475
Process design for microbial plastic factories: metabolic
engineering of polyhydroxyalkanoates
Ilana S Aldor
and Jay D Keaslingy
Implementing several metabolic engineering strategies, either
individually or in combination, it is possible to construct microbial
plastic factories to produce a variety of polyhydroxyalkanoate
(PHA) biopolymers with desirable structures and material
properties. Approaches include external substrate manipulation,
inhibitor addition, recombinant gene expression, host cell
genome manipulation and, most recently, protein engineering of
PHA biosynthetic enzymes. In addition, mathematical models
and molecular methods can be used to elucidate metabolically
engineered systems and to identify targets for performance
improvement.
Addresses
Department of Chemical Engineering, 201 Gilman Hall, University of
California, Berkeley, 94720-1462, USA


e-mail: aldor@gene.com
y
e-mail: keasling@socrates.berkeley.edu
Current Opinion in Biotechnology 2003, 14:475–483
This review comes from a themed issue on
Biochemical engineering
Edited by Jeremy S Edwards and Kenneth J Kauffman
0958-1669/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2003.09.002
Abbreviations
3HB 3-hydroxybutyrate
3HHX 3-hydroxyhexanoate
3HO 3-hydroxyoctanoate
3HV 3-hydroxyvalerate
3MB 3-mercaptobutyrate
3MP 3-mercaptopropionate
PHA polyhydroxyalkanoate
PHB poly(3-hydroxybutyrate)
P(3HB-co-3HHX) poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
P(3HB-co-3HV) poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
Introduction
Polyhydroxyalkanoates (PHAs) form a class of natural
polyesters that many organisms in the environment accu-
mulate in the form of intracellular granules to store carbon
and reducing equivalents. Although the most well-stu-
died PHA is poly(3-hydroxybutyrate) (PHB), a polymer
of 3-hydroxybutyrate (3HB), there are over 140 possible
constituent monomers [1], which have been traditionally
classified as short chain length (C4 and C5) and medium
chain length ( C6) hydroxyalkanoates. This wide vari-
ety of monomers yields PHAs with diverse material
properties that depend on polymer composition. These
material properties, which have proven useful in such
www.current-opinion.com
varied applications as the manufacture of shampoo bot-
tles, heat seal resins and heart valves, have made PHA
polymers prime candidates for commodity and speciality
commercial plastic production.
Besides the commercial applications of PHAs as bioplas-
tics that are biodegradable and made from renewable
resources, from the standpoint of an academic meta-
bolic pathway engineer, PHAs are model compounds
for metabolic engineering. Several bio-based polyesters,
including polylactic acid and 3GT (a polymer of 1,3-
propanediol and terephthalic acid), can have some or all
of their monomeric constituents produced by microbial
fermentation. However, unlike these bio-based poly-
mers which are chemically synthesized, PHAs and their
unnatural polythioester analogs (described below) are
unique in that both polymer assembly and accumulation
occur in vivo and can be manipulated by metabolic
engineering. The monomeric composition of the biopo-
lymer depends on the host’s PHA synthase (polymerase)
and on the hydroxyacyl-CoA thioester precursors sup-
plied to the enzyme, which in turn depend on the meta-
bolic pathways operating in the cell and on the external
carbon source. The biosynthetic routes to PHA mono-
mers compete with and/or rely on important pathways
such as the tricarboxylic acid (TCA) cycle, fatty acid
degradation (b-oxidation) and fatty acid biosynthesis
for precursors, and involve central metabolites such as
acetyl-CoA and cofactors such as NADPH (Figure 1).
Ultimately, the monomeric composition of the PHA
biopolymer provides the metabolic engineer with con-
siderable insight into the metabolism that was involved
in polyester biosynthesis.
The primary aims of the metabolic engineering of PHAs
include controlling different factors that determine poly-
mer material properties, such as monomeric composition,
chain length and copolymer microstructure, as well as
optimizing yield. Pathway engineering for PHA produc-
tion offers the opportunity to synthesize novel polymers
with desirable properties in low-cost, high-productivity
fermentations.
In the history of metabolic engineering, PHAs were
among the first target compounds and there is substantial
literature on this topic including some excellent recent
reviews [2–4,5,6]. Plants may be the most economical
and environmentally friendly hosts for mass production of
biodegradable, commodity plastic [7]. The metabolic
engineering of PHAs in planta was recently reviewed
[5]. However, microbial hosts provide many more
Current Opinion in Biotechnology 2003, 14:475–483
476 Biochemical engineering

Figure 1

GTP
NADH Succinyl-CoA

Succinate
α-Ketoglutarate FADH

NADH
Citric acid Fumarate
Isocitrate cycle

Malate Carbohydrates

Citrate
NADH
Oxaloacetate

Fatty acids
Acetyl-CoA ATP

Acyl-ACP Acyl-CoA
PhaA
NADPH FADH

Fatty acid
trans-2- 3-Keto- Acetoacetyl-CoA 3-Keto- trans-2-
de novo
Enoyl-ACP acyl-ACP acyl-CoA Enoyl-CoA
synthesis
NADPH

PhaB
NADPH
NADH
(R)-3-Hydroxy- (S)-3-Hydroxy-
acyl-ACP acyl-CoA
PhaJ
PhaG
(R)-3-Hydroxyacyl-CoA

PhaC

Polyhydroxyalkanoate
Current Opinion in Biotechnology

PHA biosynthesis in the context of microbial metabolism. The major enzymes involved in PHA biosynthesis are in red. Abbreviations: PhaA,
3-ketothiolase; PhaB, (R)-3-ketoacyl-CoA reductase (for PHB biosynthesis, this enzyme is acetoacetyl-CoA reductase); PhaC, PHA synthase or
polymerase; PhaG, (R)-3-hydroxyacyl ACP:CoA transacylase; PhaJ, (R)-specific enoyl-CoA hydratase. Dotted lines represent reactions where
intermediate metabolic steps are not included. PhaC is specific for enantiomeric monomers in the (R) configuration.

possibilities for precisely engineering the monomeric
composition and the properties of PHAs; the controlled
environment of a bioreactor, as compared with an outdoor
farm field, the ease of microbial genetic manipulation,
and the option to use external carbons sources other than
carbon dioxide are clear advantages. The focus of the
present article is PHA production in bacteria.
Strategies for the metabolic engineering of
bacteria for PHA production
To benefit from the advantages of metabolically engi-
neered microbes for PHA production, several strategies
can be implemented either individually or in combination
(Figure 2). Illustrative examples from the recent litera-
ture are described.
External substrate manipulation
The simplest metabolic engineering strategy is to manip-
ulate the carbon source(s) supplied to the host to control
and direct carbon flux through the precursor and polymer
biosynthesis enzymes. This strategy has traditionally
been exploited to modulate polymer composition by
varying the feed ratio of different substrate precursors.
Recently, there have been novel applications of this
strategy to generate unusual sulfur-containing polymers.

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 Metabolic engineering of polyhydroxyalkanoates Aldor and Keasling 477
Figure 2
Evolved protein
Substrate precursor
addition
Heterologous gene
expression
Schematic of various strategies for the metabolic engineering of PHAs. These approaches can be implemented individually or in combination.
In one instance, recombinant Ralstonia eutropha was fed
alkylthioalkanoates (thia fatty acids) to produce poly(3-
hydroxy-S-propyl-o-thioalkanoate) copolymers contain-
ing thioether linkages in their sidechains [8]. In another
creative example of substrate feeding, previously
unknown microbial polythioesters (which have C(O)S
bonds in contrast to the C(O)O bonds of analogous
PHA polyoxoesters), were incorporated into a copolymer
by cultivating wild-type R. eutropha in medium containing
gluconate plus, for example, 3-mercaptopropionate
(3MP) [9] or 3-mercaptobutyrate (3MB) [10]. The organ-
ism accumulated the polythioester-polyoxoester copoly-
mers P(3MP-co-3HB) and P(3MB-co-3HB), respectively,
with sulfur in their backbones and a nonrandom micro-
structure [9,11].
In another clever example of external substrate manip-
ulation, block copolymers of PHB and poly(3-hydroxy-
butyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) were
generated in wild-type R. eutropha by intermittent addi-
tion of valerate (precursor substrate of 3HV) to cultiva-
tion medium containing an excess of fructose [12]. Core
and shell (diblock copolymer) or multilayered granules
accumulated.
Inhibitor addition
Connected with the idea of a feeding strategy for external
substrates, is the addition of inhibitors that channel pre-
cursors to PHA synthesis by inhibiting competing and
undesirable pathways or that limit chain elongation. Such
compounds have proven particularly useful for incorpor-
ating medium chain length monomers derived from b-
oxidation into PHAs.
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Inhibitor addition
Host cell genome
manipulation
Current Opinion in Biotechnology
For example, acrylate was used to inhibit b-oxidation in
wild-type R. eutropha during growth on octanoate such that
the bacteria accumulated a copolymer of 3-hydroxypropio-
nate, 3HB, 3-hydroxyhexanoate and 3-hydroxyoctanoate
(P(3HP-co-3HB-co-3HHX-co-3HO)) containing both short
and medium chain length monomers [13]. Normally, this
organism accumulates PHAs with only short chain length
monomers.
Recombinant gene expression
Key genes and hosts for recombinant PHA biosynthesis
The main PHA biosynthetic enzymes are shown in
Figure 1 and the corresponding genes can be expressed
in recombinant microbes for the metabolic engineering
of PHAs. The most common microbes used as heterolog-
ous hosts and in fermentation development include R.
eutropha, Pseudomonas putida, Pseudomonas oleovorans and
Escherichia coli [14].
E. coli, which lacks a native PHA synthase and enzymes
that mediate the formation of various hydroxyacyl-CoA
precursors with high specificity, deserves special mention
because of the organism’s numerous advantages as a host
(summarized elsewhere [15,16]). This microbe has well-
understood genetics and biochemistry and can be easily
manipulated for the biosynthesis of PHAs and for high-
productivity fermentation. In E. coli, PHA synthesis may
be induced at an optimized point in the process and is not
tied to natural regulation, which often involves induction
by nutrient limitation. E. coli also does not harbor native
machinery for polymer degradation, unlike organisms that
can retrieve the stored carbon and reducing equivalents.
Finally, E. coli cells are relatively weak, which is an
Current Opinion in Biotechnology 2003, 14:475–483
478 Biochemical engineering
advantage for recovering recombinant granules that tend
to be large in this microbe.
Gene dosage
One approach to metabolically engineer PHA synthesis
involves increasing the copy number of relevant genes,
often genes that are identical or homologous to those
already expressed on the host’s chromosome, to augment
PHA content or to modify polymer composition. Histori-
cally, this strategy has had mixed success [3].
Fukui et al. [17] found that Aeromonas punctata (formerly
known as Aeromonas caviae) harboring additional copies of
its own gene cluster phaPCJ (encoding an uncharacter-
ized, granule-associated ‘phasin’, PHA synthase and
enoyl-CoA hydratase, respectively), accumulated P(3HB-
co-3HHX) at higher levels and with an increased 3HHX
fraction than the wild-type strain. Another group found
that R. eutropha harboring additional phaC copies accu-
mulated significantly more 3HV in P(3HB-co-3HV) than
the parent strain, but total copolymer levels were un-
changed [18].
A similar strategy was attempted by expressing R. eutro-
pha PHA synthesis genes (phaCAB) in a Synechocystis strain
that could naturally accumulate PHAs when grown on
carbon dioxide and various carbon sources [19]. Only a
marginal change in PHA accumulation was observed,
although synthase activity increased twofold. Clearly,
the effectiveness of a strategy involving increased gene
dosage depends on the limiting factor for polymer synth-
esis in the individual case.
Introduction of heterologous precursor production
pathways
Metabolic engineers use heterologous precursor produc-
tion pathways to facilitate the synthesis of polymers that
would not naturally accumulate in the host of interest, or
possibly anywhere in nature, and which may have desir-
able structures and material properties. Another major
goal is to develop novel pathways for efficient precursor
production using unrelated, simple and inexpensive car-
bon sources, which may also facilitate fast growth to a high
cell density.
Historically, the random copolymer P(3HB-co-3HV) has
been the most extensively studied and was sold com-
mercially under the tradename BiopolTM. Production
traditionally involved feeding propionate to produce
intracellular propionyl-CoA, the precursor of 3HV [20].
We metabolically engineered a pathway for the formation
of propionyl-CoA from succinyl-CoA, derived from the
TCA cycle, in recombinant Salmonella enterica serovar
Typhimurium using the recently discovered E. coli
enzymes, sleeping beauty mutase (Sbm) and (R)-methyl-
malonyl-CoA decarboxylase (YgfG) [21]. This facilitated
the production of P(3HB-co-3HV) from glycerol when
Current Opinion in Biotechnology 2003, 14:475–483
phaBCA from Acinetobacter was co-expressed (Figure 3a).
This proof-of-concept study showed how it is possible to
use a novel pathway to generate the copolymer from a
simple carbon source without the need for external pro-
pionate, a costly chemical.
In another recent example of producing a PHA copolymer
from an unrelated carbon source, P(3HB-co-3HHX) was
made from fructose in recombinant R. eutropha harboring
the crotonyl-CoA reductase gene (crr) from Streptomyces
cinnamonensis and phaCJ from A. punctata [22]. This work
also took advantage of several native host enzyme activ-
ities. The pathway functioned sufficiently to incorporate
1.5 mol% 3HHX in the copolymer, resulting in noticeable
improvement of its material properties.
In another example of an engineered multistep pathway,
butyrate kinase (Buk) and phosphotransbutyrylase (Ptb)
from Clostridium acetobutylicum and PHA synthase from
Thiocapsa pfennigii were recently applied to the innovative
production of homopolythioesters in E. coli (Figure 3b)
[23]. 3-Mercaptoalkanoates were supplied in a polymer
production phase that followed the glucose-fed growth
phase of the fermentation. P(3MP), P(3MB) or P(3MV)
were formed depending on the 3-mercaptoalkanoate
feed, and these polymers have unique thermoplastic
properties compared with PHAs and petrochemical-
derived polymers. Such homopolythioesters cannot be
produced in a natural PHA accumulator like R. eutropha,
because in this organism the PHB precursor also accu-
mulates and polythioester-polyoxoester copolymers are
formed (as described above).
Choosing the source of genes
With ongoing genome sequencing projects, newly iden-
tified genes continuously become available. It is common
in PHA work to utilize homologous genes encoding the
same enzyme, but with different activities or specificities,
for various metabolically engineered systems. Together
with the use of diverse, heterologous pathways for the
synthesis of precursors, this facilitates a combinatorial
biosynthetic approach to PHA metabolic engineering.
As PHA synthase specificity is a crucial parameter for
determining polymer composition, in the example given
above for producing polythioesters, the heterodimeric
‘type III’ PhaEC from T. pfennigii was used because of
its broad substrate range [24]. It is also becoming clear
that certain enoyl-CoA hydratases are more useful than
others for transferring medium chain length precursors
from b-oxidation to PHA synthase, owing to the enantio-
selectivity and chain length specificity of various enoyl-
CoA hydratases [25].
Bioprospecting for novel genes and pathways
Metabolic engineers can take advantage of natural bio-
diversity and bioprospect among organisms, including
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 Metabolic engineering of polyhydroxyalkanoates Aldor and Keasling 479

Figure 3

(a)
O O
(b)

O O
x y O
P(3HB-co-3HV)
Glycerol
HS OH
5
3MV
3-Hydroxy- 3-Hydroxy- PEP ATP
valeryl-CoA butyryl-CoA Butyrate kinase
(Buk) ADP
4 Pyruvate
3MV-Pi
3-Keto- Aceto- Phospho- CoA
valeryl-CoA acetyl-CoA transbutyrylase
3 (Ptb) Pi
Acetyl-CoA 3MV-CoA
6
2-Methylcitrate
PHA synthase
Citrate Oxaloacetate (PhaEC)
Propionyl-CoA CoA
Malate 2-Methyl-
Isocitrate Citric
citric 2-Methyl-cis-
acid O
CO2 acid aconitate
cycle Fumarate
2-Ketoglutarate cycle
Succinate S
2 2-Methyl-
Succinyl-CoA isocitrate x
(R)-Methyl- P(3MV)
Pyruvate
malonyl-CoA 1

Current Opinion in Biotechnology

Examples of recently engineered pathways using heterologous precursor production pathways for the biosynthesis of PHA and related biopolymers.
(a) Production of P(3HB-co-3HV) from an unrelated carbon source. The key precursor of 3HV, propionyl-CoA, is synthesized from succinyl-CoA
derived from the TCA cycle. Enzymes are numbered: (1) sleeping beauty mutase (Sbm); (2) (R)-methylmalonyl-CoA decarboxylase (YgfG); (3) 3-
ketothiolase (PhaA); (4) acetoacetyl-CoA reductase (PhaB); (5) PHA synthase (PhaC); (6) 2-methylcitrate synthase (PrpC). The gene encoding PrpC was
deleted from the chromosome of the S. enterica host to channel propionyl-CoA to 3HV by preventing propionyl-CoA catabolism via the 2-methylcitric
acid cycle. Dotted lines represent reactions where intermediate metabolic steps are not included. (b) Production of an unnatural polythioester P(3MV),
a sulfur analog to the polyhydroxyalkanoate (polyoxoester) P(3HV), in recombinant E. coli.

uncultivable species, in a variety of environments to
find PHA genes and pathways suitable for different
applications.
For example, a couple of studies used genes from Aero-
monas species isolated from raw sewage for recombinant
PHA production in E. coli [26,27]. Furthermore, techniques
like degenerate PCR and screening procedures such as
restriction fragment length polymorphism (RFLP) permit
the isolation of novel, PHA-related genes from community
DNA extracted from various environments (S Yilmaz, IS
Aldor, KD McMahon, JD Keasling and D Jenkins, unpub-
lished data), facilitating the discovery of PHA synthesis
genes that are homologous to known sequences.
Exploration of the sequence adjacent to well-character-
ized open reading frames involved in PHA biosynthesis
may also reveal useful genes for polymer production. For
example, upstream of phaC (encoding PHA synthase) on
the Aeromonas genome, orf1 (phaP) was discovered, a gene
which has proved useful for the production of P(3HB-co-
3HHX) with a high 3HHX fraction [27] and facilitated the
incorporation of 3HV in the terpolymer, P(3HB-co-3HV-
co-3HHX) [26].
An interesting recent example of the elucidation of a novel
pathway from an environmental organism involved the use
of 13C labeling and in vivo nuclear magnetic resonance
(NMR) to describe propionate metabolism by uncultiva-
ble, phosphorus-accumulating organisms from activated
sludge that performed enhanced biological phosphorus
removal [28]. The microbes accumulate a copolymer of
3HB, 3HV, 3-hydroxy-2-methylbutyrate and 3-hydroxy-2-
methylvalerate (P(3HB-co-3HV-co-3H2MB-co-3H2MV)),

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:475–483

480 Biochemical engineering
which is not found anywhere else in nature and whose
synthesis would be an interesting pathway to reconstitute
in a heterologous host.
Host cell genome manipulation
Host cell genome manipulation can eliminate competing
pathways or modify native regulation for improved func-
tion of desirable pathways. This strategy was involved in
the study described above for the production of P(3HB-
co-3HV) using Sbm and YgfG. The host was a prpC strain
of S. enterica, in which 2-methylcitrate synthase was
deleted to prevent catabolism of propionyl-CoA via the
2-methylcitric acid cycle and to increase the pool of this
precursor available to 3-ketothiolase [21] (Figure 3a).
In addition, in a separate study, we utilized a mutant S.
enterica prpE, ackA and acs host, which was unable to
activate propionate to propionyl-CoA [29]. We recon-
stituted prpE (encoding propionyl-CoA synthetase)
expression under the control of an isopropyl-b-D-thioga-
lactopyranoside (IPTG)-inducible promoter, indepen-
dent from the promoter expressing the Acinetobacter
PHA synthesis operon. Thus, by eliminating native pro-
pionate activation activity in the host, we could modulate
the composition of the P(3HB-co-3HV) copolymer at a
fixed, external propionate concentration by varying IPTG
addition, creating a ‘dial-a-composition’ system that
facilitated adjustment of material properties according
to inducer level.
Protein engineering of PHA biosynthetic enzymes
Protein engineering via mutagenesis and molecular evo-
lution allows metabolic engineers to optimize enzyme
performance to obtain desirable polymer properties and
yield. The recent target of this strategy has been PHA
synthase, the key enzyme that catalyzes the polymeriza-
tion of hydroxyacyl-CoA thioesters, provided by precursor
pathways, into water-insoluble, PHA granules. A full
structural and mechanistic characterization of this protein
from different microbes is ongoing [30–37].
In general, the use of molecular evolution for enhanced
PHA production has lagged behind the utilization of this
technique for the production of other metabolic engineer-
ing targets, such as carotenoids, because of the lack of a
good screening procedure for desirable mutations. The
screen used in the examples described below involves
Nile red, a lipophilic dye that imparts a pinkish stain to
PHA-containing cells cultured on plates upon exposure to
ultraviolet light. This method selects for high PHA con-
tent and traditionally has not been able to discriminate
between polymers with different compositions [38]. A
recent report, however, suggests that, at least, it is pos-
sible to distinguish between PHAs composed of short
chain length versus medium chain length monomers
using spectrofluorometry with Nile red-stained colonies
suspended in water [39].
Current Opinion in Biotechnology 2003, 14:475–483
In the case of A. punctata PHA synthase, applying single
gene shuffling to a targeted region and screening for
higher PHB-accumulators yielded two mutants that
demonstrated increased activity towards 3HB-CoA and
led to enhanced accumulation of PHB and P(3HB-co-
3HHX) [40]. In addition, both mutants incorporated a
markedly increased 3HHX fraction compared with wild
type. In a separate study, A. punctata PHA synthase was
elegantly engineered in vivo using the mutator strain
E. coli XL1-Red, and mutants were again screened for
promoting enhanced PHB accumulation in recombinant
E. coli [41]. Overproducing mutants with higher in vitro
specific activity (up to fivefold) and yielding higher PHA
content (up to 126% of wild type) were isolated, although
in vitro and in vivo activity did not directly correlate. In
this work, mutants synthesized PHAs with increased
weight average molecular mass, but in contrast to the
previous study the 3HHX fraction was only slightly
different from wild-type composition.
In the latest example of PHA synthase engineering,
Pseudomonas sp. 61-3 PHA synthase was engineered to
enhance PHB accumulation in recombinant E. coli [42].
This polymerase, which synthesizes copolymers consist-
ing of C4 to C12 units, was targeted for improved PHB
accumulation because it possesses low in vitro substrate
specificity towards 3HB-CoA compared to precursors
with longer sidechains. Error-prone PCR generated sin-
gle mutants that indicated two positions where mutations
would lead to increased PHB accumulation. The amino
acids at these respective locations were optimized using
saturation mutagenesis, in which all possible substitu-
tions at the selected sites were tested. A dramatic
increase in PHB content of up to 400-fold was achieved
by generating double mutants with optimal residues at
the two positions.
Protein engineering for metabolic engineering of PHAs
has a promising future. All the examples above utilized
single-gene shuffling techniques. However, given the
large number of cloned PHA synthase genes [15], it will
be interesting to apply family gene shuffling to synthase
engineering as well [43]. Furthermore, we expect other
PHA enzymes to be targets in the near future. Two
candidates include the recently characterized (R)-3-
hydroxyacyl ACP:CoA transacylase (PhaG) from P. putida
[44] and (R)-specific enoyl-CoA hydratase (PhaJ) from A.
punctata [45] (see Update).
Analysis of metabolically engineered, PHA-
producing systems using mathematical
models and molecular techniques
Once the above strategies have been implemented to
create microbial plastic factories, mathematical models,
genetics, microarray analysis, proteomics and metabolo-
mics can be used to study the manipulated biological
systems and suggest new metabolic engineering targets
www.current-opinion.com
 Metabolic engineering of polyhydroxyalkanoates Aldor and Keasling 481
and approaches. PHA researchers are taking advantage of
the mathematical and molecular tools available for such
purposes.
Stoichiometric flux analysis of recombinant E. coli
recently predicted the importance of the Entner–Dou-
doroff pathway in PHB production [46]. This was con-
firmed experimentally [46] and was consistent with
results obtained from a proteomic analysis using two-
dimensional polyacrylamide gel electrophoresis [47].
Mathematical models have also been used to describe
the dynamics of PHA copolymer structure and properties
during its in vivo accumulation. A population balance
model identified an optimal carbon source switching
strategy for the production of block copolymers in the
R. eutropha system described above [48,49].
Also, a recent study utilizing genetics and enzyme activ-
ity assays provides a good example of how a metabolically
engineered system can yield insight into native host
metabolism and enzyme function [50]. The finding that
YfcX (a subunit of a putative fatty acid oxidation com-
plex) is crucial for metabolic engineering of PHAs com-
posed of medium chain length monomers from fatty
acids in E. coli fadB strains preceded work that described
the enzyme’s participation in a previously unknown,
anaerobic b-oxidation pathway and provided evidence
for various aspects of enzyme function in the novel
metabolism [51].
Conclusions
This review covers the recent, published work on PHA
metabolic engineering involving both rational manipula-
tion of pathways and random protein engineering tech-
niques; however, it is also noteworthy that industry
continues to invest in PHAs. In the past, BiopolTM was
produced by ICI, Zeneca and Monsanto. Metabolix
recently acquired the BiopolTM patents from Monsanto
and is producing several PHA polymers in bacteria and
plants. Also, Proctor and Gamble developed PHA copo-
lymers of short and medium chain length monomers
under the tradename NodaxTM, and licensed the tech-
nology to the Kaneka Corporation (a Japanese manufac-
turer of plastics and resins that is focusing on the
production of P(3HB-co-3HHX)). Current, industrial pro-
duction systems extensively utilize metabolically engi-
neered bacteria to produce specialized polymers with
specific, desirable properties. This attests to the value
of PHAs as commercially viable products and to the utility
of metabolic engineering strategies for their production.
We can expect that many more microbial plastic factories
will be constructed in the near future.
Update
In recent work, Tsuge et al. [52] used information
obtained from a crystallographic structure analysis with
site-directed mutagenesis to engineer the chain length
www.current-opinion.com
substrate specificity of (R)-enoyl-CoA hydratase from A.
punctata. Increased fractions of 3HO and 3-hydroxyde-
canoate (3HD) could be incorporated into the PHA
copolymer composed of short and medium chain length
monomers, which was formed from dodecanoate in
recombinant E. coli expressing the mutant hydratase
and PHA synthase from Pseudomonas sp. strain 61-3.
In a second recent paper, a different strategy utilizing
gene dosage and host cell genome manipulation ap-
proaches was taken to modulate the composition of PHA
copolymer composed of medium chain length monomers
for material property improvement [53]. Amplification of
E. coli fadD and fadE, encoding acyl-CoA synthetase and
acyl-CoA dehydrogenase, respectively, in a fadA and/or
fadB mutant of recombinant E. coli that harbored a PHA
synthase from Pseudomonas sp. strain 61-3 (different from
the synthase from this strain mentioned above), led to
enrichment of 3-hydroxyalkanoate monomers of the
same carbon number as the fatty acid carbon source.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Steinbuchel A, Valentin HE: Diversity of bacterial
polyhydroxyalkanoic acids. FEMS Microbiol Lett 1995,
128:219-228.
2. Witholt B, Kessler B: Perspectives of medium chain length
poly(hydroxyalkanoates), a versatile set of bacterial
bioplastics. Curr Opin Biotechnol 1999, 10:279-285.
3. Madison LL, Huisman GW: Metabolic engineering of
poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol
Biol Rev 1999, 63:21-53.
4. Steinbuchel A: Perspectives for biotechnological production
and utilization of biopolymers: metabolic engineering of
polyhydroxyalkanoate biosynthesis pathways as a successful
example. Macromol Biosci 2001, 1:1-24.
5. Snell KD, Peoples OP: Polyhydroxyalkanoate polymers and
 their production in transgenic plants. Metab Eng 2002,
4:29-40.
A comprehensive review of the current status of the production of PHAs in
transgenic plants, a fascinating area not covered in this review. This
publication is from scientists in industry currently working on the meta-
bolic engineering of PHAs and contains interesting practical insights.
6. Steinbuchel A, Lutke-Eversloh T: Metabolic engineering and
 pathway construction for biotechnological production of
relevant polyhydroxyalkanoates in microorganisms.
Biochem Eng J 2003, 3734:1-16.
The latest review by academic leaders in the PHA field with interesting
perspectives on the metabolic engineering of specific PHAs. The focus is
on P(3HB-co-3HV), PHAs composed of medium chain length monomers,
P(4HB) and P(4HV) homopolyesters, copolymers containing 4HB and
copolymers composed of short and medium chain length monomers.
7. Kurdikar D, Fournet L, Slater S, Paster M, Gruys KJ, Gerngross TU,
Coulon R: Greenhouse gas profile of a plastic material derived
from a genetically modified plant. J Ind Ecol 2000, 4:107-122.
8. Ewering C, Lutke-Eversloh T, Luftmann H, Steinbuchel A:
Identification of novel sulfur-containing bacterial polyesters:
biosynthesis of poly(3-hydroxy-S-propyl-x-thioalkanoates)
containing thioether linkages in the side chains. Microbiol 2002,
148:1397-1406.
9. Lutke-Eversloh T, Bergander K, Luftmann H, Steinbuchel A:
Identification of a new class of biopolymer: bacterial synthesis
Current Opinion in Biotechnology 2003, 14:475–483
482 Biochemical engineering
of a sulfur-containing polymer with thioester linkages.
Microbiol 2001, 147:11-19.
10. Lutke-Eversloh T, Bergander K, Luftmann H, Steinbuchel A:
Biosynthesis of poly(3-hydroxybutyrate-co-3-
mercaptobutyrate) as a sulfur analogue to poly(3-
hydroxybutyrate) (PHB). Biomacromolecules 2001,
2:1061-1065.
11. Lutke-Eversloh T, Kawada J, Marchessault RH, Steinbuchel A:
Characterization of microbial polythioesters: physical
properties of novel copolymers synthesized by Ralstonia
eutropha. Biomacromolecules 2002, 3:159-166.
12. Kelley AS, Mantzaris NV, Daoutidis P, Srienc F: Controlled
synthesis of polyhydroxyalkanoic (PHA) nanostructures in
R. eutropha. Nano Lett 2001, 1:481-485.
13. Green PR, Kemper J, Schechtman L, Guo L, Satkowski M,
 Fiedler S, Steinbuchel A, Rehm BHA: Formation of short chain
length/medium chain length polyhydroxyalkanoate
copolymers by fatty acid b-oxidation inhibited Ralstonia
eutropha. Biomacromolecules 2002, 3:208-213.
A thought-provoking piece of work that challenges the idea that R.
eutropha PHA synthase is only capable of incorporating short chain
length monomers into PHAs, a long-held belief that originated with in
vitro enzyme assays. Upon treatment with the fatty acid b-oxidation
inhibitor acrylate, during growth on octanoate, wild-type R. eutropha
accumulated P(3HP-co-3HB-co-3HHX-co-3HO).
14. Lee SY, Choi J: Production of microbial polyester by
fermentation of recombinant microorganisms. Biopolyesters.
Adv Biochem Eng Biotechnol, vol 71. Edited by Steinbuchel A,
Babel W. Berlin: Springer-Verlag; 2001:185-207.
15. Steinbuchel A, Hein S: Biochemical and molecular basis of
microbial synthesis of polyhydroxyalkanoates in
microorganisms. Biopolyesters. Adv Biochem Eng Biotechnol, vol
71. Edited by Steinbuchel A, Babel W. Berlin: Springer-Verlag;
2001:82-123.
16. Lee SY: E. coli moves into the plastic age. Nat Biotechnol 1997,
15:17-18.
17. Fukui T, Kichise T, Iwata T, Doi Y: Characterization of 13 kDa
granule-associated protein in Aeromonas caviae and
biosynthesis of polyhydroxyalkanoate with altered molar
composition by recombinant bacteria. Biomacromolecules
2001, 2:148-153.
18. Choi JC, Shin HD, Lee YH: Modulation of 3-hydroxyvalerate
molar fraction in poly(3-hydroxybutyrate-3-hydroxyvalerate)
using Ralstonia eutropha transformant co-amplifying phbC
and NADPH generation-related zwf genes. Enzyme Microb
Technol 2003, 32:178-185.
19. Sudesh K, Taguchi K, Doi Y: Effect of increased PHA synthase
activity on polyhydroxyalkanoates biosynthesis in
Synechocystis sp. PCC6803. Int J Biol Macromol 2002,
30:97-104.
20. Byrom D: Polymer synthesis by microorganisms: technology
and economics. Trends Biotechnol 1987, 5:246-250.
21. Aldor IS, Kim SW, Prather KLJ, Keasling JD: Metabolic
 engineering of a novel propionate-independent pathway for the
production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
in recombinant Salmonella enterica serovar Typhimurium.
Appl Environ Microbiol 2002, 68:3848-3854.
This paper demonstrates an effective route to the 3HV monomer in
P(3HB-co-3HV) from an unrelated carbon source using the recently
discovered E. coli enzymes Sbm and YgfG, which form part of a novel
pathway to convert succinyl-CoA to propionyl-CoA. Further work with the
recombinant S. enterica host, which can synthesize vitamin B12 de novo
under anaerobic conditions, might eliminate the requirement for exogen-
ous cyanocobalamin addition for the B12-dependent mutase.
22. Fukui T, Abe H, Doi Y: Engineering of Ralstonia eutropha for
 production of poly(3-hydroxybutyrate-co-3-
hydroxyhexanoate) from fructose and solid-state properties of
the copolymer. Biomacromolecules 2002, 3:618-624.
A sophisticated pathway design utilizing the heterologous expression of
PHA synthase and enoyl-CoA hydratase (PhaCJ) from A. punctata,
crotonyl-CoA reductase (Ccr) from Streptomyces cinnamonensis and
native 3-ketothiolase, acetoacetyl-CoA reductase and (S)-3HB-CoA
dehydrogenase activities to make P(3HB-co-3HHX) from fructose in
Current Opinion in Biotechnology 2003, 14:475–483
recombinant R. eutropha. This is the first example of the synthesis of
3HHX from an unrelated carbon source and further development of this
design will hopefully generate higher 3HHX fractions.
23. Lutke-Eversloh T, Fischer A, Remminghorst U, Kawada J,
 Marchessault RH, Bogershausen A, Kalwei M, Eckert H, Reichelt R,
Liu S-J et al.: Biosynthesis of novel thermoplastic polythioesters
by engineered Escherichia coli. Nat Mater 2002, 1:236-240.
E. coli harboring genes for phosphotransbutyrylase (Ptb) and butyrate
kinase (Buk) from C. acetobutylicum and type III PHA synthase (PhaEC)
from T. pfennigii converts 3-mercaptoalkanoates into polythioesters,
which are relatives of PHAs that have sulfur in their backbones. The
homopolythioesters formed in this work are the first to be synthesized in
bacteria, and the authors demonstrate that these polymers have unique
properties compared with PHAs and petrochemical-derived polymers.
PHA synthases can clearly have a surprising and fascinating lack of
specificity.
24. Liu SJ, Steinbuchel A: A novel genetically engineered pathway
for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli.
Appl Environ Microbiol 2000, 66:739-743.
25. Tsuge T, Taguchi K, Taguchi S, Doi Y: Molecular characterization
and properties of (R)-specific enoyl-CoA hydratases from
Pseudomonas aeruginosa: metabolic tools for synthesis of
polyhydroxyalkanoates via fatty acid b-oxidation. Int J Biol
Macromol 2003, 31:195-205.
26. Park SJ, Ahn WS, Green PR, Lee SY: Biosynthesis of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-
hydroxyhexanoate) by metabolically engineered Escherichia
coli strains. Biotechnol Bioeng 2001, 74:81-86.
27. Park SJ, Ahn WS, Green PR, Lee SY: Production of
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by
metabolically engineered Escherichia coli strains.
Biomacromolecules 2001, 2:248-254.
28. Lemos PC, Serafim LS, Santos MM, Reis MAM, Santos H:
 Metabolic pathway for propionate utilization by
phosphorus-accumulating organisms in activated sludge:
13
C labeling and in vivo nuclear magnetic resonance.
Appl Environ Microbiol 2003, 69:241-251.
A ground-breaking example of the use of 13C in vivo NMR to elucidate the
complex carbon metabolism in activated sludge, composed primarily of
uncultivable organisms, performing the wastewater treatment process
known as enhanced biological phosphorus removal (EBPR). An accurate
model for EBPR metabolism has been the subject of debate for many
years. This paper is a significant contribution towards understanding this
complicated metabolism, including the associated PHA accumulation
that involves a novel PHA copolymer and that has an important role in the
treatment process.
29. Aldor I, Keasling JD: Metabolic engineering of poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) in recombinant
Salmonella enterica serovar Typhimurium. Biotechnol Bioeng
2001, 76:108-114.
30. Zhang S, Kolvek S, Lenz RW, Goodwin S: Mechanism of the
polymerization reaction initiated and catalyzed by the
polyhydroxybutyrate synthase of Ralstonia eutropha.
Biomacromolecules 2003, 4:504-509.
31. Hezayen FF, Steinbuchel A, Rehm BHA: Biochemical and
enzymological properties of the polyhydroxybutyrate synthase
from the extremely halophilic archaeon strain 56. Arch Biochem
Biophys 2002, 403:284-291.
32. Jia Y, Yuan W, Wodzinska J, Park C, Sinskey AJ, Stubbe J:
Mechanistic studies on class I polyhydroxybutyrate (PHB)
synthase from Ralstonia eutropha: class I and III synthases
share a similar catalytic mechanism. Biochemistry 2001,
40:1011-1019.
33. Yuan W, Jia Y, Tian JM, Snell KD, Muh U, Sinskey AJ, Lambalot RH,
Walsh CT, Stubbe J: Class I and III polyhydroxyalkanoate
synthases from Ralstonia eutropha and Allochromatium
vinosum: characterization and substrate specificity studies.
Arch Biochem Biophys 2001, 394:87-98.
34. Rehm BHA, Antonio RV, Spiekermann P, Amara AA, Steinbuchel A:
Molecular characterization of the poly(3-hydroxybutyrate)
(PHB) synthase from Ralstonia eutropha: in vitro evolution,
site-specific mutagenesis and development of a PHB synthase
protein model. Biochim Biophys Acta 2002, 1594:178-190.
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 Metabolic engineering of polyhydroxyalkanoates Aldor and Keasling 483
35. Liebergesell M, Rehalkar S, Steinbuchel A: Analysis of the
Thiocapsa pfennigii polyhydroxyalkanoate synthase:
subcloning, molecular characterization and generation of
hybrid synthases with the corresponding Chromatium vinosum
enzyme. Appl Microbiol Biotechnol 2000, 54:186-194.
36. Zhang S, Kamachi M, Takagi Y, Lenz RW, Goodwin S:
Comparative study of the relationship between monomer
structure and reactivity for two polyhydroxyalkanoate
synthases. Appl Microbiol Biotechnol 2001, 56:131-136.
37. Qi Q, Steinbuchel A, Rehm BHA: In vitro synthesis of
poly(3-hydroxydecanoate): purification and enzymatic
characterization of type II polyhydroxyalkanoate synthases
PhaC1 and PhaC2 from Pseudomonas aeruginosa.
Appl Microbiol Biotechnol 2000, 54:37-43.
38. Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D,
Steinbuchel A: A sensitive, viable-colony staining method using
Nile red for direct screening of bacteria that accumulate
polyhydroxyalkanoic acids and other lipid storage compounds.
Arch Microbiol 1999, 171:73-80.
39. Wu H, Sheu D, Lee C: Rapid differentiation between short-chain-
length and medium-chain-length polyhydroxyalkanoate-
accumulating bacteria with spectrofluorometry. J Microbiol
Methods 2003, 53:131-135.
40. Kichise T, Taguchi S, Doi Y: Enhanced accumulation and
 changed monomer composition in polyhydroxyalkanoate
(PHA) copolyester by in vitro evolution of Aeromonas caviae
PHA synthase. Appl Environ Microbiol 2002, 68:2411-2419.
An in vitro approach (error-prone PCR) was used for the molecular
evolution of A. punctata PHA synthase for enhanced accumulation of
P(3HB-co-3HHX) in recombinant E. coli harboring phaABJP. Interest-
ingly, in contrast to other work [41], the 3HHX fraction was affected by
evolution of the synthase. The mutant generation and screening meth-
odology is nicely illustrated in a figure in this paper.
41. Amara AA, Steinbuchel A, Rehm BHA: In vivo evolution of the
 Aeromonas punctata polyhydroxyalkanoate (PHA) synthase:
isolation and characterization of modified PHA synthases with
enhanced activity. Appl Microbiol Biotechnol 2002, 59:477-482.
A pioneering protein engineering effort to use the XL1-Red mutator strain
to evolve A. punctata PHA synthase in vivo. All other attempts to engineer
the enzyme to date have relied on in vitro mutagenesis. Mutant synthases
showed higher in vivo specific activity, resulted in higher P(3HB-co-3HHX)
accumulation in recombinant E. coli harboring phaAB and treated with
acrylate, and also generated polymers with increased weight average
molecular mass. However, insignificant changes were seen in composi-
tion compared with wild type.
42. Takase K, Taguchi S, Doi Y: Enhanced synthesis of poly(3-
hydroxybutyrate) in recombinant Escherichia coli by means of
error-prone PCR mutagenesis, saturation mutagenesis, and
in vitro recombination of the type II polyhydroxyalkanoate
synthase gene. J Biochem (Tokyo) 2003, 133:139-145.
43. Crameri A, Raillard SA, Bermudez E, Stemmer WPC: DNA shuffling
of a family of genes from diverse species accelerates directed
evolution. Nature 1998, 391:288-291.
44. Hoffmann N, Amara AA, Beermann BB, Qi Q, Hinz HJ, Rehm BHA:
Biochemical characterization of the Pseudomonas putida
www.current-opinion.com
3-hydroxyacyl ACP:CoA transacylase, which diverts
intermediates of fatty acid de novo biosynthesis. J Biol Chem
2002, 277:42926-42936.
45. Hisano T, Tsuge T, Fukui T, Iwata T, Miki K, Doi Y: Crystal structure
of the (R)-specific enoyl-CoA hydratase from Aeromonas
caviae involved in polyhydroxyalkanoate biosynthesis. J Biol
Chem 2003, 278:617-624.
46. Hong SH, Park CJ, Moon SY, Park JP, Lee SY: In silico prediction
and validation of the importance of the Entner-Doudoroff
pathway in poly(3-hydroxybutyrate) production by
metabolically engineered Escherichia coli. Biotechnol Bioeng
2003, 83:854-863.
47. Han M, Yoon SS, Lee SY: Proteome analysis of metabolically
engineered Escherichia coli producing poly(3-
hydroxybutyrate). J Bacteriol 2001, 183:301-308.
48. Mantzaris NV, Kelley AS, Srienc F, Daoutidis P: Optimal carbon
source switching strategy for the production of PHA
copolymers. AIChE J 2001, 47:727-743.
49. Mantzaris NV, Kelley AS, Daoutidis P, Srienc F: A population
 balance model describing the dynamics of molecular weight
distributions and the structure of PHA copolymer chains.
Chem Eng Sci 2002, 57:4643-4663.
For the mathematically inclined, a rigorous population balance model is
presented that describes the dynamics of the synthesis of P(3HB-co-
3HV) and PHB-P(3HB-co-3HV) block copolymers in nongrowing R.
eutropha cells. Predicted optimal conditions for di-block and tri-block
copolymer synthesis fell in the range of feasible bioprocessing manipula-
tions.
50. Snell KD, Feng F, Zhong L, Martin D, Madison LL: YfcX enables
 medium-chain-length poly(3-hydroxyalkanoate) formation
from fatty acids in recombinant Escherichia coli fadB strains.
J Bacteriol 2002, 184:5696-5705.
An excellent example of reverse engineering to understand why fadB
mutants of E. coli, with a disrupted fatty acid b-oxidation complex, can
still incorporate monomer units with a chain length shorter than that of the
fatty acid carbon source. YfcX (subunit of fatty acid oxidation complex)
was shown to be crucial for the synthesis of PHAs composed of medium
chain length monomers in these strains and this resolves a long-standing
ambiguity regarding recombinant PHA biosynthesis in E. coli.
51. Campbell JW, Morgan-Kiss RM, Cronan JE: A new Escherichia
coli metabolic competency: growth on fatty acids by a novel
anaerobic b-oxidation pathway. Mol Microbiol 2003,
47:793-805.
52. Tsuge T, Hisano T, Taguchi S, Doi Y: Alteration of chain length
 substrate specificity of Aeromonas caviae R-enantiomer-
specific enoyl-coenzyme A hydratase through site-directed
mutagenesis. Appl Environ Microbiol 2003, 69:4830-4836.
An interesting recent paper that illustrates the potential for rational design
in protein engineering of PHA biosynthetic enzymes. This approach is
feasible for engineering (R)-enoyl-CoA hydratase, because its crystal
structure has been determined.
53. Park SJ, Park JP, Lee SY, Doi Y: Enrichment of specific monomer
in medium-chain-length poly(3-hydroxyalkanoates) by
amplification of fadD and fadE genes in recombinant
Escherichia coli. Enzyme Microb Technol 2003, 33:62-70.
Current Opinion in Biotechnology 2003, 14:475–483