of rubber and its fructose-based storage sugar, inulin,
which accumulates at 25–40 % of root dry weight and could be
used in bioethanol production [26].
Proteins
Proteins can be considered polymers of amino acids that are
combined in various combinations that confer function on the
basis of their side-chain structure and on the arrangement
of the amino acid monomers within a protein for tertiary/
quaternary structure. Although plants are being used to synthesize
novel proteins (see the subsequent section), a number of naturally
occurring proteins have been exploited as plastics. For example,
proteins from wheat (Triticum aestivum), maize (Zea mays) and
soybean (Glycine max) , particularly zeins and glutens, have been
used as the basis for biodegradable polymers.
Gluten is a composite of the proteins glutenin and gliadin (with
other globulin and albumin components). Two-dimensional gelelectrophoretic analysis shows multiple spots corresponding to
multiple isoforms of glutenin [28]. The gluten fraction comprises
about 80 % (w/w) of the wheat seed protein and about 8–15 %
of seed dry weight. Gluten is easily harvested from seed by
washing away soluble components (mainly starch) to produce
an essentially pure protein isolate [28]. Gluten is used as a protein
source in a number of food products, for example in preparing
fibrous meat analogues composed of gluten, soy protein and
starch [29]. Gluten confers elastic properties on dough, owing
to the presence of disulfide-linked glutenin chains [28], gliadin
intercalating with the glutenin chains (Figure 2B). This property
has led to research in using gluten and zein/gluten composites
as plastics. Gluten coated with zein has been used to produce a
plastic that is biodegradable and yet has a compressive strength
similar to that of polypropylene. Production of this polymer is
very simple; gluten and zein are mixed in ethanol and then formed
in moulds. Essentially the zein forms a ‘glue’, which binds the
matrix (gluten) and causes aggregation; pressure moulding then
removes the ethanol and allows the polymer to form [30]. Maize
gluten has been used in the manufacture of wood composites. In
this process, wood fibres are mixed with maize gluten, plasticized
using glycerol, water and ethanol, and then extrusion-moulded
into pots that are reasonably water-resistant and biodegradable
[31]. Gluten-based polymers are very efficiently degraded in
soil and in liquid environments, being degraded completely after
50 days in soil and about ten times more quickly in liquid [32].
Medical uses for gluten-based polymers also exist. When gliadin
is purified from wheat gluten using ethanol extraction, it can
be spun into fibres that allow adhesion and growth of muscle
cells. Using plant proteins for this purpose (rather than collagen)
is postulated to be superior, owing to the mechanical properties
c The Authors Journal compilation c 2009 Biochemical Society
of the gliadin fibre and because it obviates the risk of disease
transmission [33].
Zein is an alcohol-soluble protein extracted from maize gluten
meal. Zein accounts for approx. 65 % of the protein content of
the meal. Zein is a member of the prolamin family, which are
proteins characterized by a high percentage of hydrophobic amino
acid residues, including proline [34]. Its purification scheme
and its use as a plastic resulted in the award of a number of
patents in the late 19th and early 20th Centuries. Commercial
production of zein started in the 1930s and was used in the
manufacture of buttons, fibres, adhesives etc., with production
peaking in the 1950s at around 6.8 million kg (15 million lb)/year
(reviewed in [35]). More recently, production has fallen to about
0.45 million kg (1 million lb)/year, and it is produced by two
companies, one in the United States (Freeman Industries) and
a second in Japan (Showa Sangyo Corp.). Its current price
ranges from $10 to $40/kg, depending on the purity and so is
considered a ‘value-added’ by-product of maize [36]. Zein fibres
were used for clothing and furniture stuffing, and were marketed
under the brand name VicaraTM in the 1950s (reviewed in [36]).
Zein has also been used for film production, for example for
use as a grease-resistant coating for paper that is resistant to
microbial attack [37,38]. Addition of food-grade antimicrobial
agents, such as lysozyme and the peptide nisin, enhanced the
application for food-packaging films [39]. Pure zein polymers
tend to be brittle and hygroscopic [40], but the addition of
various plasticizers has allowed production of useful plastics.
PCLH [polycaprolactone (polyhexanolactone)/hexamethylene diisocyanate] was used to coat a zein matrix by the addition
of 10 % zein to about 50 % PCLH. Through interactions
with glutamate, glutamine, tyrosine and histidine side chains,
a polymer with superior mechanical properties (specifically
flexibility) was produced [41]. Porous zein three-dimensional
scaffolds with the potential for use in bone-tissue engineering
have been produced by mixing zein with NaCl crystals. Once
this polymer is pressure-moulded, the salt is removed in hot
water. Following freeze-drying, the zein forms a porous polymer
with interconnecting pores; this interconnection allows bloodvessel proliferation during bone growth, but the mechanical
properties (i.e. brittleness) of the polymer would limit it to
non-weight-bearing applications [42]. An improved polymer was
subsequently achieved by coating the zein with hydroxyapatite
[43]. Multiple uses of zein microspheres for sustained or targeted
drug delivery have also been reported [44–46]. These reports state
that simply mixing the drug and purified zein in aqueous ethanol
solutions results in the production of microspheres that have an
average diameter of about 1 μm and allow gradual release of the
drug.
An early use of plasticized soy protein was by the Ford Motor
Company. In 1940, Henry Ford applied for patent protection on
his invention for automobile body construction, which stated that
“the object of my invention is to provide a body construction in
which plastic panels are employed, not only for the doors and side
panels, but also for the roof, hood and all other exposed panels
on the body.” The panels were made from soy meal (about 50 %
protein) that was cross-linked with formaldehyde with the addition
of phenol or urea to increase strength and resistance to moisture.
The panels were layered over a unique (at the time) tubular
steel cage that provided the structural rigidity for the car, which
was actually the focus of the patent application. The patent was
issued on 13 January 1942 (Automobile body construction, U.S.
Patent 2269451). Henry Ford was photographed demonstrating
the strength of the panels by swinging an axe at the car
(although the photograph fails to show the impact or any damage
associated with it). The prototype was never put into production,
Plant-based biodegradable plastics
Figure 3
223
Natural polymers: cellulose and starch
Cellulose and starch can be modified to produce biodegradable polymers with various R groups in place of hydrogen. Nitrocellulose is cast as films/paper, and cellulose acetate and CAP have
been used for both films and nanospheres. Derivatization is achieved by chemical means from the isolated plant material. Starch has been used directly for polymer production and also through
derivatization with caprolactone, cellulose and various vinyl alcohols. The degradation properties of starch polymers are dependent on the relative proportion of pure starch to secondary compounds.
The degradation of the Mater-BiTM starch polymers is shown in the bottom left-hand panel.
partly because the plastic was susceptible to microbial degradation
(it was biodegradable!) and was not adequately moisture resistant,
and apparently the car smelled strongly of formaldehyde [47].
SPI (soy protein isolate) is a minimum of 90 % protein (by
dry weight) and is purified from defatted soy flour. The glycinin
and conglycinin seed storage proteins are the bulk of the protein
content. Although SPI has been widely used as a food ingredient, it
also has been used as a basis for plastic production. Heat-induced
cross-linking of the proteins creates a thermoplastic polymer
based on three-dimensional networks of disulfide bonds, as well
as hydrophobic interactions and hydrogen bonding. Addition of
plasticizers, such as glycerol, improves the structural properties
of the film. Combining 5 % (w/v) SPI and 3 % (w/v) glycerol
in water and then adjusting the pH outside the pI of the major
storage proteins to prevent aggregation/coagulation (< pH 3 or
> pH 6), followed by heating to 80 ◦C, allowed casting of films
on a levelling table. Alkaline-cast films are more flexible than
acid-cast ones. Although these films are poor moisture barriers,
they are good oxygen barriers and so can be used as a layer in
a multilayer sheet to prevent oxidation of packaged food [48].
Using γ -irradiation for protein cross-linking in film production
has the added benefit of being a common sterilization technique.
γ -Irradiation causes the production of free radicals in the protein
solution, and cross-linking through biphenolic compounds is
achieved. These films have superior puncture strength and deformation properties than have the thermoplastic SPI films, and
this could be further improved by the inclusion of carboxymethylcellulose or poly(vinyl alcohol) [49]. More recently, composites
of SPI and chitin or cellulose microfibres (‘whiskers’) have
been produced that have superior properties compared with
pure SPI-based plastics [50,51]. Cellulose whiskers with average
dimensions of 1.2 μm long × 90 nm diameter were prepared from
cotton by hydrolysis in sulfuric acid. Mixing SPI, whiskers and
glycerol in water was followed by heating and pressure moulding
to produce the polymer. The SPI–cellulose composites have a
superior moisture resistance, tensile strength, thermal stability
and flexibility to SPI-based polymers, but retain biodegradability
[51]. Similar improvements in the quality of SPI-based plastics
have been achieved by using polylactide in the composite [52].
Cellulose
Although much research using starch and cellulose reserves is
based on their use for biofuels (reviewed in [53]), they have also
been used for plastic production through derivatization.
Cellulose is a linear polymer of β-1,4-linked D-glucose
(Figure 3). Although cellulose cannot be thermally processed
into plastics, owing to decomposition of its hydrogen-bonded
structure, derivatized cellulose has been employed for plastics
production. ParkesineTM (named after its inventor, Alexander
Parkes) was an early pressure-mouldable form of nitrocellulose
(Figure 3) used as a replacement for ivory in the late 19th Century.
The cellulose was derived from cotton fibres solubilized with
nitric acid and ethanol. This solution, called ‘collodion’, could
be cast in sheets or pressure-moulded. Parkesine was eventually
updated and replaced by celluloid, which used camphor, a tough
gummy volatile aromatic crystalline compound (C10 H16 O), as
a plasticizer. The addition of the camphor plasticizer made
celluloid more flexible and mouldable and less likely to fracture
compared with Parkesine. Celluloid was also used as a substitute
for ivory, specifically for billiard balls and, towards the end
of the 19th Century, was used as a photographic film for still
photographs and movie films and even as windshields [47].
Cellulose acetate (Figure 3), another derivative of cellulose,
is prepared by dissolving cellulose in acetic acid and acetic
anhydride in the presence of sulfuric acid. Sheets are then cast, or
c The Authors Journal compilation c 2009 Biochemical Society
224
B. P. Mooney
fibres spun, typically from an acetone solution. Cellulose acetate
was widely used as film stock in the early 20th Century.
There are a number of medical uses reported for a further
derivative of cellulose acetate, namely CAP (cellulose acetate
phthalate) (Figure 3). CAP is widely used as a coating for pills, but
more specialized uses also exist. For example, CAP microspheres
have been used to improve the retention of anti-diabetic drugs
in the gut. The microspheres float in the stomach and so are
not evacuated as quickly and therefore aid drug release [54].
Controlled release of drugs at sites of bone regeneration is also an
application of CAP [55]. Additionally, there are reports of the use
of CAP as an anti-HIV1 agent [56,57]. Cellophane is restructured
cellulose prepared from wood pulp using NaOH to break down the
crystalline structure, followed by gelling with acid [47]. Despite
being mostly replaced by synthetics, it is still used in some foodpackaging applications, where, for example, the cellophane is
impregnated with an antibacterial peptide, nisin, and used to wrap
fresh meat [58], and some specialized medical uses, for example
by preventive adhesion of intestine and abdominal wall tissues to
allow proper healing [59]. Additionally, cellophane is still widely
used for packaging.
Figure 4
Starch
Starch is one of the cheapest and most abundant agricultural
products and is completely degradable in a number of environments [60]. These properties have lead to exploring starch as a
polymer for various applications. Thermoplastic starch is prepared
by temperature and pressure extrusion and/or moulding. This
pure-starch polymer degrades very quickly in a composting
environment (the process lasting about a month), but it tends to age
poorly and is not moisture-resistant [61]. Production of various
thermoplastic starch composites is based on mixing starch with
vinyl alcohols, and these types of polymers tend to be more stable.
However, the biodegradability of these composites is inversely
proportional to their starch content [62]. An Italian company,
Novamont, has commercialized four starch-based biodegradable
plastic composites that vary in their additives and are trademarked
Mater-BiTM (Figure 3). Their output is about 36 300 metric tons
(40 000 short tons) of biodegradable plastic pellets/year, which
are supplied to a number of packaging companies. Their Vclass polymer is about 85 % thermoplastic starch, degrades very
quickly in compost and is used as a polystyrene replacement.
Their Z-class polymer is composed of starch and polycaprolactone
(a synthetic polyester), is biodegradable in composting
environments (20–45 days) and is mainly used for bags and films.
The Y-class polymer can be injection-moulded, is composed
of starch and modified cellulose and lasts about 4 months in
composting environments. Finally the A-class polymer, which is
a mixture of starch and ethylene vinyl alcohol, is their most stable
form of polymer, which is not compostable, but does biodegrade
(in about 2 years) in a simulated sewage- sludge treatment
plant. The A-class polymer can be used for rigid and expanded
items [63]. The Mater-BiTM -based products include garden-waste
(leaves, twigs etc.) and rubbish bags, nappies (diapers) and organic
food packaging used by the U.K. supermarket chain Sainsbury’s.
Medical uses for starch-based polymers are also being explored,
for example as scaffolds for bone-tissue engineering because of
their biocompatibility, biodegradability and porous nature, which
allows blood-vessel proliferation during bone growth. The SEVAC type polymer used is a 1:1 (w/w) blend of cornstarch and
ethyl vinyl alcohol, which is mouldable, non-toxic and does
not inhibit cell growth [64]. Additionally, implantation of the
SEVA-C polymer in rats did not produce a severe inflammatory
c The Authors Journal compilation c 2009 Biochemical Society
Polymers produced by bacteria using plant materials
Using plant carbon reserves for industrial biotechnology feedstocks – typically sugar, starch,
or oils – various polymers can be produced by bacterial fermentation. (A) PLA is produced
by bacteria of the genus Lactobacillus , and often D-glucose (maize dextrose) is the carbon
source. It is a high-molecular-mass polymer (> 1 MDa) that can be used for films and moulded.
(B) PHB and (C) PHBV are in the class of PHAs that naturally occur in many bacteria as carbon
reserves. Typically, highly refined sources of glucose derived from plants are used in batch-fed
bacterial cultures; however, use of industrial wastewater and plant oils have also shown promise
for PHA production through fermentation.
response, demonstrating it as a good candidate for implants for
bone regeneration [65].
BIOCONVERSION OF PLANT POLYMERS: FERMENTATION
Currently, bioethanol production is a major focus for the use
of plant starch and sugar reserves. Attempts at increasing the
amounts of reserves accumulated have been addressed through
altering the activity of the ADP-glucose pyrophosphorylase
enzyme, altering source-sink allocation of carbon or altering
carbon partitioning within storage organs in favour of starch
synthesis. These and other approaches were reviewed recently
by Smith [66]. Use of plant carbon reserves for plastics has
also been addressed through fermentation using various strains
of bacteria for different types of polymer production, such as
PLA [poly(lactic acid)] and polyhydroxyalkanoates.
PLA [Poly(lactic acid)]
PLA (also known as polylactide) is a polymer of lactic acid
(2-hydroxypropionic acid) that was first developed by Dow
Chemicals in the 1950s. However, because of the high cost of
production, its use was limited to specialized medical devices
such as sutures, soft-tissue implants etc. Figure 4(A) shows
the structure of the PLA polymer. More recent advances in
fermentation have resulted in a dramatic cost reduction to
the point that PLA is widely used in packaging. PLA is
produced by naturally occurring lactic acid bacteria of the
genus Lactobacillus, which ferment hexose sugars. Lactate
dehydrogenase converts pyruvate into lactate, with concomitant
oxidation of NADH (reviewed in [67]). Production of PLA is
classed as heterofermentative or homofermentative. The former
Plant-based biodegradable plastics
method produces less than 1.8 mol of lactic acid/mol of hexose,
along with a number of other metabolites, such as acetic acid,
ethanol, glycerol, mannitol and CO2 . The latter homofermentative
method produces an average of 1.8 mol of lactic acid/mol of
glucose and only minor amounts of other metabolites, which
allows about a 90 % conversion rate of glucose into lactic acid
(reviewed in [68]). Polymerization of lactic acid to PLA by
chemical synthesis produces a large amount of racemic D,L-lactic
acid, but is very expensive. The method employed by Dow–
Cargill to produce their NatureWorks® PLA product involves
condensation of lactic acid to lactide (circularized lactic acid; two
monomers both D-lactic acid, both L-lactic acid or a mixture of the
two enantiomers), followed by ring-opening polymerization [69]
to produce high-molecular-mass (> 100 kDa) PLA. NatureWorks
LLC uses dextrose maize sugar (D-glucose), from Cargill, as a
carbon source for fermentation. They produce over 136 million kg
(300 million lb)/year in their plant in Blair, Nebraska, U.S.A. Over
70 companies are using their PLA for various film and moulded
applications, because of its high gloss, high transparency and
physical properties similar to those of poly(ethylene terephthalate)
(source: http://www.natureworksllc.com). PLA is compostable
and degrades in a two-stage process that involves reduction in molecular mass by hydrolysis, followed by biodegradation by microorganisms [68]. For example, a number of filamentous fungi
naturally present in soil can biodegrade hydrolysed PLA [70].
In the United States, NatureWorks LLC also runs a ‘buy-back’
program for PLA-based products as part of their commitment to
producing an environmentally friendly plastic. A recent advance
in PLA production has been made by utilizing a ‘microbial
factory’ instead of ring-opening polymerization for generation
of high-molecular-mass PLA. A Japanese group (including
researchers for the Toyota Motor Corporation) has modified a
polyhydroxyalkanoate-synthesizing enzyme to facilitate polymerization of lactic acid, thereby allowing polymer synthesis within
the microbe without the need for chemical polymerization [71].
PHAs
PHAs are hydroxyalkanoate polyesters of various chain
lengths. PHAs are carbon and energy reserves stored as insoluble
inclusion bodies in most bacteria under nutrient-limiting and
carbon-excess conditions [72]. The inclusion bodies were first
observed in the late 19th Century, and the composition of
PHA was first examined in the 1920s by Maurice Lemoigne
at the Pasteur Institute in Paris (reviewed in [73]). PHB
(polyhydroxybutyrate; Figure 4B) has properties that resemble
those of polypropylene and is amenable to injection moulding,
extrusion blow moulding and fibre spray-gun moulding. p(3HBco-3HV) or simply PHBV, the co-polymer of hydroxybutyrate and
hydroxyvalerate (Figure 4C), is similar to the PHB homopolymer,
although more flexible [72]. PHAs have been used for the
manufacture of films, coated paper and board, compost bags and
disposable flatware and can also be moulded for the production
of bottles and razors and are completely biodegradable (to CO2
and water). These properties make PHAs an attractive source of
non-polluting renewable plastics and elastics [74]. A considerable
body of research has gone into production of various PHAs in
transgenic plants (see the subsequent section), but the use of plant
products as carbon sources for bacterial bioreactors has also been
explored.
Fermentative production of PHAs using the bacteria Ralstonia
eutropha and Alcaligenes latus has been established for almost
40 years. The R. eutropha PHA synthase operon encodes a
thiolase (phaA), a reductase (phaB) and a synthase (or polymerase,
phaC) [75]. Although wild-type R. eutropha can only use fructose
225
as a carbon source, a glucose-utilizing mutant of R. eutropha was
engineered and used in the production of Biopol® . Biopol®
was originally developed by ICI (Imperial Chemical Industries),
which began worldwide commercialization of a family of
PHBV polymers in the 1980s. In 1990, the agricultural and
pharmaceutical divisions of ICI were spun off as Zeneca Ltd,
and then Biopol® was bought by Monsanto in 1996. However,
soon thereafter, Monsanto closed its bioplastics division as
it refocused on agricultural applications of biotechnology and
moved away from industrial applications. The patent rights were
then bought by a Cambridge, (MA, U.S.A.) company called
Metabolix (source: press release 16 May 2001, Metabolix.com).
In addition to the numerous naturally occurring PHA-producing
prokaryotes, other bacteria that do not normally produce PHAs
can be modified to do so. For example, cloning the PHA
operon into E. coli has also allowed various types of PHAs to
be produced. Specifically, the co-polymer PHBV is produced
by adding glucose and propionic acid to the culture medium
(reviewed in [76]). Figure 4(C) shows the structure of the copolymer. Growing modified E. coli strains under specific growth
conditions can be used to alter the structure of the co-polymer,
specifically the relative abundances of the HB (hydroxybutyrate)
and HV (hydroxyvalerate) monomers. Adding valine to the
glucose-based culture medium results in 2 % HV co-polymer,
and including threonine along with valine results in 4 % HV
[77]. Typically PHAs are produced using pure cultures of defined
bacterial isolates and supplying highly purified carbon sources
like glucose. This type of production yields an average cost of
$14/kg (€9/kg; £7.9), considerably more than petroleum-based
plastics (e.g. polyethylene). However, production of PHA from
mixed cultures using various wastestreams has been proposed
as an effective way to reduce these costs [78]. Yu et al. [79]
described using industrial food wastewater as a carbon source
for production of PHA by A. latus. They investigated the use of
soy wastes from a milk dairy and malt wastes from a brewery
as carbon sources. The malt-waste-fed bacteria produced 70 %
polymer (on a cell weight basis), whereas the soy-waste-fed
and sucrose-fed cultures produced a little over 30 % polymer.
A recombinant strain of E. coli was used to produce PHB with
whey and maize steep liquor as the main carbon and nitrogen
sources respectively. The whey liquor is a by-product of the
cheese industry and maize steep liquor is a by-product of wetmilled maize. E. coli was transformed with a plasmid containing
the phaABC genes expressed from the phage T5 promoter and
two lac operator sequences. PHB polymer accumulated to over
70 % of the cell dry weight, although this amount tended to decrease as the cultures aged, a phenomenon attributable to plasmid
loss during growth. The PHB obtained was very similar to
the naturally produced PHB, with a high molecular mass and
a similar crystalline structure [80]. Interestingly, Metabolix
and ADM (Archer Daniels Midland) have co-operated to produce
a bacterially produced PHA with the trade name MirelTM . They
are in the process of constructing a commercial-scale production
plant adjacent to ADM’s wet maize mill in Clinton, Iowa,
and plan to provide polymer in the second quarter of 2009 with a
production capacity of 49.8 million kg (110 million pounds)/year
(source: http://www.mirelplastics.com/).
Plant oils have also been successfully used to produce the
typical thermoplastic PHAs. PHB and the PHBV co-polymer
can be produced by wild-type A. eutrophus and a modified
strain containing the synthase gene from Aeromonas caviae
respectively. Wild-type A. eutrophus accumulates 80 % of its cell
weight as PHB when fed olive, maize or palm oils, and similar
amounts of PHBV are produced by the recombinant strain using
the same plant-oil carbon sources. Using these cheap oils as a
c The Authors Journal compilation c 2009 Biochemical Society
226
B. P. Mooney
renewable carbon source could increase the economic viability of
fermentative PHA production [81].
Despite these advances in using relatively cheap plant oils
and carbohydrates or industrial effluents as carbon sources,
the limitations of bioreactor production still exist. PHA or
PLA production in bioreactors requires careful monitoring of
the conditions within the bioreactor, including the cell density,
nutrient reserves, accumulation of waste products etc. Bioreactors
also have certain size and production capacity limits [82].
Additionally, pure culture production of PHA is thought to
consume more fossil-fuel resources than petroleum-based plastics
[83,84]. Although mixed-culture PHA fermentative production
is proposed to have significantly less environmental impact
than traditional petroleum-based plastics such as high-density
polyethylene [85], using plants for de novo synthesis of polymers
could theoretically prove an economically viable option, either as
a value-added co-product of food crop plants or perhaps as a novel
use for some marginal land species such as switchgrass (Panicum
virgatum L.).
DE NOVO SYNTHESIS OF POLYMERS WITHIN PLANTS
Traditional plant breeding to produce valuable traits has been
employed since prehistory, when selection of visual phenotypes
led to the domestication of the first crop plants. Wheat, for
example, is thought to have been domesticated about 10 000 years
ago in the Near East [86]. However, it was not until the late
20th Century that molecular manipulation to produce the first
transgenic plants was conducted. Altering plant metabolism to
produce value-added products by transgenic methods has slowly
begun to yield some promising results for plastics production.
Proteins
A number of hurdles need to be tackled before commercialscale production of value-added heterologous proteins (and other
polymers) can be achieved in plants. The DNA encoding the
foreign protein must be stable across a number of generations,
and synthesis of the protein from mRNA, through processing,
translation and, addition of post-translation modifications, must
be considered. Additionally, the type of crop used and the site of
accumulation must be carefully considered to allow high levels
of expression and ease of purification (reviewed in [87]).
Among the number of novel proteins being expressed in
transgenic plants, many of these confer traits on the plant itself, for
example the Bacillus thuringiensis cry toxin for insect resistance
(see [88]). However, in the context of polymer production,
proteins with an inherent commercial value following purification
from the plant are also being produced. Typically, these are highvalue proteins with specialized uses, for example as vaccines.
Production of heterologous proteins within plants has numerous
advantages over production in animal or bacterial systems. Plantbased therapeutic proteins, for example, are theoretically safer
from a disease point of view than those purified from animal
sources (whether native or recombinant). Also, incorporation
of post-translational modifications such as glycosylation can
be achieved by in planta production, in contrast with bacterial
systems. Although eukaryotic culture systems such as insect and
mammalian cell cultures can be used in many cases to produce
viable proteins, an additional advantage of plants is that they are
renewable and scaleable and require less energy and nutrient input
than bioreactors, which should make plants cost-effective for bulk
applications (reviewed in [89]).
Heterologous protein accumulation within plants can be
achieved through standard transgenic approaches (i.e. nuclear
c The Authors Journal compilation c 2009 Biochemical Society
transformation), through plastid transformation and also through
viral vectors. The latter approach typically leads to transient
expression most commonly targeted to the leaves [90,91] and,
thus, cannot be considered a sustainable approach. Nuclear
transformation, typically mediated by Agrobacterium infection,
allows incorporation of the heterologous genes into the nuclear
genome, (often) followed by stable expression and heritability of
the trait. A number of crop plants, including maize, soybean,
rice and cabbage (Brassica) species, have been successfully
transformed using this technique. In terms of heterologous
protein expression for purification purposes, the goal, obviously,
is to achieve high levels of expression. Avidin was produced
in transgenic maize using a constitutive ubiquitin promoter to
levels exceeding 2 % of aqueous-soluble protein from seed,
where more than half of the protein accumulated within the
embryo. The extracted avidin could be highly purified using
affinity chromatography and was identical with the native eggwhite protein. The study reported that 150 –300 mg of purified
avidin could be extracted/kg of seed, and one high-expressing line
contained 20 g of avidin in 100 kg of maize seed, which was the
equivalent of 900 kg of eggs. The authors postulated that with
the levels of accumulation, the stability and viability of the protein,
and the heritability of the trait, that commercial-scale production
was quite feasible [92]. Nandi et al. [93] reported production in
rice grain of human lactoferrin, a protein which is involved
in iron absorption and has properties ranging from antimicrobial
to immune-system modulation and promotion of cell growth. It
was demonstrated that stable expression over nine generations
could be achieved with production amounts ranging from 4.5 to
5.5 g/kg (about 5 % of the rice flour dry weight). The recombinant
protein was very similar to the native protein from human milk,
although the glycosylation patterns were not identical. Process
simulations suggested that 1 g of lactoferrin would cost $6 (€4.64,
£4.06) to produce, assuming a 5 % (dry weight) level of expression
and using a single cation-exchange purification step [93]. There
are potential problems with nuclear transformation, however; for
example, in the case of avidin production in maize, the plants
exhibited male sterility [92]. Additionally, silencing of transgenes
is also a frequent problem in plants. Gene silencing can occur from
position effects – the influence of the local environment on the
chromosome around the site of transgene insertion, which can
affect, for example, the rates of transcription. Additionally, when
the transgene and native genes are similar in sequence, homologydependent gene silencing of the transgene and/or co-suppression
of transgene and native gene can occur. Achieving high
levels of heterologous protein expression that is stable over a
number of generations can be severely hampered by these events,
although there are strategies to mitigate silencing of nucleartransformed plants (reviewed in [94]).
Production of spider silk proteins in plants genetically modified
by Agrobacterium-mediated nuclear transformation has been
explored. Spider silk proteins used in web construction tend to
be elastic Lycra® -like fibres, whereas the dragline (lifeline) silks
are Kevlar® -like and are stronger than steel on a per-weight basis.
Silk proteins are composed of GPGXX and GGX (one-letter
amino acid code) motifs, along with [GA]n repeat sequences that
confer elasticity and high tensile strength respectively. Transgenic
tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and
thale cress (Arabidopsis thaliana) have been used for silk
production (reviewed in [95]). Yang et al. [96] reported production
of DP1B, a synthetic analogue of spider dragline silk protein, in
A. thaliana. Expression was driven from the CaMV (cauliflower
mosaic virus) 35 S promoter introduced by the standard floraldip method for nuclear transformation by Agrobacterium. When
protein accumulation was targeted to the ER (endoplasmic
Plant-based biodegradable plastics
reticulum) lumen of leaf tissue, up to 6.7 % total soluble protein
was produced. This level could be increased to 8.5 % in the
leaf apoplast and as high as 18 % in seed-targeted expression.
In tobacco and potato, the CaMV 35 S promoter was used to
drive expression in nuclear-transformed plants and expression was
targeted to the ER using the KDEL (one-letter amino acid code)
ER retention signal [97]. These workers concentrated on a silk–
elastin fusion protein and achieved laboratory-scale production
of 80 mg of pure protein/kg of tobacco leaves using a simple
buffer-extraction procedure followed by heating to 95 ◦C for 1 h to
cause aggregation of most proteins, leaving the silk protein in the
supernatant. Purification of the protein, however, does not confer
the structural properties of the silks, as this is accomplished by the
correct assembly of the proteins during the process of spinning in
specialized organs in the spider (i.e. spinnerets), and this natural
process has not been replicated for the recombinant proteins [98].
Generating transgenic plants through plastid genome
(plastome) transformation has a number of advantages. High
levels of transgene expression are typically achieved due to the
plastome copy number and the presence of multiple chloroplasts
per cell [99]. For example, cholera toxin B was expressed at very
low levels by nuclear transformation. However, 400–3000-fold
increases were observed when chloroplasts were used as the site
of transformation/expression [100]. Another advantage of the
transplastomic approach is that multiple genes can be inserted in
a single event [101] without problems of gene silencing, since
genes are targeted to a specific locus on the plastid genome
by homologous recombination. Plastome transformation has the
added advantage of maternal inheritance, so that transmission of
transgenes through pollen is eliminated, resulting in improved
containment of the transgene in the environment [102]. Tobacco
has been widely used for expression of proteins from the plastome
as have soybean, cotton (Gossypium hirsutum) and carrot (Daucus
carota) [103]. Lutz et al. [104] produced a guide to aid selection
of vectors for plastid transformation that include marker genes to
allow selection of transformants and also to follow insertion and
expression of the transgene of interest.
Human growth hormone, or somatotropin, is one of the many
proteins produced in tobacco through plastid transformation.
Chloroplasts were shown to accumulate soluble active and
disulfide-bonded somatotropin. Additionally, use of a fusionprotein approach (N-terminal ubiquitin) facilitated production of
a functional protein lacking the N-terminal methionine residue;
the native protein starts with a phenylalanine residue following
signal sequence removal in the pituitary gland. High levels of
somatotropin protein accumulation within tobacco chloroplasts
was achieved by driving expression of the gene from the plastid
psbA promoter and other domains in the UTRs (untranslated
regions) [105]. The psbA gene encodes the D1 protein, part of the
reaction centre of Photosystem II. Using the psbA promoter and
other sequences in the gene’s UTRs was previously demonstrated
to confer high expression of foreign genes in light-exposed leaves
[106]. The amount of somatotropin produced was > 7 % of
total soluble protein, which corresponded to a 300-fold increase
relative to levels achieved by nuclear transformation [105].
Attempts at producing an anthrax vaccine have also been
made by plastid transformation of tobacco leaves. The Bacillus
anthracis PA (protective antigen) was expressed in chloroplasts,
also under the control of the psbA promoter. The widely used
anthrax vaccine derived from B. anthracis itself has other minor
components in addition to PA that cause side effects when
administered. Using tobacco leaves as a source for a recombinant
PA should obviate these reactions. During normal day/night light
regimes PA was produced at between 1.7 and 2.7 % of total soluble
protein from mature leaves. However, using a continuous light
227
regime up to a ten-fold increase in expression was observed. About
170 mg was harvested from a single plant, equivalent to 2.5 mg/g
fresh weight. The authors extrapolated this expression level to a
supply of about 990 million doses of vaccine/hectare (400 million
doses/acre) using their experimental cultivar and possibly more
than 18-fold higher using a commercial cultivar, which would be
the equivalent of a world supply of vaccine from just a few acres
of transgenic tobacco [107].
Although extensive research has gone into using plants for
heterologous-protein expression, apart from those that impart a
specific valuable trait on the crop plant itself, few have made the
leap to commercial/agricultural-scale production. In fact, this can
be said of transgenic plants as a whole [108].
PHAs
The chemical properties of various types of PHAs impart
multiple potential applications. Additionally, these polymers are
completely biodegradable. As a result, PHAs have been proposed
as alternatives to conventional petroleum-based plastics. The
bacterial genes responsible for polymer synthesis have been
studied in great detail, and numerous recombinant bacterial strains
have been generated to facilitate PHA production. However,
production of PHAs in transgenic plants has been proposed as an
economically viable alternative to bioreactor-based or chemical
synthesis.
Production of the PHB homopolymer in transgenic plants was
first achieved by Chris Somerville’s group (then at Michigan State
University in East Lansing, U.S.A.). These workers generated
A. thaliana plants that were transformed with the R. eutropha
genes encoding the acetoacetyl-CoA reductase and PHB synthase.
Polymer synthesis was initiated by a cytosolic β-oxothiolase
naturally present in A. thaliana along with the introduced bacterial
enzymes (under the control of the CaMV 35 S promoter) that
facilitated production of high-molecular-mass PHB polymer
in the cytosol, nucleus or vacuoles. However, the amount of
PHB produced was quite low (about 0.1 % dry weight) [109],
and this level of accumulation was not improved by cytosolic
production in other transgenic plants such as tobacco [110].
Additionally, the transgenic plants showed serious side effects
of PHB accumulation, namely a notable decrease in growth and
seed production. The precursor for PHB production is acetyl-CoA
(Figure 5), and diversion of much of this toward PHB production
and away from natural pathways, such as those for flavanoid and
phytosterol production and fatty acid elongation, has been
postulated as the likely cause of the deleterious side effects [95].
Plastids were considered as a good site for polymer production
for a number of reasons. Plastids contain large quantities of
acetyl-CoA destined for fatty acid and amino acid synthesis,
so the metabolic flux should be sufficient for PHA production.
Plastids also naturally accumulate bulky starch grains; therefore
they should tolerate accumulation of PHAs with less deleterious
effects. However, plastids do not contain the β-oxothiolase, so
this must be included in the generation of transgenic plants. As
discussed above, nuclear-transformation-mediated heterologousprotein production has been demonstrated to be far inferior to
plastome transformation in terms of accumulation levels. Lössl
et al. [111] introduced the R. eutropha PHA-synthesis operon
into the plastome under the control of the psbA promoter by
biolistic (‘gene gun’) transformation. The average amount of
PHB synthesized by their transplastomic tobacco lines was
715 p.p.m. on a dry-weight basis (< 0.01 %), although one line
accumulated 1.7 % (by dry weight) polymer. Unfortunately, there
was a strong correlation between PHB levels and reduced plant
c The Authors Journal compilation c 2009 Biochemical Society
228
Figure 5
B. P. Mooney
Scheme for production of PHBV in plants
Accumulation of PHBV within plastids uses the endogenous PDC for generation of the two
precursors of the polymer – acetyl-CoA and propionyl-CoA. Introduction of the bacterial PHA
synthase operon comprising three enzymes (blue italics) allows accumulation of the polymer
within the plastid. The plastidial α-oxobutyrate (α-ketobutyrate) is produced from threonine by
an introduced threonine deaminase (not shown).
growth. Additionally, the transformed plants were male-sterile,
and maintenance of the transgenes could only be achieved through
fertilization with wild-type pollen [111]. Unlike the numerous
success stories for protein production by transplastomic plants,
in this case PHA production was poor when driven from the
plastome.
An alternative to plastome transformation is to target the
enzymes to the plastids post-translationally by incorporating Nterminal plastid-transit peptides. Nawrath et al. [112] inserted
a plastid-transit peptide sequence derived from the pea (Pisum
sativum) small subunit of RuBisCO (ribulose 1,5-bisphosphate
carboxylase/oxygenase) at the N-terminus of the three bacterial
genes necessary for PHA synthesis. The three chimaeric
enzymes were introduced into A. thaliana plants separately and
then a triple-hybrid plant generated by cross-pollination. PHB
production in the hybrids of 14 % (by dry weight; 10 mg/g
by fresh weight) was achieved without affecting growth or
seed set. Additionally, it appeared that the amount of PHB
produced was limited by the (bacterial) enzyme activities rather
than limiting acetyl-CoA [112]. In addition to leaf chloroplasts,
seed leucoplasts in rapeseed (canola; Brassica napus) were
used as the site of PHB accumulation by introduction of all
three bacterial genes on a single multigene vector. The authors
postulated that, because B. napus produces large amounts of oil,
the flux of acetyl-CoA should be more than sufficient to support
PHB accumulation and perhaps prove superior to the previous
A. thaliana experiments. Although their highest producing line
accumulated 7.7 % (by fresh weight), over 90 % of their lines
produced < 3 %. They did confirm, by electron microscopy, that
the ultrastructure of the plastids changed in response to PHB
accumulation (they expanded), proving that plastids are suitable
as a site of accumulation of PHAs and suggesting that expansion
of plastids is a direct result of accumulation of polymer and
c The Authors Journal compilation c 2009 Biochemical Society
not a specific response to starch-grain growth [113]. The highest
accumulation of PHB reported to date was achieved in transgenic
A. thaliana shoots, in which 4 % (by fresh weight; approx. 40 %
by dry weight) was PHB polymer [114]. Numerous other plants
(e.g. cotton, potato, maize and tobacco) have been examined
for PHB production, both cytoplasmic and plastidic, but none
has resulted in improving the levels of accumulation seen in
A. thaliana leaf chloroplasts [115–119].
Most recently, switchgrass has been employed as a source
of the PHB polymer. Switchgrass is a warm-season grass and
one of the dominant species of the central North American
tall-grass prairie. Switchgrass is seen as a valuable biomass
crop with the potential for use in bioethanol production with
concomitant reduction in greenhouse-gas emissions compared
with gasoline/petrol [120]; it was recently targeted for funding
as one of the useful bioenergy crops by the U.S. Department of
Energy [121]. In addition to their bacterially produced polymer,
researchers at Metabolix are also exploring plant-based PHAs.
They used both constitutive and light-inducible promoters to drive
expression of PHB synthesis genes appended with N-terminal
chloroplast transit peptides. Greenhouse-grown switchgrass was
found to accumulate almost 4 % (by dry weight) PHB in the leaves
and 1.2 % (by dry weight) in whole tillers. Even their highest
producing lines [3.72 % (by dry weight) PHB in leaves] developed
normally and accumulated biomass at levels similar to those of the
wild-type. Additionally, stable high-level PHB accumulation was
maintained in the subsequent generation. Although the amounts
of PHB produced were below commercially viable levels, this was
the first successful expression of a functional multigene pathway
in switchgrass and bodes well for future engineering of the plants
for value-added-product formation [122]. The hope eventually
would be to produce a ‘double-crop’ for both PHAs and cellulosic
ethanol production.
Polymers consisting of PHB tend to be crystalline and brittle,
whereas PHBV, the co-polymer of HB and HV, is considerably
more flexible. PHBV precursors are acetyl-CoA and propionylCoA, and, although acetyl-CoA is present in the cytosol and
plastids, propionyl-CoA is restricted to the mitochondria. An
alternative route for production of propionyl-CoA is introduction
of a threonine deaminase, which converts threonine into α-oxobutyrate. The α-oxobutyrate can then be utilized by the
endogenous plastid PDC (pyruvate dehydrogenase complex) to
produce propionyl-CoA (Figure 5). Slater et al. [123] employed
this route to propionyl-CoA synthesis in plastids of B. napus
and A. thaliana. This required introduction of the three bacterial
PHA synthesis genes, along with the threonine deaminase – the
ilvA gene from E. coli. Although the presence of the threonine
deaminase did raise the levels of propionyl-CoA relative to plants
without it, the amounts were still 4–6-fold less than acetyl-CoA.
Additionally, the poor efficiency of the plastid PDC for conversion
of α-oxobutyrate into propionyl-CoA resulted in accumulation of
< 3 % (by dry weight) polymer in rapeseed and about 1.6 % in
A. thaliana leaves. The relative amounts of the HV monomer
in the PHA produced also decreased with increasing levels of
accumulation, again pointing to inefficiency in generating HV.
An encouraging point, however, is that no deleterious phenotypes
were linked to the presence of the transgenes or PHBV content,
although the authors concede that this might be due to the low
levels of accumulation [123].
It is clear from the research to date that plastids are the
most useful and widely used site for PHA accumulation in
a number of crop plants, including alfalfa (Medicago sativa),
cotton, potato, canola, tobacco, sugar crops [beet (Beta vulgaris)
and cane (Saccharum officinarum) and fibre crops such as flax
(Linum usitatissimum) [95]. A goal of 7.5 % (by dry weight)
Plant-based biodegradable plastics
Figure 6
229
Using plants for production of biodegradable polymers
The present review discusses three main themes for synthesis of biodegradable polymers. There are a number of naturally occurring plant polymers that have been exploited by direct harvesting
for biodegradable plastics – rubber, starch, proteins and cellulose – sometimes followed by derivatization. Industrial or ‘white’ biotechnology using plant extracts as carbon sources for bioreactor
fermentative production of PLA and PHA polymers is an alternative. Through engineering of bacteria, a number of valuable polymers can be produced with various monomers based on the nutrient
input. Finally, using transgenic technologies, plants can be engineered to produce novel polymers, including value-added proteins and PHAs, and therefore function as solar-driven biofactories. In
all cases, the polymers produced are biodegradable, so the carbon cycle is maintained – the transgenic route to plastics is considered the closest to a carbon-neutral method for polymer production.
polymer has been set by Metabolix as a commercially viable
level of accumulation [122], and some of the research into the
slightly less desirable PHB polymer have clearly shown accumulation in excess of this (e.g. 40 %; [114]). Unfortunately
accumulation is only part of the process; extraction of the polymer
is probably an even greater component for commercialization in
a cost-effective and environmentally friendly manner to allow
plant-based biodegradable polymers to replace petroleum-based
plastics. Bacterial culture systems for PHA can accumulate 50–
85 % (by dry weight) polymer, with 80 g/litre and 2 g/h per
litre capacities relatively easily met in fed-batch cultures [125].
Laboratory-scale purification of PHAs from bacterial cells can be
achieved by simple mechanical (20 h constant stirring) organicsolvent extraction using chloroform and methanol, which exploits
the solubility of PHA in chloroform and insolubility in methanol,
allowing fractionation of the PHA [126]. An alternative approach
is to use a ‘cocktail’ of enzymes and detergents to remove
proteins, nucleic acids and cell walls, leaving the PHA intact
[127]. However, a low-cost, highly efficient and environmentally
friendly commercial-scale PHA extraction process has yet to
be implemented. Two cost-effective and more environmentally
friendly approaches have been developed recently, namely
supercritical CO2 extraction and cell-mass dissolution by protons
in aqueous solution (sulfuric acid extraction at high temperature).
Recovery efficiencies of 90–95 % with very high purity were
achieved with these methods respectively (reviewed in [82]).
Whether these methods can be used for PHA extraction from
plants has not been tested. There are a number of patent filings
for PHA extraction from plants based on non-halogenated solvent
extraction (U.S. Patent 6043063) at high-temperature (U.S. Patent
6087471) and those that allow simultaneous extraction of PHAs
and oils from oilseed crops using differential solvent extraction
(U.S. Patent 6709848). None of these methods seem particularly
environmentally friendly, and none have been published to date
for commercial-scale extraction from plants.
CONCLUDING REMARKS
As discussed in the present review, the production of plant-based
polymers can be approached in a number of ways (Figure 6).
Some progress has been made in commercialization of plantbased materials, for example, the starch-based Mater-BiTM range
from the Italian company Novamont and U.S. NatureWorks
LLC PLA polymer. However, commercialization of transgenic
plants specifically designed to synthesize polymers has not been
achieved. Parameters affecting commercialization of transgenic
plants for bioplastics include the time and costs associated with
their development (often 10–12 years and tens of millions of U.S.
dollars in total) [128], the typically poor accumulation levels of
novel polymers (compared with bacterial systems), the lack
of proven processing and extraction methods, the price per
unit of polymer from plants, and the stigma associated with GMOs
in many parts of the world.
Many of these problems can be addressed by more research.
Building on current seed stocks of plants that have already
been developed for PHB production, such as those from
Metabolix, for example, through cross-pollination or further
genetic manipulation, could conceivability reduce the timeline
for development and might, in part, reduce the costs associated
with regulatory testing if the original plants already have a GRAS
(‘generally recognized as safe’) designation. As more knowledge
c The Authors Journal compilation c 2009 Biochemical Society
230
B. P. Mooney
is gained on carbon flux in plants, perhaps, through emerging
metabolomics methods for example, this will give us a better
understanding of how to increase polymer accumulation. Judging
from the numerous patent awards, processing technologies are
being developed for transgenic plant polymers. In terms of
price per unit polymer, fermentative PHA production could
produce $2 (€1.55, £1.35)/kg prices, which is still about twice
the price of polyethylene [129], but using crop plants in a
‘double-cropping’ value-added manner could allow exploitation
of agronomic plants for polymer production. Additionally the
scalability and renewable nature of plants should address price
issues. Finally the acceptance of GMOs is still a point of
contention. The StarLink maize incident in 2000 gained national
and worldwide attention for the problems of segregation of food
crops [130]. Utilizing non-food crops, like switchgrass, might
ameliorate the stigma associated with transgenic crops, although
food-chain contamination is only one of the issues that are used
to argue against GMOs. Having said all this, the pressures on
fossil fuels, seen in rising oil prices and environmental concerns,
could soon precipitate a revolution against traditional plastics
in favour of more environmentally conscious and, theoretically
carbon-neutral, plastics such as those derived from plants.
ACKNOWLEDGMENTS
We thank Melody Kroll (Interdisciplinary Plant Group, University of Missouri, Columbia,
MO, U.S.A.), Emeritus Professor Douglas D. Randall (Department of Biochemistry,
University of Missouri, Columbia, MO, U.S.A.) and Dr Jan Miernyk (Plant Genetics
Unit, USDA/ARS, Columbia, MO, U.S.A., and Department of Biochemistry, University
of Missouri, Columbia, MO, U.S.A.) for critical reading of the manuscript before its
submission.
FUNDING
This research received no specific grant from any funding agency in the public, commercial
or not-for-profit sectors.
REFERENCES
1 McCoy, M. (1998) Commodity plastics in downhill year. Chem. Eng. News 7, 15
2 Zan, L., Fa, W. and Wang, S. (2006) Novel photodegradable low-density
polyethylene–TiO2 nanocomposite film. Environ. Sci. Technol. 40, 1681–1685
3 Hiatt, A. and Pauly, M. (2006) Monoclonal antibodies from plants: a new speed record.
Proc. Natl. Acad. Sci. U.S.A. 103, 14645–14646
4 Stoger, E., Sack, M., Nicholson, L., Fischer, R. and Christou, P. (2005) Recent progress
in plantibody technology. Curr. Pharm. Des. 11, 2439–2457
5 Dyer, J. M., Stymne, S., Green, A. G. and Carlsson, A. S. (2008) High-value oils from
plants. Plant J. 54, 640–655
6 Damude, H. G. and Kinney, A. J. (2008) Enhancing plant seed oils for human nutrition.
Plant Physiol. 147, 962–968
7 Wackernagel, M., Schulz, N. B., Deumling, D., Linares, A. C., Jenkins, M., Kapos, V.,
Monfreda, C., Loh, J., Myers, N., Norgaard, R. and Randers, J. R. (2002) Tracking the
ecological overshoot of the human economy. Proc. Natl. Acad. Sci. U.S.A. 99,
9266–9271
8 Johnson, K. E., Pometto, III, A. L. and Nikolov, Z. L. (1993) Degradation of degradable
starch–polyethylene plastics in a compost environment. Appl. Environ. Microbiol. 59,
1155–1161
9 Garg, A., Smith, R., Hill, D., Simms, N. and Pollard, S. (2007) Wastes as co-fuels:
the policy framework for solid recovered fuel (SRF) in Europe, with UK implications.
Environ. Sci. Technol. 41, 4868–4874
10 Kim, D., Shin, S., Sohn, S., Choi, J. and Ban, B. (2002) Waste plastics as supplemental
fuel in the blast furnace process: improving combustion efficiencies. J. Haz. Mat. 94,
213–222
11 Royte, E. (2008) Bottlemania: how water went on sale and why we bought it.
Bloomsbury, London
12 Park, C.-H., Jeon, H.-S., Yu, H.-S., Han, O.-H. and Park, J.-K. (2008) Application of
electrostatic separation to the recycling of plastic wastes: separation of PVC, PET, and
ABS. Environ. Sci. Technol. 42, 249–255
c The Authors Journal compilation c 2009 Biochemical Society
13 De Sena, G., Nardi, C., Cenedese, A., La Marca, F., Massacci, P. and Moroni, M. (2008)
The hydraulic separator Multidune: preliminary tests on fluid-dynamic features and
plastic separation feasibility. Waste Manag. 28, 1560–1571
14 Ashori, A. (2008) Wood–plastic composites as promising green-composites for
automotive industries! Bioresour. Technol. 99, 4661–4667
15 Panyakapo, P. and Panyakapo, M. (2008) Reuse of thermosetting plastic waste for
lightweight concrete. Waste Manag. 28, 1581–1588
16 Riggle, D. (1998) Moving towards consensus on degradable plastics. BioCycle 39,
64–70
17 Perry, G. H., Dominy, N. J., Claw, K. G., Lee, A. S., Fiegler, H., Redon, R., Werner, J.,
Villanea, F. A., Mountain, J. L., Misra, R. et al. (2007) Diet and the evolution of human
amylase gene copy number variation. Nat. Genet. 39, 1256–1260
18 Cornish, K. (2001) Similarities and differences in rubber biochemistry among plant
species. Phytochemistry 57, 1123–1134
19 Lieberei, R. (2007) South American leaf blight of the rubber tree (Hevea spp.): new steps
in plant domestication using physiological features and molecular markers. Ann. Bot.
100, 1125–1142
20 Dennis, M. S. and Light, D. R. (1989) Rubber elongation factor from Hevea brasiliensis .
Identification, characterization, and role in rubber biosynthesis. J. Biol. Chem. 264,
18608–18617
21 Asawatreratanakul, K., Zhang, Y.-W., Wititsuwannakul, D., Wititsuwannakul, R.,
Takahashi, S., Rattanapittayaporn, A. and Koyama, T. (2003) Molecular cloning,
expression and characterization of cDNA encoding cis -prenyltransferases from Hevea
brasiliensis . A key factor participating in natural rubber biosynthesis. Eur. J. Biochem.
270, 4671–4680
22 Van Beilen, J. and Poirier, Y. (2007) Establishment of new crops for the production of
natural rubber. Trends Biotechnol. 25, 522–529
23 Da Costa, B. M. T., Keasling, J. D. and Cornish, K. (2005) Regulation of rubber
biosynthetic rate and molecular weight in Hevea brasiliensis by metal cofactor.
Biomacromolecules 6, 279–289
24 Charous, B. L., Blanco, C., Tarlo, S., Hamilton, R. G., Baur, X., Beezhold, D., Sussman,
G. and Yunginger, J. W. (2002) Natural rubber latex allergy after 12 years:
recommendations and perspectives. J. Allergy Clin. Immunol. 190, 31–34
25 Bousquet, J., Flahault, A., Vandenplas, O., Ameille, J., Duron, J.-J., Pecquet, C., Chevrie,
K. and Annesi-Maesano, I. (2006) Natural rubber latex allergy among health care
workers: a systematic review of the evidence. J. Allergy Clin. Immunol. 118, 447–454
26 van Beilen, J. B. and Poirier, Y. (2007) Guayule and Russian dandelion as alternative
sources of natural rubber. Crit. Rev. Biotechnol. 27, 217–231
27 Siler, D. J., Cornish, K. and Hamilton, R. G. (1996) Absence of cross-reactivity of IgE
antibodies from subjects allergic to Hevea brasiliensis latex with a new source of natural
rubber latex from guayule (Parthenium argentatum ). J. Allergy Clin. Immunol. 98,
895–902
28 Shewry, P. R., Halford, N. G., Belton, P. S. and Tatham, A. S. (2002) The structure and
properties of gluten: an elastic protein from wheat grain. Philos. Trans. R. Soc. London B
357, 133–142
29 Liu, K. and Hsieh, F.-H. (2008) Protein–protein interactions during high-moisture
extrusion for fibrous meat analogues and comparison of protein solubility methods
using different solvent systems. J. Agric. Food Chem. 56, 2681–2687
30 Kim, S. (2008) Processing and properties of gluten/zein composite. Bioresour. Technol.
99, 2032–2036
31 Wu, Q., Sakabe, H. and Isobe, S. (2003) Processing and properties of low cost corn
gluten meal/wood fiber composite. Ind. Eng. Chem. Res. 42, 6765–6773
32 Domenek, S., Feuilloley, P., Gratraud, J., Morel, M.-H. and Guilbert, S. (2004)
Biodegradability of wheat gluten based bioplastics. Chemosphere 54, 551–559
33 Reddy, N. and Yang, Y. (2008) Self-crosslinked gliadin fibers with high strength and
water stability for potential medical applications. J. Mater. Sci. Mater. Med. 19,
2055–2061
34 Shewry, P. R. and Tatham, A. S. (1990) The prolamin storage proteins of cereal seeds:
structure and evolution. Biochem. J. 267, 1–12
35 Lawton, J. W. (2002) Zein: a history of processing and use. Cereal Chem. 79, 1–18
36 Shukla, R. and Cheryan, M. (2001) Zein: the industrial protein from corn.
Ind. Crops Prod. 13, 171–192
37 Parris, N., Vergano, P. J., Dickey, L. C., Cooke, P. H. and Craig, J. C. (1998) Enzymatic
hydrolysis of zein–wax-coated paper. J. Agric. Food Chem. 46, 4056–4059
38 Trezza, T. A. and Vergano, P. J. (1994) Grease resistance of corn zein coated paper.
J. Food Sci. 59, 912–915
39 Padgett, T., Han, I. Y. and Dawson, P. L. (1998) Incorporation of food-grade antimicrobial
compounds into biodegradable packaging films. J. Food Proteins 61, 1330–1335
40 Parris, N. and Coffin, D. R. (1997) Composition factors affecting the water vapor
permeability and tensile properties of hydrophilic zein films. J. Agric. Food Chem. 45,
1596–1599
Plant-based biodegradable plastics
41 Wu, Q., Yoshino, T., Sakabe, H., Zhang, H. and Isobe, S. (2003) Chemical modification
of zein by bifunctional polycaprolactone (PCL). Polymer 44, 3909–3919
42 Gong, S., Wang, H., Sun, Q., Xue, S.-T. and Wang, J.-Y. (2006) Mechanical properties
and in vitro biocompatibility of porous zein scaffolds. Biomaterials 27, 3793–3799
43 Qu, Z.-H., Wang, H.-J., Tang, T.-T., Zhang, X.-L., Wang, J.-Y. and Dai, K.-R. (2008)
Evaluation of the zein/inorganics composite on biocompatibility and osteoblastic
differentiation. Acta Biomater. 4, 1360–1368
44 Liu, X., Sun, Q., Wang, H., Zhang, L. and Wang, J.-Y. (2005) Microspheres of corn
protein, zein, for an ivermectin drug delivery system. Biomaterials 26, 109–115
45 Muthuselvi, L. and Dhathathreyan, A. (2006) Simple coacervates of zein to encapsulate
Gitoxin. Colloids Surf. B Biointerfaces 51, 39–43
46 Liu, L., Fishman, M. L., Hicks, K. B., Kende, M. and Ruthel, G. (2006) Pectin/zein beads
for potential colon-specific drug delivery: synthesis and in vitro evaluation. Drug Deliv.
13, 417–423
47 Stevens, E. S. (2002) Green plastics: an introduction to the new science of
biodegradable plastics, Princeton University Press, Princeton
48 Gennadios, A., Weller, C. L. and Testin, R. F. (1993) Modification of physical and barrier
properties of edible wheat gluten-based films. Cereal Chem. 70, 426–429
49 Sabato, S. F., Ouattara, B., Yu, H., D’Aprano, G., Le Tien, C., Mateescu, M. A. and
Lacroix, M. (2001) Mechanical and barrier properties of cross-linked soy and whey
protein based films. J. Agric. Food Chem. 49, 1397–1403
50 Lu, Y., Weng, L. and Zhang, L. (2004) Morphology and properties of soy protein isolate
thermoplastics reinforced with chitin whiskers. Biomacromolecules 5, 1046–1051
51 Wang, Y., Cao, X. and Zhang, L. (2006) Effects of cellulose whiskers on properties of soy
protein thermoplastics. Macromol. Biosci. 6, 524–531
52 Zhang, J., Jiang, L. and Zhu, L. (2006) Morphology and properties of soy protein and
polylactide blends. Biomacromolecules 7, 1551–1561
53 Sticklen, M. B. (2008) Plant genetic engineering for biofuel production: towards
affordable cellulosic ethanol. Nat. Rev. Genet. 9, 433–443
54 Choudhury, P. K., Kar, M. and Chauhan, C. S. (2008) Cellulose acetate microspheres as
floating depot systems to increase gastric retention of antidiabetic drug: formulation,
characterization and in vitro in vivo evaluation. Drug Dev. Ind. Pharm. 34, 349–354
55 Jeon, J. H. and Puleo, D. A. (2008) Alternating release of different bioactive molecules
from a complexation polymer system. Biomaterials 29, 3591–3598
56 Neurath, A. R., Strick, N., Li, Y.-Y. and Debnath, A. (2001) Cellulose acetate phthalate, a
common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding
site on the virus envelope glycoprotein gp120. BMC Infect. Dis. 1, 17
57 Neurath, A. R., Strick, N., Jiang, S., Li, Y.-Y. and Debnath, A. (2002) Anti-HIV-1 activity
of cellulose acetate phthalate: synergy with soluble CD4 and induction of “dead-end”
gp41 six-helix bundles. BMC Infect. Dis. 2, 6
58 Guerra, N. P., Macias, C. L., Agrasar, A. T. and Castro, L. P. (2005) Development of a
bioactive packaging cellophane using Nisaplin as biopreservative agent.
Lett. Appl. Microbiol. 40, 106–110
59 Roche-Nagle, G., O’Sullivan, M., McGreal, G. and O’Sullivan, G. (2007) Reconstructing
the abdominal wall with a biocompatible patch. Can. J. Surg. 50, E15–E16
60 Whistler, R. H. (1984) History and future expectation of starch use. In Starch Chemistry
and Technology (Whistler, R., Bemiller, J. and Paschall, E., eds), pp. 1–9, Academic
Press, San Franciso
61 Glenn, G. M., Klamczynski, A. P., Ludvik, C., Shey, J., Imam, S. H., Chiou, B. S.,
McHugh, T., DeGrandi-Hoffman, G., Orts, W., Wood, D. and Offeman, R. (2006)
Permeability of starch gel matrices and select films to solvent vapors. J. Agric.
Food Chem. 54, 3297–3304
62 Glenn, G. M., Klamczynski, A. K., Holtman, K. M., Shey, J., Chiou, B. S., Berrios, J.,
Wood, D., Orts, W. J. and Imam, S. H. (2007) Heat expanded starch-based
compositions. J. Agric. Food Chem. 55, 3936–3943
63 Bastioli, C. (1998) Properties and applications of Mater-Bi starch-based materials.
Polym. Degrad. Stabil. 59, 263–272
64 Salgado, A. J., Coutinho, O. P. and Reis, R. L. (2004) Novel starch-based scaffolds for
bone tissue engineering: cytotoxicity, cell culture, and protein expression. Tissue Eng.
10, 465–474
65 Marques, A. P., Reis, R. L. and Hunt, J. A. (2005) An in vivo study of the host response
to starch-based polymers and composites subcutaneously implanted in rats.
Macromol. Biosci. 5, 775–785
66 Smith, A. M. (2008) Prospects for increasing starch and sucrose yields for bioethanol
production. Plant J. 54, 546–558
67 Doran-Peterson, J., Cook, D. M. and Brandon, S. K. (2008) Microbial conversion of
sugars from plant biomass to lactic acid or ethanol. Plant J. 54, 582–592
68 Auras, R., Harte, B. and Selke, S. (2004) An overview of polylactides as packaging
materials. Macromol. Biosci. 4, 835–864
69 Albertsson, A. C. and Varma, I. K. (2003) Recent developments in ring opening
polymerization of lactones for biomedical applications. Biomacromolecules 4,
1466–1486
231
70 Torres, A., Li, S. M., Roussos, S. and Vert, M. (1996) Screening of microorganisms for
biodegradation of poly(lactic acid) and lactic acid-containing polymers.
App. Environ. Microbiol. 62, 2393–2397
71 Taguchi, S., Yamada, M., Matsumoto, K. I., Tajima, K., Satoh, Y., Munekata, M., Ohno,
K., Kohda, K., Shimamura, T., Kambe, H. and Obata, S. (2008) A microbial factory for
lactate-based polyesters using a lactate-polymerizing enzyme. Proc. Natl. Acad.
Sci. U.S.A. 105, 17323–17327
72 Steinbüchel, A. and Füchtenbusch, B. (1998) Bacterial and other biological systems for
polyester production. Trends Biotechnol. 16, 419–427
73 Braunegg, G., Lefebvre, G. and Genser, K. F. (1998) Polyhydroxyalkanoates,
biopolyesters from renewable resources: physiological and engineering aspects.
J. Biotechnol. 65, 127–161
74 Lee, S. Y. and Choi, J.-I. (2001) Production of microbial polyester by fermentation of
recombinant microorganisms. Adv. Biochem. Eng. Biotechnol. 71, 183–207
75 Schubert, P., Steinbuchel, A. and Schlegel, H. G. (1988) Cloning of the Alcaligenes
eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of
PHB in Escherichia coli . J. Bacteriol. 170, 5837–5847
76 Steinbuchel, A. and Schlegel, H. G. (1991) Physiology and molecular genetics of
poly(p -hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus. Mol. Microbiol. 5,
535–542
77 Eschenlauer, A. C., Stoup, S. K., Srienc, F. and Somers, D. A. (1996) Production of
heteropolymeric polyhydroxyalkanoate in Eschericha coli from a single carbon source.
Int. J. Biol. Macromol. 19, 121–130
78 Reis, M. A. M., Serafim, L. S., Lemos, P. C., Ramos, A. M., Aguiar, F. R. and
Van Loosdrecht, M. C. M. (2003) Production of polyhydroxyalkanoates by mixed
microbial cultures. Bioprocess Biosyst. Eng. 25, 377–385
79 Yu, P., Chua, H., Huang, A.-L. and Ho, K.-P. (1999) Conversion of industrial food wastes
by Alcaligenes latus into polyhydroxyalkanoates. Appl. Biochem. Biotechnol. 78,
445–454
80 Nikel, P. I., de Almeida, A., Melillo, E. C., Galvagno, M. A. and Pettinari, M. J. (2006)
New recombinant Escherichia coli strain tailored for the production of
poly(3-hydroxybutyrate) from agroindustrial by-products. Appl. Environ. Microbiol. 72,
3949–3954
81 Fukui, T. and Doi, Y. (1998) Efficient production of polyhydroxyalkanoates from plant
oils by Alcaligenes eutrophus and its recombinant strain. Appl. Microbiol. Biotechnol.
49, 333–336
82 Dias, J. M. L., Lemos, P. C., Serafim, L. S., Oliveira, C., Eiroa, M., Albuquerque,
M. G. E., Ramos, A. M., Oliveira, R. and Reis, M. A. M. (2006) Recent advances in
polyhydroxyalkanoate production by mixed aerobic cultures: from the substrate to the
final product. Macromol. Biosci. 6, 885–906
83 Gerngross, T. U. (1999) Can biotechnology move us toward a sustainable society?
Nat. Biotechnol. 17, 541–544
84 Kim, S. and Dale, B. (2005) Life cycle assessment study of biopolymers
(polyhydroxyalkanoates) – derived from no-tilled corn. Int. J. LCA 10, 200–210
85 Gurieff, N. and Lant, P. (2007) Comparative life cycle assessment and financial analysis
of mixed culture polyhydroxyalkanoate production. Bioresour. Technol. 98, 3393–3403
86 Tanno, K.-I. and Willcox, G. (2008) How fast was wild wheat domesticated? Science
311, 1886
87 Kusnadi, A. R., Nikolov, Z. L. and Howard, J. A. (1997) Production of recombinant
proteins in transgenic plants: practical considerations. Biotechnol. Bioeng. 56, 473–484
88 Romeis, J., Meissle, M. and Bigler, F. (2006) Transgenic crops expressing Bacillus
thuringiensis toxins and biological control. Nat. Biotechnol. 24, 63–71
89 Streatfield, S. J. (2007) Approaches to achieve high-level heterologous protein
production in plants. Plant Biotechnol. J. 5, 2–15
90 Porta, C. and Lomonossoff, G. P. (2002) Viruses as vectors for the expression of foreign
sequences in plants. Biotechnol. Genet. Eng. Rev. 19, 245–291
91 Canizares, M. C., Nicholson, L. and Lomonossoff, G. P. (2005) Use of viral vectors for
vaccine production in plants. Immunol. Cell Biol. 83, 263–270
92 Hood, E. E., Witcher, D. R., Maddock, S., Meyer, T., Baszczynski, C., Bailey, M., Flynn, P.,
Register, J., Marshall, L., Bond, D. et al. (1997) Commercial production of avidin from
transgenic maize: characterization of transformant, production, processing, extraction
and purification. Mol. Breeding 3, 291–306
93 Nandi, S., Yalda, D., Lu, S., Nikolov, Z. L., Misaki, R., Fujiyama, K. and Huang, N. (2005)
Process development and economic evaluation of recombinant human lactoferrin
expressed in rice grain. Transgenic Res. 14, 237–249
94 De Wilde, C., Van Houdt, H., De Buck, S., Angenon, G., De Jaeger, G. and Depicker, A.
(2000) Plants as bioreactors for protein production: avoiding the problem of transgene
silencing. Plant Mol. Biol. 43, 347–359
c The Authors Journal compilation c 2009 Biochemical Society
232
B. P. Mooney
95 Van Beilen, J. B. and Poirier, Y. (2008) Production of renewable polymers from crop
plants. Plant J. 54, 684–701
96 Yang, J., Barr, L. A., Fahnestock, S. R. and Liu, Z.-B. (2005) High yield recombinant
silk-like protein production in transgenic plants through protein targeting.
Transgenic Res. 14, 313–324
97 Scheller, J., Henggeler, D., Viviani, A. and Conrad, U. (2004) Purification of spider
silk-elastin from transgenic plants and application for human chondrocyte proliferation.
Transgenic Res. 13, 51–57
98 Huang, J., Foo, C. W. P. and Kaplan, D. L. (2007) Biosynthesis and applications of
silk-like and collagen-like proteins. Polymer Rev. 47, 29–62
99 Daniell, H., Khan, M. S. and Allison, L. (2002) Milestones in chloroplast genetic
engineering: an environmentally friendly era in biotechnology. Trends Plant Sci. 7,
84–91
100 Daniell, H., Lee, S.-B., Panchal, T. and Wiebe, P. O. (2001) Expression of the native
cholera toxin B subunit gene and assembly as functional oligomers in transgenic
tobacco chloroplasts. J. Mol. Biol. 311, 1001–1009
101 De Cosa, B., Moar, W., Lee, S.-B., Miller, M. and Daniell, H. (2001) Overexpression of
the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals.
Nat. Biotechnol. 19, 71–74
102 Daniell, H. (2002) Molecular strategies for gene containment in transgenic crops.
Nat. Biotechnol. 20, 581–586
103 Daniell, H., Kumar, S. and Dufourmantel, N. (2005) Breakthrough in chloroplast genetic
engineering of agronomically important crops. Trends Biotechnol. 23, 238–245
104 Lutz, K. A., Azhagiri, A. K., Tungsuchat-Huang, T. and Maliga, P. (2007) A guide to
choosing vectors for transformation of the plastid genome of higher plants.
Plant Physiol. 145, 1201–1210
105 Staub, J. M., Garcia, B., Graves, J., Hajdukiewicz, P. T. J., Hunter, P., Nehra, N.,
Paradkar, V., Schlittler, M., Carroll, J. A., Spatola, L. et al. (2000) High-yield production
of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18, 333–338
106 Fernández-San Millán, A., Mingo-Castel, A., Miller, M. and Daniell, H. (2003)
A chloroplast transgenic approach to hyper-express and purify human serum albumin,
a protein highly susceptible to proteolytic degradation. Plant Biotechnol. J. 1, 71–79
107 Watson, J., Koya, V., Leppla, S. H. and Daniell, H. (2004) Expression of Bacillus
anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop.
Vaccine 22, 4374–4384
108 Wenzel, G. (2006) Molecular plant breeding: achievements in green biotechnology and
future perspectives. Appl. Microbiol. Biotechnol. 70, 642–650
109 Poirier, Y., Dennis, D. E., Klomparens, K. and Somerville, C. (1992) Polyhydroxybutyrate,
a biodegradable thermoplastic, produced in transgenic plants. Science 256, 520–523
110 Nakashita, H., Arai, Y., Yoshioka, K., Fukui, T., Doi, Y., Usami, R., Horikoshi, K. and
Yamaguchi, I. (1999) Production of a biodegradable polyester by a transgenic tobacco.
Biosci. Biotechnol. Biochem. 63, 870–874
111 Lössl, A., Eibl, C., Harloff, H.-J., Jung, C. and Koop, H.-U. (2003) Polyester synthesis in
transplastomic tobacco (Nicotiana tabacum L.): significant contents of
polyhydroxybutyrate are associated with growth reduction. Plant Cell Rep. 21, 891–899
112 Nawrath, C., Poirier, Y. and Somerville, C. (1994) Targeting of the polyhydroxybutyrate
biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of
polymer accumulation. Proc. Natl. Acad. Sci. U.S.A. 91, 12760–12764
Received 2 September 2008/4 November 2008; accepted 5 November 2008
Published on the Internet 11 February 2009, doi:10.1042/BJ20081769
c The Authors Journal compilation c 2009 Biochemical Society
113 Houmiel, K. L., Slater, S., Broyles, D., Casagrande, L., Colburn, S., Gonzalez, K., Mitsky,
T. A., Reiser, S. E., Shah, D., Taylor, N. B. et al. (1999) Poly(β-hydroxybutyrate)
production in oilseed leukoplasts of Brassica napus . Planta 209, 547–550
114 Bohmert, K., Balbo, I., Kopka, J., Mittendorf, V., Nawrath, C., Poirier, Y., Tischendorf, G.,
Trethewey, R. N. and Willmitzer, L. (2000) Transgenic Arabidopsis plants can accumulate
polyhydroxybutyrate to up to 4 % of their fresh weight. Planta 211, 841–845
115 John, M. E. and Keller, G. (1996) Metabolic pathway engineering in cotton: biosynthesis
of polyhydroxybutyrate in fiber cells. Proc. Natl. Acad. Sci. U.S.A. 93, 12768–12773
116 Bohmert, K., Balbo, I., Steinbuchel, A., Tischendorf, G. and Willmitzer, L. (2002)
Constitutive expression of the β-ketothiolase gene in transgenic plants. A major
obstacle for obtaining polyhydroxybutyrate-producing plants. Plant Physiol. 128,
1282–1290
117 Romano, A., van der Plas, L. H. W., Witholt, B., Eggink, G. and Mooibroek, H. (2005)
Expression of poly-3-(R )-hydroxyalkanoate (PHA) polymerase and
acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a
hydrophobic polymer, presumably medium-chain-length PHAs. Planta 220, 455–464
118 Hahn, J. J., Eschenlauer, A. C., Sleytr, U. B., Somers, D. A. and Srienc, F. (1999)
Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants.
Biotechnol. Prog. 15, 1053–1057
119 Arai, Y., Nakashita, H., Doi, Y. and Yamaguchi, I. (2001) Plastid targeting of
polyhydroxybutyrate biosynthetic pathway in tobacco. Plant Biotechnol. J. 18,
289–293
120 Schmer, M. R., Vogel, K. P., Mitchell, R. B. and Perrin, R. K. (2008) Net energy of
cellulosic ethanol from switchgrass. Proc. Natl. Acad. Sci. U.S.A. 105, 464–469
121 Sanderson, M. A., Adler, P. R., Boateng, A. A., Casler, M. D. and Sarath, G. (2006)
Switchgrass as a biofuels feedstock in the USA. Can. J. Plant Sci. 86, 1315–1325
122 Somleva, M. N., Snell, K. D., Beaulieu, J. J., Peoples, O. P., Garrison, B. R. and
Patterson, N. A. (2008) Production of polyhydroxybutyrate in switchgrass, a value-added
co-product in an important lignocellulosic biomass crop. Plant Biotechnol. J. 6,
663–678
123 Slater, S., Mitsky, T. A., Houmiel, K. L., Hao, M., Reiser, S. E., Taylor, N. B., Tran, M.,
Valentin, H. E., Rodriguez, D. J., Stone, D. A. et al. (1999) Metabolic engineering of
Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer
production. Nat. Biotechnol. 17, 1011–1016
124 Reference deleted
125 Lee, S. Y. (1996) Plastic bacteria? Progress and prospects for polyhydroxyalkanoate
production in bacteria. Trends Biotechnol. 14, 431–438
126 Serafim, L. S., Lemos, P. C., Torres, C., Reis, M. A. M. and Ramos, A. M. (2008)
The influence of process parameters on the characteristics of polyhydroxyalkanoates
produced by mixed cultures. Macromol. Biosci. 8, 355–366
127 Byrom, D. (1987) Polymer synthesis by microorganisms: technology and economics.
Trends Biotechnol. 5, 246–250
128 Devine, M. D. (2005) Why are there not more herbicide-tolerant crops? Pest Manag. Sci.
61, 312–317
129 Philip, S., Keshavarz, T. and Roy, I. (2007) Polyhydroxyalkanoates: biodegradable
polymers with a range of applications. J. Chem. Technol. Biotechnol. 82, 233–247
130 Kaiser, J. (2000) Toxicology. Panel urges further study of biotech corn. Science 290,
1867