Next Article 
J Bacteriol, April 1998, p. 1979-1987, Vol. 180, No. 8
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multiple 
-Ketothiolases Mediate
Poly(-Hydroxyalkanoate) Copolymer Synthesis in Ralstonia
eutropha
Steven
Slater,*
Kathryn L.
Houmiel,
Minhtien
Tran,
Timothy A.
Mitsky,
Nancy B.
Taylor,
Stephen R.
Padgette, and
Kenneth J.
Gruys
Sustainable Development and Agricultural
Sectors, Monsanto Company, St. Louis, Missouri 63198
Received 22 October 1997/Accepted 16 February 1998
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ABSTRACT |
Polyhydroxyalkanoates (PHAs) are a class of carbon and energy
storage polymers produced by numerous bacteria in response to environmental limitation. The type of polymer produced depends on the
carbon sources available, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA
biosynthetic enzymes. Ralstonia eutropha produces both the homopolymer poly-
-hydroxybutyrate (PHB) and, when provided with the
appropriate substrate, the copolymer
poly(-hydroxybutyrate-co--hydroxyvalerate) (PHBV). A required
step in production of the hydroxyvalerate moiety of PHBV is the
condensation of acetyl coenzyme A (acetyl-CoA) and propionyl-CoA to
form -ketovaleryl-CoA. This activity has generally been attributed
to the -ketothiolase encoded by R. eutropha phbA.
However, we have determined that PhbA does not significantly contribute
to catalyzing this condensation reaction. Here we report the cloning
and genetic analysis of bktB, which encodes a
-ketothiolase from R. eutropha that is capable of
forming -ketovaleryl-CoA. Genetic analyses determined that BktB is
the primary condensation enzyme leading to production of
-hydroxyvalerate derived from propionyl-CoA. We also report an
additional -ketothiolase, designated BktC, that probably serves as a
secondary route toward -hydroxyvalerate production.
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INTRODUCTION |
Polyhydroxyalkanoates (PHAs) are a
class of naturally occurring polymers which serve as a carbon and
energy reserve in numerous bacterial species. Ralstonia
eutropha (formerly designated Alcaligenes eutrophus
[41]) produces the homopolymer
poly(-hydroxybutyrate) (PHB) and, when provided with propionate in
the feedstock, the copolymer
poly(-hydroxybutyrate-co--hydroxyvalerate) (PHBV). R. eutropha is used commercially to produce PHBV, which is a
biodegradable thermoplastic.
The PHB biosynthetic pathway requires three enzymatic activities: a
-ketothiolase (PhbA), an NADPH-dependent acetoacetyl coenzyme A
(acetoacetyl-CoA) reductase (PhbB) and a PHB synthase (PhbC). The first
step in production of the homopolymer PHB is catalyzed by
-ketothiolase which condenses two acetyl-CoA molecules to form
acetoacetyl-CoA. Formation of the copolymer PHBV requires the
additional condensation of acetyl-CoA with propionyl-CoA to form
-ketovaleryl-CoA (Fig. 1).
Subsequently, the acetoacetyl-CoA and -ketovaleryl-CoA are converted
into a polymer by the activities of the reductase and synthase. The
genes encoding these proteins in R. eutropha reside in an
operon which has been well characterized (10, 21, 22, 31,
37).
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FIG. 1.
Pathway for production of PHBV from acetyl-CoA and
propionyl-CoA. -Ketothiolase performs the condensation reactions to
generate either acetoacetyl-CoA or -ketovaleryl-CoA. These are
reduced by acetoacetyl-CoA reductase (PhbB) and polymerized by PHB
synthase (PhbC).
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The substrate specificities of these three enzymes are reportedly
adequate for production of PHBV copolymer (7-9), but
propionate-fed Escherichia coli harboring the R. eutropha phb operon produces essentially PHB homopolymer
(35). Moreover, PHBV copolymer can be produced in E. coli after induction of the fatty acid -oxidation complex,
which contains a -ketothiolase with broad substrate specificity
(26, 27, 35). These data suggest that the R. eutropha PHB pathway is capable of producing copolymer, but only in the context of a second -ketothiolase with broad substrate specificity.
R. eutropha is known to produce at least two
-ketothiolases (7), and at least two distinct plasmid
clones which express -ketothiolase have been isolated from R. eutropha (37). In this work, we analyzed the substrate
specificity of the PhbA -ketothiolase and demonstrated that this
enzyme catalyzes thiolysis of -ketovaleryl-CoA very poorly. We
determined that R. eutropha expresses at least two
-ketothiolases in addition to PhbA and that these additional enzymes, which we designate BktB and BktC, efficiently utilize -ketovaleryl-CoA. We also report the isolation and characterization of bktB (-ketothiolase B), which encodes the BktB
-ketothiolase required for efficient production of PHBV in R. eutropha.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The bacterial
strains utilized in this study are summarized in Table
1. Luria-Bertani (LB) medium contained
10 g of tryptone (Difco), 5 g of yeast extract (Difco), and
5 g of NaCl. All plates contained 1.5% Bacto Agar (Difco).
LB-sucrose plates used to select against pJQ200SK and pLO2 derivatives
had no NaCl and contained 5% sucrose. M9 minimal medium was prepared
according to the method of Miller (18). R. eutropha was grown in Schlegel's minimal medium (29)
containing 0.1% nutrient broth powder (Difco), with the exception that
Hoagland's trace element solution was replaced with SL6 trace element
solution (23). Schlegel's nitrogen-complete (SNC) medium
also contained 1 g of NH4Cl per liter, whereas
Schlegel's nitrogen-limited (SNL) medium also contained 0.1 g of
NH4Cl per liter. Antibiotics were used at the following
levels: E. coli, carbenicillin, 100 µg/ml;
chloramphenicol, 25 µg/ml; and kanamycin, 50 µg/ml; R. eutropha, chloramphenicol, 100 µg/ml for broth and 100 to 250 µg/ml for plates; kanamycin, 350 µg/ml; and gentamicin, 10 µg/ml.
Cultures for polymer analysis were grown in 25 ml of the appropriate
medium in 250-ml flasks and shaken at 220 rpm for the
desired time.
Each flask was inoculated with 0.5 ml of a separate
5-ml seed culture
containing the appropriate antibiotics.
R. eutropha seed
cultures were grown in SNC medium containing 0.4% fructose,
and
E. coli seed cultures were grown in M9 medium containing
0.2%
glucose.
E. coli was incubated at 37°C, and
R. eutropha was incubated
at 30°C.
For polymer production,
R. eutropha was grown in SNL medium
containing 3% fructose and 0.1% sodium propionate. At 24-h intervals,
each culture was supplemented with 1 ml of 2.5% sodium propionate,
and
the cells were harvested 72 h after inoculation.
For polymer production in
E. coli, strains were grown in M9
medium containing 1% glucose, the desired amount of sodium propionate,
and 100 µg of carbenicillin where appropriate. Cultures were
incubated
for approximately 40 h prior to harvesting.
DNA manipulations.
The plasmids utilized in this study are
described in Table 2. All cloning was
performed by standard procedures (28). The location of
bktB was mapped within plasmid pBK6 (37) by
sequential deletion of restriction fragments (Fig.
2). Plasmid pMON25728 contains
bktB on a 3.9-kb DNA fragment extending from a
BglII site about 0.5 kb upstream of bktB to an
NcoI site approximately 2 kb downstream of bktB.
pMON25765 contains a DNA fragment extending from the aforementioned
BglII site to a SalI site 98 bp downstream of
bktB, and the R. eutropha DNA is flanked by
HindIII sites. Plasmid pMON25901 harbors the
bktB-containing HindIII fragment of pMON25765
cloned into the HindIII site of pBBR1MCS
(13). Plasmid pMON25831 harbors DNA extending from the
BglII site just upstream of bktB to the
BglII site within the phb operon.
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FIG. 2.
(A) Restriction map of the region surrounding the
phb operon and bktB in pAE175 (37) and
the regions of DNA harbored by plasmid vectors pertinent to this work.
B, BglII; E, EcoRI; X, XhoI. Beyond
the phbCAB-bktB region, the structure of pBK6 does not
accurately represent that of the R. eutropha chromosome.
(See "DNA sequencing and chromosomal analysis" in Materials and
Methods for details.) (B) Restriction map and locations of ORFs within
the phbCAB-bktB chromosomal region of R. eutropha. The PstI site marked with an asterisk is the
termination site of the previously published sequence (21)
and the initiation site of the sequence reported in this work. P,
PstI; S, SmaI.
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Plasmid pMON25636 harbors a derivative of
phbA in which an
NcoI site has been engineered via PCR to overlap the
initiation
ATG codon and a
HindIII site has been
engineered immediately downstream
of
phbA. Plasmid pMON25966
contains the
phbA NcoI-
HindIII fragment
of
pMON25636 fused to the
bktB promoter of pMON25765 at the
BspHI
site that overlaps the
bktB initiation
codon in the native gene.
The resulting construct contains
phbA under
bktB promoter control,
and the entire
gene is flanked by
HindIII sites.
Plasmids used for PHA production in
E. coli were constructed
with the
HindIII fragments from pMON25765 and pMON25966
cloned
into plasmid pPHA
A. Plasmid pPHA
A (obtained from D. Dennis)
contains the
R. eutropha phb operon from plasmid
pJM9238 (
11)
in pBBR1MCS, but with a deletion of a 995-bp
StuI fragment. The
deletion inactivates
phbA, but
both
phbC and
phbB are expressed.
The
-ketothiolase-containing
HindIII fragments of
pMON25765 and
pMON25966 were cloned into the unique
HindIII site of pPHA
A (located
upstream of the
phb promoter), and the orientation of the insert
was
determined. Plasmids pMON25968 and pMON25970 harbor the resulting
bktB and
phbA constructs, respectively, oriented
such that the
phbCB and
bktB promoters are
adjacent and divergent. Plasmids
pMON25982 and pMON25984 were
constructed by excising the entire
PHA production cassette from
pMON25968 and pMON25970, respectively,
with
EcoRI and
XhoI and cloning the resulting fragments into the
EcoRI-
XhoI sites of pBluescript KS(+)
(Stratagene).
Plasmid pMON25742 (
bktB::
kan) contains
the end-filled 1.7-kb Kan
r BamHI fragment of
Tn
10kan (
12) cloned in the end-filled
NcoI
site within
bktB of pMON25728. Plasmid
pMON25776 contains an approximately
5-kb
ApaI fragment
harboring
bktB::
kan from pMON25742
cloned in
the
ApaI site of pJQ200SK (
25). Plasmid
pMON25845 contains a
Cam
r BamHI fragment of
pFF589 (provided by R. Maurer) cloned into
the
BamHI site
within the gentamicin resistance gene of pMON25776.
The
phbA allele used for disruption of
phbA in
R. eutropha is the same allele harbored by plasmid pPHA
A
(described above
[Table
2]). The particular construct used for
deletion of
phbA in
R. eutropha was derived from
pMON25971, which is identical
to pMON25970 (described above), except
that the
HindIII fragment
harboring
phbA is
in the opposite orientation. This orientation
places an
XbaI
site upstream of
phbC, with the result that the
entire
phbA allele is flanked by
XbaI sites. The
XbaI fragment
harboring
phbC-phbA-phbB was
ligated into the
XbaI site of pLO2
(
15) to create
pMON36818, which was used for disruption of
phbA.
DNA sequencing and chromosomal analysis.
Sequencing of the
region from the PstI site downstream of phbB
through bktB was accomplished by a combination of subcloning and primer walking with both automated and manual sequencing. Automated
sequencing utilized the ABI-Prism sequencing kit with AmpliTaq FS DNA
polymerase (Perkin-Elmer), and manual sequencing used Sequenase DNA
polymerase (U.S. Biochemicals). Sequence analysis was performed with
the Wisconsin Package of molecular analysis software (Genetics Computer
Group, Madison, Wis.) and the Sequencher DNA sequencing software
package (Gene Codes Corporation).
The chromosomal structure of the
R. eutropha phbCAB-bktB
region was determined by Southern blot hybridization to match the
structure identified by DNA sequencing (Fig.
2 and
3). However,
these
analyses also determined that a region of pAE175 is the
product of a
cloning artifact. Specifically, the
bktB-containing
plasmid
pBK6 (derived from pAE175) harbors an
EcoRI fragment of
approximately 12 kb, whereas
bktB from the
R. eutropha chromosome
is harbored on an
EcoRI fragment of
approximately 4 kb. Two other
cosmid clones from
R. eutropha, pAE537 and pAE683 (
37), also
harbor
bktB on a 4-kb
EcoRI fragment. Additional
Southern blots
determined that the pBK6 sequence diverges from the
wild-type
sequence immediately downstream from
bktB, at the
SalI site at
bp 5592 of Fig.
3. Since pAE175 was constructed
with a partial
SalI digestion of
R. eutropha
chromosomal DNA, it appears that
the
phbCAB-bktB region was
linked to another portion of the
R. eutropha chromosome
during the ligation reaction.
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FIG. 3.
Nucleotide sequence and predicted translation products
of the R. eutropha chromosomal region downstream of the
phb operon through bktB. The sequence shown
initiates from the PstI site downstream of phbB
in the published phbAB sequence (21). Possible
ribosome-binding (RB) sequences upstream of each ORF are underlined.
The predicted amino acids underlined within the bktB ORF
correspond precisely to those determined by N-terminal sequence
analysis of BktB.
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Disruption of bktB and phbA.
For
disruption of bktB, E. coli S17-1 (34)
was transformed with pMON25845 (Table 2), and the resulting strain was
mated overnight at 30°C with wild-type R. eutropha H16
(strain EE167) on LB plates containing 0.2% fructose. Exconjugants
were purified on NaCl-free LB agar containing 350 µg of kanamycin per
ml, 10 µg of gentamicin per ml, 5% sucrose, and 0.2% fructose.
Following 3 days of growth at 30°C, several colonies were twice
purified and then checked for the absence of BktB by immunoblotting,
and the bktB genotype was confirmed by Southern blot
hybridization. Strains EE168 and EE169 were isolated from two
independent mating events (Table 1).
For disruption of
phbA in
R. eutropha,
E. coli S17-1 (
34) was transformed with pMON36818 (Table
2), and the resulting strain
was mated overnight at 30°C with
wild-type
R. eutropha H16 (strain
EE167) on LB plates
containing 0.2% fructose. Exconjugants were
twice purified on LB
medium containing 0.2% fructose and 350 µg
of kanamycin per ml and
then purified on NaCL-free LB medium containing
5% sucrose and 0.2%
fructose. Several kanamycin-sensitive strains
having reduced
-ketothiolase activity were further purified,
and the
phbA deletion was verified by Southern blot hybridization.
One strain, EE303 (Table
1), was chosen for further analysis.
Strain EE317, a
phbA bktB strain (Table
1), was produced by
integrating pMON25845 in EE303 and then isolating a
bktB::
kan derivative as described above
for construction of strains EE168
and EE169. The genotype of EE317 was
confirmed by Southern blotting.
Complementation of bktB in R. eutropha.
Mobilization of pBBR1MCS and pMON25901 (Table 2) into R. eutropha was performed by mating E. coli S17-1
(34) harboring the appropriate plasmid with the appropriate
R. eutropha recipient strain on LB plates containing 0.2%
fructose. Exconjugants were purified for isolation on LB plates
containing 250 µg of chloramphenicol per ml, 10 µg of gentamicin
per ml, and 0.2% fructose, and the plates were incubated for 4 days at
30°C. Surviving colonies were twice purified on LB-fructose plates
containing 100 µg of chloramphenicol per ml. Plasmids were purified
from each strain, and their structures were verified by digestion with
a variety of restriction endonucleases.
Purification of BktB and PhbA.
All protein concentrations
were determined by the method of Bradford (3) with reagents
obtained from Bio-Rad Laboratories. Bovine serum albumin (obtained from
Sigma) was used as a protein standard.
For purification of BktB,
E. coli EE32 (Table
1) was grown
in a 2-liter Bioflow III fermentor (New Brunswick Scientific)
at 37°C
for 16 h in Terrific Broth (
18) containing
carbenicillin.
Cells were harvested by centrifugation, washed once with
phosphate-buffered
saline (
28), and frozen at
20°C until
lysis. This produced
approximately 100 ml of concentrated cell paste.
Approximately 30 ml of cell paste was resuspended in 90 ml of lysis
buffer [10 mM Tris (pH 8.0), 1 mM dithiothreitol (DTT),
2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)] and the
cells
were disrupted by sonication. The lysate was cleared by
centrifugation
for 10 min at 31,000 ×
g in a Beckman SA-17 rotor.
Protein was precipitated from the cleared lysate (approximately
100 ml)
with 80% saturated ammonium sulfate, and the pellet was
redissolved in
50 ml of 1 M ammonium sulfate. The solution was
passed through a
0.2-µm-pore-diameter filter and then was loaded
onto a
phenyl-Sepharose fast-performance liquid chromatography
(FPLC) column
(1 by 10 cm) (Pharmacia). Buffer A was 1 mM DTT-20
mM sodium phosphate
(pH 7.0), and buffer B was 1 mM DTT-20 mM
sodium phosphate (pH 7.0)
containing 1 M ammonium sulfate. The
gradient was 100 to 20% buffer B
in 40 min and then 20 to 0% buffer
B in 20 min. The flow rate was 2 ml
per min, and one fraction
was collected per minute. Fractions 42 to 51, which contained
most of the
-ketothiolase activity and which were
pooled, were
concentrated to 5 ml with a Centriprep-10 (Amicon, Inc.),
desalted
with a PD-10 column (Pharmacia), passed through a
0.2-µm-pore-diameter
filter, and further separated on a Mono-Q HR5/5
FPLC column (Pharmacia).
Buffer C was 1 mM DTT-10 mM Tris (pH 7.8),
and buffer D was 1
mM DTT-10 mM Tris (pH 7.8) containing 1 M KCl. The
gradient was
0 to 10% buffer D in 10 min, 10 to 35% buffer D in 40 min, and
then 35 to 100% buffer D in 20 min. The flow rate was 1 ml
per
min, and 1 fraction was collected per minute. Most BktB eluted
in
fractions 18 through 24. These fractions were pooled, and the
pooled
material was at least 95% pure as estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Pro-Blue
staining (Daiichi). The final solution (6 ml containing 1.6 mg
of
protein/ml) contained BktB at 65 U/mg (assayed with acetoacetyl-CoA).
This material was used for kinetic analyses and to generate antisera
against BktB.
Material from a separate, smaller-scale purification utilizing the same
frozen cell paste was used for N-terminal sequence
analysis. In this
case, fraction 23 from the final Mono-Q chromatography
step was
concentrated in a Centricon-10 column and washed several
times with 10 mM NaHCO
3 buffer to remove Tris buffer. The final
material
(0.2 ml at 0.68 mg/ml) was directly N-terminally sequenced
by the
Monsanto protein analysis laboratory.
PhbA was purified from
E. coli DH5
harboring pMON25636, a
plasmid in which
R. eutropha phbA is under control of an
IPTG (isopropyl-
-
D-thiogalactopyranoside)-inducible
promoter (Table
2). A 200-ml shaken-flask culture of this strain
was
grown to mid-log phase (Klett reading of 45 units) at 37°C
in LB
medium containing carbenicillin, and then cells were induced
by adding
IPTG to 0.5 mM. After 2 more h of growth, the cells
were harvested by
centrifugation and frozen until use. Cells were
disrupted by
sonication, and the protein was purified as described
above, except
that the phenyl-Sepharose column was omitted. PhbA
elutes from Mono-Q
earlier than BktB, with the peak activity occurring
in fractions 12 and
13 of this preparation. These two 1-ml fractions
were combined,
providing a final solution containing 0.6 mg of
total protein per ml.
The material was at least 95% pure, as estimated
by SDS-PAGE and
Pro-Blue staining (Daiichi).
Substrate synthesis and -ketothiolase enzyme assays.
Preparation of -ketoacyl-CoA substrate precursors
trans-2,3-pentenoyl-CoA and
trans-2,3-hexenoyl-CoA was accomplished by the procedure of
Schulz (32), essentially as described previously, except on
a larger scale (100 µmol of starting trans-2,3-pentenoic or trans-2,3-hexenoic acid and 100 µmol of CoA). Another
modification to the procedure was the purification of the enoyl-CoA
products by semi-prep C8 reverse-phase chromatography.
-Ketothiolase was assayed in the thiolysis direction by two methods,
depending on whether the substrate was acetoacetyl-CoA
(method 1) or
-ketovaleryl-CoA or
-ketohexanoyl-CoA (method
2). Method 1 is
similar to that described previously (
19) and
monitors the
disappearance of acetoacetyl-CoA (40 µM) spectrophotometrically
at
304 nm in a mixture of 150 mM EPPS
(
N-[2-hydroxyethyl]piperazine-
N'-[3-propanesulfonic
acid] (pH 8.0) and 50 mM MgCl
2. CoA was used at 100 µM,
and the
total reaction volume was 1.0 ml. The reaction was initiated by
the addition of enzyme. Method 2 is principally the same; however,
-ketovaleryl-CoA or
-ketohexanoyl-CoA was generated in situ
by
quantitative conversion of the respective enoyl-CoA by using
crotonase
(3 U),
-hydroxyacyl-CoA dehydrogenase (5 U), lactate
dehydrogenase
(10 U), 1.5 mM pyruvate, and 0.4 mM NAD
+ in a reaction
similar to that described by Haywood et al. (
7).
Enoyl-CoA
at a concentration of 40 µM, 100 µM CoA, and the buffering
system
described above were used for this study. The extinction
coefficients
for acetoacetyl-CoA,
-ketovaleryl-CoA, and
-ketohexanoyl-CoA
at
304 nm were 19.5, 12.2, and 14.0 mM
1 cm
1,
respectively, in this buffer system (
36) and were used for
all activity calculations. The two
-ketothiolase assay methods
gave
equivalent results, as demonstrated by comparing the results
obtained
with the in situ generation of acetoacetyl-CoA from crotonyl-CoA
(method 2) with results obtained with acetoacetyl-CoA directly
(method
1 [
36]). One unit of enzyme activity is defined as the
amount of enzyme required to convert 1 µmol of
-ketoacyl-CoA
to
product per min at 25°C.
Polymer analysis.
Cells from each culture were harvested by
centrifugation, washed once with absolute ethanol, and then dried
overnight at 70°C. The dried pellets were weighed, and then polymer
was extracted by immersion of the cell powder in 5 ml of chloroform and
heating at 100°C for 5 h. After cooling, the chloroform was
filtered through glass wool and collected in glass tubes. The
chloroform was evaporated, and the polymer was removed and weighed.
Polymer composition was analyzed by methanolysis of the polymer,
followed by gas chromatography of the methyl ester residues.
In a
glass, screw-cap tube, 5 mg of polymer sample was dissolved
in 1 ml of
chloroform containing 3 µmol of benzoate per ml as
an internal
standard. One milliliter of 15% sulfuric acid in methanol
was added,
and then the tubes were tightly capped and incubated
at 100°C for
2.5 h. The tubes were cooled to room temperature
and then placed
on ice, and the solution was extracted with 0.5
ml of water to remove
sulfuric acid. The organic layer was transferred
to a clean tube
containing approximately 200 mg of sodium sulfate
as a drying agent,
the mixture was mixed with a Vortex for approximately
1 min, and then
solids were removed by centrifugation. The supernatant
solution was
transferred to vials for chromatography.
Gas chromatography was performed according to a variation of the method
of Braunegg et al. (
4). The gas chromatograph was
a Varian
3400 equipped with a Varian 8200 autosampler, a 1077
split/splitless
injector, a 1-ml Hamilton gas-tight syringe, a
J & W DB-5 capillary
column (0.25 mm by 30 m; 0.25-µm film thickness),
and a flame
ionization detector. The flow rate of helium carrier
gas was 2.5 ml per
min, with a 50:1 sample/split ratio at injection.
The initial column
temperature of 60°C was held for 3 min, and
then the temperature was
increased by 10°C per min up to 240°C.
Finally, the column
temperature was raised by 30°C per min to
300°C and held for 10 min. Standards were methyl-(
D)-hydroxybutyrate
and
methyl-(
D)-hydroxyvalerate (Fluka, Ronkonkoma, N.Y.).
Nucleotide sequence accession number.
The GenBank accession
number for the DNA sequence reported herein is AF026544.
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RESULTS |
Purification and enzymatic characterization of BktB and PhbA.
The R. eutropha genes sufficient for PHB biosynthesis were
previously isolated on cosmid pAE175 (37). Subcloning of
pAE175 identified two distinct EcoRI fragments, harbored in
plasmids pBK12 and pBK6, that express -ketothiolase activity.
Plasmid pBK12 harbors the phb operon, which includes the
phbA -ketothiolase gene. Plasmid pBK6 carries a second,
distinct -ketothiolase gene that we have designated bktB
(-ketothiolase B) of R. eutropha.
To compare the kinetic properties of the
-ketothiolases, recombinant
E. coli strains were used to produce BktB and PhbA
(Materials
and Methods). The proteins were purified, and the thiolysis
reaction
was performed with various substrates (Table
3). Both enzymes
efficiently catalyze the
thiolysis of acetoacetyl-CoA. However,
the specific activity of PhbA is
approximately 150-fold lower
when
-ketovaleryl-CoA is the substrate,
and no thiolysis by PhbA
of
-ketohexanoyl-CoA was detected. In
contrast, BktB prefers
-ketovaleryl-CoA and also efficiently
catalyzes the thiolysis
of
-ketohexanoyl-CoA. We infer from the
thiolysis data that PhbA
and BktB can also catalyze the condensation
reactions, and in
a separate series of experiments, we have confirmed
the ability
of these enzymes to catalyze the expected condensations
(
36).
Subcloning and sequencing of bktB and surrounding
DNA.
Starting with plasmid pBK6, serial subcloning experiments
isolated bktB on pMON25765, which harbors a 1.8-kb fragment
extending from the BglII site upstream of bktB to
a SalI site just downstream of bktB. Additional
overlapping clones derived from pAE175 linked bktB to the
phb operon (Fig. 2A).
The entire region from
bktB to the
phb operon was
sequenced. A map of this region appears in Fig.
2, and the DNA sequence
appears in Fig.
3. This sequence is contiguous with the previously
published sequence of
phbAB of
R. eutropha
(
10,
21 [GenBank
accession no.
J04987]). The
phb operon lies approximately 4.6
kb upstream of
bktB. The 16 N-terminal amino acids predicted by
the
bktB sequence match those identified by amino acid
sequencing
of purified BktB protein (Materials and Methods).
bktB is approximately
66% identical to
phbA at
the nucleotide level, and the two encoded
proteins are 53% identical
and 61% similar (Fig.
4). BktB contains
394 amino acid residues and has a calculated molecular mass of
40,903 Da.
View larger version (61K):
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|
FIG. 4.
Comparison of the predicted amino acid sequences of PhbA
(top) and BktB (bottom) -ketothiolase proteins from R. eutropha. The two proteins are 51% identical and 61% similar,
with similarity extending across the entire sequence. The cysteine
residues designated by arrows are the absolutely conserved active site
cysteines (20, 39). Comparison was performed with the Gap
program of the Wisconsin Package.
|
|
The
phbCAB-bktB intergenic region contains three additional
open reading frames (ORFs) that are likely to be expressed (Fig.
2B and
3). All three ORFs show significant homology to putative
genes from
other organisms, but none of these putative genes has
been assigned a
function (see Discussion).
bktB can complement phbA for PHA production
in E. coli.
The abilities of PhbA and BktB to produce PHA
were compared by using E. coli. A pair of plasmids was
constructed, with each plasmid harboring phbCB and with the
two differing only in the -ketothiolase ORF (Materials and Methods).
Strains harboring these plasmids were grown in M9 minimal medium
containing 1% glucose and various amounts of propionate. After about
40 h of growth, the cells were harvested and the polymer was
analyzed (Fig. 5). In these assays,
bktB and phbA were essentially equivalent in their ability to produce high levels of polymer. The strain harboring bktB incorporated -hydroxyvalerate into polymer much more
efficiently than a strain harboring phbA. However, the
amount of -hydroxyvalerate incorporated into polymer reached a
plateau when the media contained 0.05% or more propionate.
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|
FIG. 5.
Comparison of PhbA and BktB in synthesis of PHBV in
E. coli. Both strains harbored a plasmid expressing R. eutropha phbCB plus a -ketothiolase gene, either
bktB or phbA (see Materials and Methods). Polymer
accumulation and composition were monitored as a function of the
propionate in the medium. Total polymer ( ) and the
-hydroxyvalerate fraction of the polymer ( ) in strain EE245,
which expresses bktB, are shown, as are total polymer ( )
and the -hydroxyvalerate fraction of the polymer ( ) in strain
EE247, which expresses phbA.
|
|
R. eutropha bktB mutants are impaired in their ability
to produce PHBV when grown on propionate.
To determine if BktB
normally plays a role in PHA synthesis, a
bktB::kan allele was used to replace
the bktB locus of wild-type R. eutropha. Two
independent kanamycin-resistant strains, EE168 and EE169, were
characterized. Both strains were determined by Southern blotting to
carry the bktB::kan allele, and both
are negative for BktB protein as determined by immunoblotting
(36).
We compared
-ketothiolase activity and polymer production by
wild-type
R. eutropha and two independent
bktB::
kan mutants
(Table
4). When
-ketothiolase was assayed
with acetoacetyl-CoA
as a substrate, essentially no difference was seen
between extracts
from wild-type
R. eutropha and the
bktB mutants. However, the
specific activity of
-ketothiolase capable of metabolizing
-ketovaleryl-CoA
is
approximately 80% lower in the
bktB mutants than in the
wild-type
strain. This decreased ability to metabolize
-ketovaleryl-CoA
was reflected in the composition of the polymer
produced when
the various strains were grown in the presence of
propionate.
The
-hydroxyvalerate component of the polymer in
the
bktB::
kan strains was approximately
one-third that found in the wild-type
strain. This deficiency is
complemented by a plasmid, pMON25901,
that carries
bktB+. In fact, overproduction of BktB from this
plasmid led to slightly
increased incorporation of
-hydroxyvalerate
into PHA. The overexpression
of
-ketothiolase (cumulatively,
approximately threefold when
assayed on acetoacetyl-CoA) did not
significantly affect the total
amount of polymer accumulated by
R. eutropha in these fermentation
end-point assays.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
-Ketothiolase activity and polymer composition in
R. eutropha bktB::kan mutants and in
strains overexpressing bktB
|
|
An additional -ketothiolase is expressed in R. eutropha.
R. eutropha bktB mutants retain significant -ketothiolase
activity capable of degrading -ketovaleryl-CoA (Table 4). This activity is inconsistent with the substrate specificity of PhbA, so we
examined R. eutropha for the presence of additional
-ketothiolases.
Figure
6 shows anion-exchange
chromatography of extracts from wild-type
R. eutropha (Fig.
6A), a
bktB mutant (Fig.
6B), and
a
phbA bktB
double mutant (Fig.
6C), all grown on fructose. Four
peaks of
-ketothiolase activity were distinguishable in extracts
of wild-type
R. eutropha. PhbA eluted first, as a large peak containing
activity primarily restricted to acetoacetyl-CoA utilization.
Peak 2 was smaller than peak 1, but more active when assayed on
-ketovaleryl-CoA. We designated this activity as BktC, although
we
cannot rule out the presence of multiple
-ketothiolases within
this
activity peak. Peak 3 contains BktB, which provides most
of the
activity capable of metabolizing
-ketovaleryl-CoA. Peak
4 is a small
peak eluting after the primary BktB peak. We have
not yet fully
characterized the contents of peak 4, but immunoblot
analyses suggest
that BktB is a component (reference
36 and
described
below).
View larger version (13K):
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|
FIG. 6.
Mono-Q chromatography of extracts from the R. eutropha wild-type strain EE167 (A), the
bktB::kan strain EE168 (B), and the
phbA bktB::kan strain EE317 (C), all
grown on fructose. Chromatographic conditions were as described in
Materials and Methods. The enzymatic activity of -ketothiolase was
assayed in the thiolysis direction with either acetoacetyl-CoA ( ) or
-ketovaleryl-CoA ( ) as the substrate. The number designations of
the individual peaks resolved from each strain are shown within each
panel. Note that peak 1 is missing from the phbA mutant,
peak 3 is missing from the bktB mutants, and peaks 1, 3, and
4 are missing from the double mutant.
|
|
Figure
6B shows anion-exchange chromatography of extract from a
bktB::
kan mutant. The absence of BktB
(peak 3) is clear in
this chromatogram, and the relative area of peak 4 is also diminished.
Peak 2 (BktC) accounts for almost all of the
residual activity
capable of degrading
-ketovaleryl-CoA. In Fig.
6C,
the presence
of BktC is clearly demonstrated in a strain lacking both
PhbA
and BktB. Peak 4 also disappears in the double mutant, suggesting
that peak 4 normally contains BktB and PhbA, apparently in some
modified form.
PhbA, BktB, and BktC were all expressed when
R. eutropha was
grown on fructose as the sole carbon source and are distinguishable
from the
-oxidative
-ketothiolase, which is expressed only during
growth on fatty acids and prefers acyl-CoAs of longer chain lengths
(
36). The gene encoding the
-oxidative enzyme from
R. eutropha has been cloned on pAE65 (
36,
37).
| |
DISCUSSION |
R. eutropha constitutively expresses at least three
different -ketothiolases, designated PhbA, BktB, and BktC. PhbA is
catalytically the most active, with BktB and BktC activities present at
lower levels. Production of PHBV from acetyl-CoA and propionyl-CoA
requires -ketothiolase to produce both acetoacetyl-CoA and
-ketovaleryl-CoA. However, PhbA, the -ketothiolase encoded within
the phb operon, is essentially limited to production of
acetoacetyl-CoA, as inferred from the thiolysis activity results. The
genetic and biochemical evidence presented herein establishes BktB as
the -ketothiolase primarily responsible for generating
-ketovaleryl-CoA during growth on fructose and propionate. BktC
appears to be a less active contributor to -ketovaleryl-CoA
formation.
The phb operon and bktB map approximately 4.6 kb
apart on the R. eutropha chromosome, and the intervening
region contains three ORFs that are likely to be expressed. ORF1 is
highly homologous to ORFs identified near the pha genes of
Rhizobium, Thiocystis, and Chromatium
species (16, 17, 40), suggesting that the ORF1 product
participates in PHA metabolism. ORF2 and ORF3 apparently form an operon
with overlapping termination and initiation codons, respectively. ORF2
is similar to the E. coli yhdG and yohI ORFs, members of an apparently ubiquitous class of genes with homologs in
yeast and humans (14). ORF3 is highly similar to E. coli f441 and to other putative genes in both bacteria and
archaea. No function has been assigned to any ORF2 or ORF3 homolog.
Complementation studies with E. coli demonstrate that
bktB can be used to produce PHBV copolymer in a recombinant
system. The -hydroxyvalerate composition of the polymer increased
linearly up to about 0.05% propionate in the media and then reached a
plateau (Fig. 5). This plateau is probably due to saturation of
propionyl-CoA formation, since E. coli acs (acetyl-CoA
synthetase) is repressed under the growth conditions in these
experiments (5). Enhancement of propionate import and
activation should yield significantly higher incorporation of
-hydroxyvalerate into polymer (26, 27), potentially
opening E. coli as a system for copolymer production.
Our findings confirm and extend those of Haywood et al. (7),
who partially purified two -ketothiolases from R. eutropha, designated -ketothiolases A and B. These two
preparations probably correspond to PhbA and BktB, respectively,
although the relatively high level of activity on acetoacetyl-CoA
reported for -ketothiolase B suggests that their preparation also
contained an enzyme that prefers acetoacetyl-CoA, such as PhbA and/or
BktC. We also noted that the BktB-containing peak from wild-type
R. eutropha showed a preference for catalysis of
acetoacetyl-CoA (Fig. 6), whereas pure BktB preferred
-ketovaleryl-CoA (Table 3). It is possible that PhbA and/or BktC
forms mixed oligomers with BktB, resulting in this shift of substrate
specificity. Alternatively, other factors may affect the substrate
specificity of BktB.
Our data establish a role for BktB in PHA synthesis, but it is likely
that BktB and BktC also function during degradation of fatty acids
(7). Although we did not assay substrates larger than
-ketohexanoyl-CoA, the substrate flexibility of BktB probably extends at least to -ketodecanoyl-CoA (7). BktB may,
therefore, play an auxiliary role in -oxidation, including partial
degradation of short- and medium-chain fatty acids which do not induce
the -oxidative complex.
Finally, PhbA, BktB, and BktC may all play a critical role as
scavengers of carbon, allowing PHA formation when the available carbon
cannot be metabolized directly. This situation occurs under anaerobic
conditions. R. eutropha is strictly respiratory but can grow
anaerobically in the presence of an alternative electron acceptor such
as nitrate or nitrite (2). R. eutropha also
induces a number of enzymes in response to low O2,
indicating that it normally encounters anaerobic conditions (6,
24, 30, 38). Finally, anaerobic conditions induce PHA production
in R. eutropha (33). The ability to form PHA is
advantageous in anaerobic conditions for at least two reasons: (i) PHA
formation serves as an electron sink (33), and (ii) carbon
can be scavenged and stored for use when O2 becomes
available. In this respect, R. eutropha seems well adapted
to a niche at the aerobic-anaerobic boundary. Here, it would encounter
acetate and propionate produced by anaerobic fermentation, and both of
these compounds could be channeled into polymer by BktB and BktC. Being
motile, R. eutropha could potentially migrate between
environments, collecting carbon in the anaerobic zone and consuming it
in the aerobic zone. In this scenario, PHA production becomes central
to cellular survival, providing a means for a respiratory organism to
exploit the anaerobic environment.
| |
ACKNOWLEDGMENTS |
We thank Damien Rodriguez, Catharine Gunter, Greg Thorne, Jun Wu,
and Sunny Gilbert for excellent technical assistance; Bärbel Friedrich, Doug Dennis, and Russ Maurer for providing plasmids; and
Barry Goldman for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Monsanto
Company, 700 Chesterfield Parkway North, Mail Zone AA3I, St. Louis, MO
63198. Phone: (314) 737-7266. Fax: (314) 737-6759. E-mail:
steven.c.slater{at}monsanto.com.
| |
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Abstract
Introduction
