173
6
PHA Synthases: The Key
Enzymes of PHA Synthesis
Dr. Bernd H. A. Rehm1, Prof. Dr. Alexander Steinb¸chel2
1
Institut f¸r Mikrobiologie, Westf‰lische Wilhelms-Universit‰t M¸nster,
Corrensstra˚e 3, D-48149 M¸nster, Germany; Tel.: ‡ 49-251-8339848;
Fax: ‡ 49-251-8338388; E-mail: [email protected]
2
Institut f¸r Mikrobiologie, Westf‰lische Wilhelms-Universit‰t M¸nster,
Corrensstra˚e 3, D-48149 M¸nster, Germany; Tel.: ‡ 49-251-8339821;
Fax: ‡ 49-251-8338388; E-mail: [email protected]
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
2
Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
3
Cloning Strategies for PHA Synthase Genes . . . . . . . . . . . . . . . . . . . .
175
4
Organization of PHA Synthase Genes . . . . . . . . . . . . . . . . . . . . . . .
178
5
Primary Structures of PHA Synthases . . . . . . . . . . . . . . . . . . . . . . .
179
6
6.1
Secondary and Quaternary Structures of PHA Synthases . . . . . . . . . . . .
In vivo versus in vitro Substrate Specificity of PHA Synthases . . . . . . . . .
194
195
7
Development of a Topological Model for PHA Synthases and Analysis
of Site-specific Mutants of the PHA Synthases . . . . . . . . . . . . . . . . . .
199
8
The Proposed Catalytic Mechanism of PHA Synthases . . . . . . . . . . . . . .
202
9
Factors Determining the Molecular Weight and Composition of PHAs . . . .
203
10
PHA Granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
11
In vitro Synthesis of PHA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
12
Diversion of Intermediates from Central Pathways to PHA Biosynthesis . . .
207
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6 PHA Synthases: The Key Enzymes of PHA Synthesis
13
Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
209
3HB
HA
ORF
PHA
Poly(3HB )
3-hydroxybutyrate
hydroxyalkanoic acid
open reading frame
Polyhydroxyalkanoic acids
poly 3-hydroxybutyrate
1
Introduction
Polyhydroxyalkanoic acids (PHAs) represent
a rather complex class of polyesters that are
synthesized by most genera of bacteria and
members of the family Halobacteriaceae of
the Archaea (Steinb¸chel et al., 1997; Steinb¸chel and F¸chtenbusch, 1998). Most of
these prokaryotes synthesize poly(3-hydroxybutyric acid), poly(3HB ), and other PHAs as
storage compounds and deposit these polyesters as insoluble inclusions in the cytoplasm.
The number of 91 different constituents of
PHAs that were recently compiled (Steinb¸chel and Valentin, 1995) has meanwhile been
outnumbered, and approximately 150 different hydroxyalkanoic acids are now known to
occur as constituents of PHAs. These waterinsoluble PHAs exhibit rather high molecular
weights, thermoplastic and/or elastomeric
features, and some other interesting physical and material properties. Therefore, and
since they are biodegradable (Jendrossek
et al., 1996), they are considered for several
applications in the packaging industry, medicine, pharmacy, agriculture and food industry, or as raw materials for the synthesis of
enantiomerically pure chemicals and the
production of paints (Anderson and Dawes,
1990; M¸ller and Seebach, 1993; Hocking
and Marchessault; 1994 Steinb¸chel, 1996;
Williams et al., 1999; van der Walle et al.,
1999). Many prokaryotic and eukaryotic
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organisms are able to produce low-molecular
weight poly(3HB ) molecules that are complexed with other biomolecules and that
occur at concentrations which are three to
four orders of magnitude less than storage
PHAs in the cells (Reusch and Sadoff, 1988).
A few eukaryotic microorganisms such as
for example Aureobasidium pullulans are able
to synthesize the water-soluble polyester
polymalic acid which is not synthesized by
prokaryotes (Liu and Steinb¸chel, 1996).
PHA synthases are the key enzymes of
PHA biosynthesis. They use coenzyme A
(CoA ) thioesters of hydroxyalkanoic acids
(HA ) as substrates and catalyze the polymerization of HAs into PHA with the concomitant release of CoA. After the cloning of the
PHA synthase operon of Ralstonia eutropha,
approximately 12 years ago in three different
laboratories (Schubert et al., 1988; Slater
et al., 1988; Peoples and Sinskey, 1989), as
many as 54 different PHA synthases (plus
one partial PHA synthase gene from Pseudomonas sp.) from in total 44 microorganisms were cloned, and the primary structures of 44 different PHA synthases are
available. This contribution provides an
update of previously published reviews on
the knowledge of the organization of PHA
biosynthesis genes, the PHA synthase primary structures, biochemical features of
these unique enzymes and their proposed
catalytic mechanism (Rehm and Steinb¸chel, 1999; Steinb¸chel and Hein, 2001).
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3 Cloning Strategies for PHA Synthase Genes
2
Terminology
How to refer to the occurrence of homopolyesters, copolyesters and blends of two
different polyesters in biological samples
has been proposed and is now widely used as
terminology (Steinb¸chel et al., 1992). Few
publications exist which still refer to light
scattering inclusion bodies or lipophilic
polymeric materials as poly(3HB ) without
performing sufficient chemical analysis, and
not taking into account the large number of
different types of PHAs. However, PHAs
composed of short-chain-length (three to five
carbon atoms) or medium chain-length (six
to 14 carbon atoms) hydroxyalkanoic acid,
the respective CoA thioesters or the substrate range of PHA synthases are indicated
by the terms SCL or MCL, respectively, that
are used as subscripts (e. g. PHASCL, HAMCLCoA thioesters, PHASCL synthase). The
proposed term LCL (more than 14 carbon
atoms) has so far not really been required.
Suggestions for the designations of genes,
which are involved in PHA biosynthesis,
auxiliary structural proteins bound to PHA
granules and PHA-degrading enzymes, were
also accepted and are now mostly used in the
literature (Steinb¸chel et al., 1992; Rehm
and Steinb¸chel, 1999). Genes coding for
proteins involved in the biosynthesis of PHA
will be referred in alphabetical order as phaA
(b-ketothiolase), phaB (acetoacetyl-CoA reductase), phaC (PHA synthase), phaG (3hydroxyacyl-acyl carrier protein-CoA transacylase), phaJ (enoyl-CoA hydratase), etc.,
whereas the designation of genes required
for the degradation will be referred in
opposite alphabetical order such as phaZ
for PHA depolymerases, phaY, phaX, phaW,
etc. The genes for phasins and regulator
proteins should be referred to as phaP and
phaR, respectively. It is highly recommended
to continue the use of the proposed terms in
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order to refer to the respective genes or the
respective proteins. Due to the large number
of genes and proteins involved in PHA
metabolism, the large number of different
organisms currently under investigation and
the many more that will follow, it will be also
useful to indicate the origin of a gene or a
protein by the first letter of the genus and the
species designation and to add these two
letters as a subscript. The PHA synthase of
R. eutropha will, for example, be referred to
as PhaCRe.
3
Cloning Strategies for PHA Synthase Genes
To date, eight different strategies have been
applied to identify PHA synthase genes and
other genes involved in PHA biosynthesis
(Table 1, A±H). These approaches are of quite
different quality and elegance, and are distinguished with respect to the time and effort
required. The successful application depends
on several prerequisites and constellations,
and few strategies will allow the identification
of PHA synthase genes encoding enzymes
with unusual and/or novel features.
The enzymatic approach (strategy A )
simply screened clones for functional expression of PHA synthase genes involved in
PHA biosynthesis. Homologous gene
probes were obtained after transposon mutagenesis and were used to identify the
respective intact gene of the same genome
(strategy B ). Heterologous gene probes,
mostly prepared from the well characterized
R. eutropha PHA synthase gene, were used to
identify corresponding genes in genomic
libraries prepared from other bacteria (strategy C ). Similarly, short oligonucleotides
designed according to short highly conserved stretches of PHA synthases derived
from the multiple alignment of PHA syn-
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6 PHA Synthases: The Key Enzymes of PHA Synthesis
Tab. 1
Strategies for cloning of PHA synthase genes
Strategy
Screening technology
A
B
C
D
Enzymatic analysis
Homologous gene probes (hybridization) obtained by transposon mutagenesis
Heterologous gene probes (hybridization) obtained from well-characterized genes
Consensus oligonucleotides derived from the multiple alignment (hybridization or PCR
technique)
Oligonucleotides derived from N-terminal or internal amino acid sequence of PHA synthases
Opaque colonies or fluorescent colonies (in vivo staining with Nile red) in PHA-negative host
after heterologous expression
Growth after detoxification of media due to removal of fatty acids
Genome sequence analysis and functional assignment of PHA synthase genes
Growth in medium without carbon source due to the mobilization of storage polymer
E
F
G
H
I
thases were also successfully employed
(strategy D ). In one case, the PHA synthase
protein was purified, and oligonucleotides
were designed from the N-terminal amino
acids sequence to identify the corresponding
gene in a genomic library (Strategy E ).
The most successful and widely applied
strategy, was to screen genomic libraries for
phenotypic complementation of a PHAnegative mutant or for conferring the ability
to synthesize and accumulate PHA to a
PHA-negative wild-type (strategy F ). The
PHA-negative mutants PHB-4 (Schlegel
et al., 1970) of R. eutropha and GPp104 of
Pseudomonas putida (Huisman et al., 1991),
a PHA-leaky mutant of P. putida and various
strains of E. coli were most widely used.
Recently, another interesting strategy
(strategy G ) was employed to clone heterologous phaC genes in a PhaC-negative
mutant of Rhodobacter capsulatus, utilizing
the detoxification of the medium from fatty
acids due to their incorporation into PHAs.
The increasing number of genome-sequencing projects will provide access to
further PHA synthase genes. Since hitherto
known PHA synthases are distinguished
from other proteins but have a rather high
degree of homology to each other (see
below), a homology search in the databases
will result in the identification of genes
encoding homologous proteins which are
subsequently cloned by employing the PCR
technique (strategy H ) (see also Tables 2 and
Bacteria from which PHA synthase genes were cloned and assigned (updated version of Rehm and
Steinb¸chel, 1999; Steinb¸chel and Hein, 2001)
Tab. 2
Bacterium
Accession
no.
Cloning Reference
strategy
Acinetobacter sp. RA3849
Aeromonas caviae
Alcaligenes latus
Alcaligenes sp. SH-69
Allochromatium vinosum D
Azorhizobium caulinodans
Bacillus megaterium
Burkholderia sp. DSMZ9242
Caulobacter crescentus
L37761
D88825
AF078795
U78047
L01112
AJ006237
AF109909
AF153089
AY007313
F
D
F
?
C
C
E
F
H
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Schembri et al. (1994)
Fukui and Doi (1997)
Choi et al. (1998)
Lee et al. (1996), unpublished results
Liebergesell and Steinb¸chel (1992)
Mandon et al. (1998)
McCool and Cannon (1999)
Rodrigues et al. (2000a)
Qi and Rehm (2001)
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3 Cloning Strategies for PHA Synthase Genes
Tab. 2
(cont.)
Bacterium
Accession
no.
Cloning Reference
strategy
Chromobacterium violaceum
Comamonas acidovorans
Ectothiorhodospira shaposhnikovii N1
Lamprocystis roseopersicina 3112
Methylobacterium extorquens IBT6
Nocardia corallina
Paracoccus denitrificans
Pseudomonas acidophila
Pseudomonas aeruginosa
Pseudomonas citronellolis
Pseudomonas fluorescens
Pseudomonas mendocina
Pseudomonas putida KT2442
Pseudomonas putida U
Pseudomonas putida BM01
Pseudomonas sp. DSMZ1650
Pseudomonas sp. GP4BH1
Pseudomonas sp. 61-3
C
C
C
F
F
?
C
F
D
D
?
D
F
B
E
D
D
C
Kolibachuk et al. (1999)
Sudesh et al. (1998)
S. Zhang et al. (2000), unpublished results
Liebergesell and Steinb¸chel (1993)
Valentin and Steinb¸chel (1993)
B. Hall et al., unpublished results
Ueda et al. (1996)
Umeda et al. (1998)
Timm and Steinb¸chel (1992)
Timm et al. (1994)
D. Dennis, unpublished results
Timm et al. (1994)
Huisman et al. (1991)
Garcia et al. (1999)
Valentin et al. (1998)
Timm et al. (1994)
Timm et al. (1994)
Matsusaki et al. (1998)
Pseudomonas sp.
Pseudomonas oleovorans
Pseudomonas resinovorans
Ralstonia eutropha H16
AF061446
AB009273
AF307334
±
L07893
AF019964
D43764
±
X66592
±
±
±
±
AF150670
AF042276a
±
±
AB014757
AB014758
Z80158b
M58445
AF129396
A34341
?
F
H
A,B,C
Rhizobium etli
Rhodobacter capsulatus
Rhodobacter sphaeroides
Rhodococcus ruber PP2
Rhodospirillum rubrum Ha
Rhodospirillum rubrum ATCC25903
Rickettsia prowazekii
Sinorhizobium meliloti 41
Synechocystis sp. PCC6803
Syntrophomonas wolfei
Thiocystis violacea 2311
Thiocapsa pfennigii 9111
Vibrio cholerae
Zoogloea ramigera
U30612
±
X97200
X66407
AJ245888
AF178117
AJ235273
AF031938
Slr1830/29
±
L01113
X93599
AE004398
U66242
C
G
F
F
F
F
±
?
H
F
C
C
±
?
J. K. Shin et al. (1996), unpublished results
Huisman et al. (1991)
Solaiman (2000)
Slater et al. (1988); Schubert et al. (1988);
Peoples and Sinskey (1989)
Cevallos et al. (1996)
Kranz et al. (1997)
Hustede and Steinb¸chel (1993)
Pieper and Steinb¸chel (1992)
Clemente et al. (2000)
Clemente et al. (2000)
Andersson et al. (1998)
L. Willis and G. Walker, unpublished results
Kaneko et al. (1996); Hein et al. (1998)
McInerney et al. (1992)
Liebergesell and Steinb¸chel (1993)
Liebergesell et al. (2000)
Heidelberg et al. (2000)
I. Lee et al. (1996), unpublished results
a
The 533 C-terminal amino acids of PHA synthase PhaC2 are available. b The 90 N-terminal amino acids of
the PHA synthase are available. c Assignment was carried out based on amino acid sequence homology.
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6 PHA Synthases: The Key Enzymes of PHA Synthesis
Bacteria, which partial genomic DNA sequence showed ORFs with strong homology to PHA
synthases
Tab. 3
Bacterium
Accession
Homology (% Identity)a
Pseudomonas syringae
www.tigr.com
- colocalized ORFs
Burkholderia cepacia
www.jgi.doe.gov
contig630, contig318 (not colocalized ORFs)
Burkholderia pseudomallei
www.sanger.ac.uk
- shotgun (not colocalized ORFs)
Rhodopseudomonas palustris
www.jgi.doe.gov
contig59, contig58 (not colocalized ORFs)
Magnetospirillum magnetotacticum
www.jgi.doe.gov
1. PhaC1 from Pseudomonas
aeruginosa (75%)
2. PhaC2 from Pseudomonas
aeruginosa (60%)
1. PhaC from Azorhizobium
caulinodans (39%)
2. PhaC from Burkholderia sp.
DSMZ 9242 (80%)
1. PhaC from Rhodococcus ruber
(40%)
2. PhaC from Burkholderia sp.
DSMZ 9242 (90%)
1. PhaC from Ralstonia eutropha
H16 (42%)
2. PhaC from Azorhizobium
caulinodans (59%)
1. PhaC from Ralstonia eutropha
H16 (41%)
a
The high scores of the amino acid sequence homology search are presented. The numbers indicate the
different ORFs.
3). Table 2 summarizes all currently available PHA synthase genes, for which an
accession number has been provided, and
which were functionally assigned except the
PHA synthase genes from Rickettsia prowazekii and Vibrio cholerae. The latter PHA
synthase genes were only assigned based on
amino acid sequence homology. In Table 3, a
list of putative PHA synthase genes (ORFs)
is presented, which were obtained by homology search of the currently available
partial microbial genome sequences. One of
the first examples are the cloning of the class
I and class III PHA synthase structural
genes from Synechocystis sp. PCC 6803 and
Caulobacter crescentus (Hein et al., 1998; Qi
and Rehm, 2001). Strategy I has not been
applied to clone PHA synthase genes, but
might be useful in future to isolate PHA
synthase mutants with modified properties
such as, for example, changed substrate
specificity.
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4
Organization of PHA Synthase Genes
The PHA synthase genes and genes for other
proteins related to the metabolism of PHA
are often clustered in the bacterial genomes
(Figures 1 to 4). In R. eutropha, which has
been studied in most detail with respect to
PHA biosynthesis (Steinb¸chel and Schlegel, 1991), the genes for PHA synthase
(phaC ), b-ketothiolase (phaA ) and NADPdependent acetoacetyl-CoA reductase (phaB )
constitute the phaCAB operon (Schubert
et al., 1988; Slater et al., 1988; Peoples and
Sinskey, 1989). Approximately 4 kbp downstream of this operon, a second b-ketothiolase gene (bktB ) was recently identified
(Slater et al., 1998). BktB is in contrast to
PhaA able to synthesize 3-ketovaleryl-CoA.
Besides R. eutropha, also Alcaligenes latus,
Burkholderia sp. DSMZ9242, Chromobacterium violaceum and Comamonas acidovorans
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4 Organization of PHA Synthase Genes
seem to possess a phaCAB operon, whereas
in Acinetobacter sp., Pseudomonas sp. 61-3
and V. cholerae these genes are also clustered,
but organized in a different array (Figure 1).
In contrast, phaC seems to be separated
from other pha genes in Zoogloea ramigera,
Methylobacterioum extorquens, Sinorhizobium
meliloti, Nocarda corallina, Rhodococcus ruber,
Paracoccus denitrificans, Rhodobacter sphaeroides, Rhodospirillum rubrum, Rhodobacter
capsulatus, Caulobacter crescentus, Rickettsia
prowazekii and Aeromonas caviae. PhaA,
phaB or other genes related to PHA metabolism are not directly linked to the phaCs in
these genomes (Figure 2). The only exception is A. caviae. In this bacterium the gene
encoding an enoyl-CoA hydratase (see below) is located downstream of phaC.
Pseudomonas oleovorans, Pseudomonas sp.
61 ± 3, P. aeruginosa, P. resinovorans, P. putida
U and P. mendocina possess two different
phaC genes which are in the genome
separated by the structural gene for an
intracellular PHA depolymerase (Figure 3).
Pseudomonas sp. 61 ± 3 contains these two
phaC genes in addition to another phaC
gene, which is co-localized with phaB and
phaA (Figures 1 and 3).
In all bacteria, which posses a two-component PHA synthase, phaC and phaE are
directly linked in the genomes constituting
most probably single operons. In A. vinosum,
phaA and phaB are located on the opposite
strand in a gene cluster related to PHA
metabolism. The organization of the genes
is most probably similar if not identical in
Thiocystis violacea and Thiocapsa pfennigii,
whereas in Synechocystis sp. PCC 6803
further pha genes do definitely not map
close to the phaEC locus (Figure 4).
It should be emphasized that in R. ruber, P.
denitrificans, Acinetobacter sp., Ectothiorhodospira shaposhnikovii and Allochromatium vinosum the structural genes for phasin proteins (ORF3, ORF5, and phaP respectively;
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see below) map close to the respective phaC
loci (Figures 2 and 4).
Homology search analysis (tBlastN ) of
the currently available microbial genome
sequences strongly suggested that some
species harbor a completely new constellation and organization of PHA synthase
genes (Table 3). For instance, in Burkholderia
cepacia, Burkholderia pseudomallei and Rhodopseudomonas palustris two not colocalized
ORFs, respectively, were identified, which
deduced amino acid sequences show strong
homology to class I PHA synthases.
Interestingly, PHA-accumulating bacteria
belonging to the a-proteobacteria, such as
Caulobacter crescentus, Azorhizobium caulinodans, Rhizobium etli, Sinorhizobium meliloti,
P. denitrificans and Methylobacterium extorquens, contained the class I PHA synthase
gene not colocalized with other PHA biosynthesis genes ( Valentin and Steinb¸chel,
1993; Tombolini et al., 1995; Cevallos et al.,
1996; Mandon et al., 1998; Maehara et al.
1999; Qi and Rehm, 2001). Only a few
exceptions, such as Zoogloea ramigera (bproteobacterium), A. caviae (g-proteobacterium) and Nocardia corallina (a firmicute),
not belonging to a-proteobacteria have been
described, which do not contain colocalized
PHA biosynthesis genes. Some species such
as P. denitrificans possessed adjacent to the
PHA synthase further genes like phaP
(encoding phasin) and phaR (encoding
regulator protein) related to PHA biosynthesis. Among the b-proteobacteria PHAaccumulating bacteria, such as R. eutropha,
Burkholderia sp., A. latus and C. acidovorans
(Schubert et al., 1991; Choi et al., 1998;
Sudesh et al., 1998; Rodrigues et al.,
2000a), an operonic organization of PHA
biosynthesis genes, which are related to the
short-chain-length PHA biosynthesis (class I
PHA synthase gene) is found.
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