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JOURNAL OF BACTERIOLOGY,

Apr. 1998, p. 1979–1987

Vol. 180, No. 8

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0021-9193/98/$04.00 0 Copyright © 1998, American Society for Microbiology

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

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 -hy- droxyvalerate derived from propionyl-CoA. We also report an additional -ketothiolase, designated BktC, that probably serves as a secondary route toward -hydroxyvalerate production.

Polyhydroxyalkanoates (PHAs) are a class of naturally oc- curring polymers which serve as a carbon and energy reserve in numerous bacterial species. Ralstonia eutropha (formerly des- ignated Alcaligenes eutrophus [41]) produces the homopolymer poly( -hydroxybutyrate) (PHB) and, when provided with pro- pionate in the feedstock, the copolymer poly( -hydroxybu- tyrate-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 con- denses 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 -ke- tovaleryl-CoA (Fig. 1). Subsequently, the acetoacetyl-CoA and -ketovaleryl-CoA are converted into a polymer by the activ- ities 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). The substrate specificities of these three enzymes are report- edly adequate for production of PHBV copolymer (7–9), but propionate-fed Escherichia coli harboring the R. eutropha phb operon produces essentially PHB homopolymer (35). More- over, PHBV copolymer can be produced in E. coli after induc- tion 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 capa- ble 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

* 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 @monsanto.com.

(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 -ke- tothiolase and demonstrated that this enzyme catalyzes thioly- sis 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 ( -ketothio- lase B), which encodes the BktB -ketothiolase required for efficient production of PHBV in R. eutropha.

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 NH 4 Cl per liter, whereas Schlegel’s nitro- gen-limited (SNL) medium also contained 0.1 g of NH 4 Cl per liter. Antibiotics were used at the following levels: E. coli, carbenicillin, 100 g/ml; chloramphen- icol, 25 g/ml; and kanamycin, 50 g/ml; R. eutropha, chloramphenicol, 100 g/ml for broth and 100 to 250 g/ml for plates; kanamycin, 350 g/ml; and gentamicin, 10 g/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 contain- ing 1% glucose, the desired amount of sodium propionate, and 100 g of carbenicillin where appropriate. Cultures were incubated for approximately 40 h prior to harvesting.

1979

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1980 SLATER ET AL.

J. BACTERIOL.

Downloaded from 1980 SLATER ET AL. J. B ACTERIOL . FIG. 1. Pathway for production of

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).

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 frag- ment 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. 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

TABLE 1. E. coli and R. eutropha strains used in this study

Strain

Genotype or description

Source or reference

E.

coli

DH5

supE44 (lacZYA-argF)U169 ( 80 lacZ M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 deoR supE44 supF58 hsdR514 galK2 galT22 metB1 trpR55 lacY1 C600: recA thi pro hsdR RP-4

28

LE392

28

S17-1

34

 

2-tet::Mu::kan::Tn7

 

EE32

LE392 harboring pBK6 DH5 harboring pMON25982 DH5 harboring pMON25984

D. Dennis

EE245

This study

EE247

This study

R.

eutropha

EE167

R. eutropha DSM428 (wild type)

Deutsche Sammlung

 

von

Mikroorganismen,

Go¨ttingen,

Germany

 

EE168

bktB::kan bktB::kan EE167 harboring pBBR1MCS EE167 harboring pMON25901 EE168 harboring pBBR1MCS EE168 harboring pMON25901 EE169 harboring pBBR1MCS EE169 harboring pMON25901 phbA phbA bktB::kan

This study

EE169

This study

EE234

This study

EE235

This study

EE236

This study

EE237

This study

EE238

This study

EE239

This study

EE303

This study

EE317

This study

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 con- structed by excising the entire PHA production cassette from pMON25968 and pMON25970, respectively, with EcoRI and XhoI and cloning the resulting frag- ments 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 Tn10kan (12) cloned in the end-filled NcoI site within bktB of

pMON25728. Plasmid pMON25776 contains an approximately 5-kb ApaI frag- ment 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 genta-

micin 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 con-

struct 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 combi- nation 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 per- formed with the Wisconsin Package of molecular analysis software (Genetics Computer Group, Madison, Wis.) and the Sequencher DNA sequencing soft- ware package (Gene Codes Corporation). The chromosomal structure of the R. eutropha phbCAB-bktB region was de- termined 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 har- bored 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 di-

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MULTIPLE -KETOTHIOLASES OF R. EUTROPHA

TABLE 2. Plasmids used in this study

1981

Plasmid

Description a

Reference

pBluescript KS( )

General-utility E. coli cloning vector, Amp r Broad-host-range cloning vector, Cam r E. coli expression vector Suicide vector for gene replacement, Gen r Suicide vector for gene replacement, Kan r R. eutropha phbCAB-bktB region harbored in cosmid pVK102 pUC13 harboring 12-kb EcoRI fragment from pAE175, bktB R. eutropha phbCB in pBBR1MCS R. eutropha phbA expressed from trc promoter of pSE280 3.9-kb BglII-NcoI subclone of pBK6 in pBluescript KS( ), bktB bktB::kan derivative of pMON25728 1.8-kb BglII-SalI subclone of pBK6 in pBluescript KS( ), bktB bktB::kan harbored in pJQ200SK Cam r Gen s derivative of pMON25776 1.8-kb bktB fragment from pMON25765 cloned into pBBR1MCS R. eutropha phbA under control of bktB promoter in pBluescript KS( ) R. eutropha phbCB and bktB genes in pBBR1MCS R. eutropha phbCB and phbA genes in pBBR1MCS R. eutropha phbCB and bktB genes in pBluescript KS( ) R. eutropha phbCB and phbA genes in pBluescript KS( ) phbA harbored in pLO2

Stratagene

pBBR1MCS

13

pSE280

1

pJQ200SK

25

pLO2

15

pAE175

37

pBK6

37

pPHA A

D. Dennis

pMON25636

This study

pMON25728

This study

pMON25742

This study

pMON25765

This study

pMON25776

This study

pMON25845

This study

pMON25901

This study

pMON25966

This study

pMON25968

This study

pMON25970

This study

pMON25982

This study

pMON25984

This study

pMON36818

This study

a Resistance abbreviations are as follows: Amp r , ampicillin resistant; Cam r , chloramphenicol resistant; Gen r , gentamicin resistant; Kan r , kanamycin resistant.

verges 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. 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 g of kanamycin per ml, 10 g 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 g of kanamycin per ml and then purified on NaCL-free LB medium containing 5% sucrose and 0.2% fructose. Several kanamycin-sensitive strains

and 0.2% fructose. Several kanamycin-sensitive strains FIG. 2. (A) Restriction map of the region surrounding the

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.

having reduced -ketothiolase activity were further purified, and the phbA de- letion 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 recip-

ient strain on LB plates containing 0.2% fructose. Exconjugants were purified for isolation on LB plates containing 250 g of chloramphenicol per ml, 10 g 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 g of chloramphenicol per ml. Plasmids were purified from each strain, and

their

structures were verified by digestion with a variety of restriction endonucle-

ases.

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 pro-

duced 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)ben-

zenesulfonyl 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- m-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- m-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

1982 SLATER ET AL.

J. BACTERIOL.

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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.

(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 se- quenced by the Monsanto protein analysis laboratory. PhbA was purified from E. coli DH5 harboring pMON25636, a plasmid in

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MULTIPLE -KETOTHIOLASES OF R. EUTROPHA

1983

. 180, 1998 MULTIPLE -KETOTHIOLASES OF R. EUTROPHA 1983 FIG. 3— Continued. which R. eutropha phbA

FIG. 3—Continued.

which R. eutropha phbA is under control of an IPTG (isopropyl- -D-thiogalac- topyranoside)-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 (Daii- chi). Substrate synthesis and -ketothiolase enzyme assays. Preparation of -ke- toacyl-CoA substrate precursors trans-2,3-pentenoyl-CoA and trans-2,3-hex- enoyl-CoA was accomplished by the procedure of Schulz (32), essentially as described previously, except on a larger scale (100 mol of starting trans-2,3- pentenoic or trans-2,3-hexenoic acid and 100 mol of CoA). Another modifica- tion to the procedure was the purification of the enoyl-CoA products by semi- prep C 8 reverse-phase chromatography. -Ketothiolase was assayed in the thiolysis direction by two methods, depend- ing 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 M) spectrophotometrically at 304 nm in a mixture of 150 mM EPPS (N-[2-hydroxy- ethyl]piperazine-N -[3-propanesulfonic acid] (pH 8.0) and 50 mM MgCl 2 . CoA was used at 100 M, 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 M, 100 M CoA, and the buffering system described above were used for this study. The extinction coefficients for aceto-

acetyl-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 equiva- lent results, as demonstrated by comparing the results obtained with the in situ generation of acetoacetyl-CoA from crotonyl-CoA (method 2) with results ob- tained with acetoacetyl-CoA directly (method 1 [36]). One unit of enzyme ac- tivity is defined as the amount of enzyme required to convert 1 mol of -ke- toacyl-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 mol 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,aJ&W DB-5 capillary column (0.25 mm by 30 m; 0.25- m 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

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1984 SLATER ET AL.

TABLE 3. Thiolytic cleavage by PhbA and BktB

-Ketothiolase sp act (U/mg) for substrate a :

-Ketothiolase

 

C4

C5

C6

PhbA

162

1.2

ND b

BktB

65

177

87

a One unit is equal to 1 mol of substrate degraded per min. C4, acetoacetyl- CoA; C5, -ketovaleryl-CoA; C6, -ketohexanoyl-CoA. b ND, no activity was detected.

per min to 300°C and held for 10 min. Standards were methyl-(D)-hydroxybu- tyrate and methyl-(D)-hydroxyvalerate (Fluka, Ronkonkoma, N.Y.). Nucleotide sequence accession number. The GenBank accession number for the DNA sequence reported herein is AF026544.

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 ac- tivity. 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 sub- strates (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 iso- lated 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 de- rived from pAE175 linked bktB to the phb operon (Fig. 2A). The entire region from bktB to the phb operon was se- quenced. 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 ap- proximately 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% iden- tical and 61% similar (Fig. 4). BktB contains 394 amino acid residues and has a calculated molecular mass of 40,903 Da. The phbCAB-bktB intergenic region contains three addi- tional open reading frames (ORFs) that are likely to be ex- pressed (Fig. 2B and 3). All three ORFs show significant ho- mology to putative genes from other organisms, but none of these putative genes has been assigned a function (see Discus- sion).

J. BACTERIOL.

been assigned a function (see Discus- sion). J. B ACTERIOL . FIG. 4. Comparison of the

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 con- served active site cysteines (20, 39). Comparison was performed with the Gap program of the Wisconsin Package.

bktB can complement phbA for PHA production in E. coli. The abilities of PhbA and BktB to produce PHA were com- pared 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 -hydroxyval- erate incorporated into polymer reached a plateau when the media contained 0.05% or more propionate. 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 produc- tion by wild-type R. eutropha and two independent bktB::kan mutants (Table 4). When -ketothiolase was assayed with ace- toacetyl-CoA as a substrate, essentially no difference was seen between extracts from wild-type R. eutropha and the bktB mu- tants. 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 re- flected 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

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guest http://jb.asm.org/ Downloaded from V OL . 180, 1998 FIG. 5. Comparison of PhbA and BktB

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 (Œ) in strain EE247, which expresses phbA.

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 -ke- tothiolase (cumulatively, approximately threefold when as- sayed on acetoacetyl-CoA) did not significantly affect the total amount of polymer accumulated by R. eutropha in these fer- mentation end-point assays. An additional -ketothiolase is expressed in R. eutropha. R. eutropha bktB mutants retain significant -ketothiolase activity capable of degrading -ketovaleryl-CoA (Table 4). This activ- ity is inconsistent with the substrate specificity of PhbA, so we examined R. eutropha for the presence of additional -keto- thiolases. 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 fruc- tose. Four peaks of -ketothiolase activity were distinguishable in extracts of wild-type R. eutropha. PhbA eluted first, as a large

MULTIPLE -KETOTHIOLASES OF R. EUTROPHA

1985

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 con- tains 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 character- ized the contents of peak 4, but immunoblot analyses suggest that BktB is a component (reference 36 and described below). 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 re- sidual 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 pri- marily 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 con- tains 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 metab-

TABLE 4. -Ketothiolase activity and polymer composition in R. eutropha bktB::kan mutants and in strains overexpressing bktB

R. eutropha

strain

bktB

genotype

Plasmid a

-Ketothiolase activity with substrate b

PHA formation c

Sp act ( mol degraded/min/mg of protein)

C5

C4

C5/C4

ratio

% (dry wt) PHA

% C5 in polymer

EE234

Wild type

pBBR1MCS

0.78 0.1

2.5 0.3

0.31

66 3

11.9 2.6

EE236

bktB::kan

pBBR1MCS

0.19 0.2

2.4 0.6

0.08

63 7

3.9 0.9

EE238

bktB::kan

pBBR1MCS

0.12 0.04

2.3 0.3

0.05

64 5

3.7 0.7

EE235

Wild type

pMON25901

16.4 1.8

6.7 1.2

2.45

67 6

16.9 2.5

EE237

bktB::kan

pMON25901

18.8 5.3

7.5 3.8

2.50

70 6

19.3 3.3

EE239

bktB::kan

pMON25901

17.2 4.7

8.7 3.9

1.97

60 11

15.9 4.0

a pMON25901 harbors bktB in the broad-host-range plasmid pBBR1MCS.

b Specific activities were derived from cleared-cell extracts of at least four independent cultures. C5, -ketovaleryl-CoA; C4, acetoacetyl-CoA.

c Polymer production data were derived from six independent cultures grown in 3% fructose plus 0.1% propionate.

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1986 SLATER ET AL.

guest http://jb.asm.org/ Downloaded from 1986 SLATER ET AL. FIG. 6. Mono-Q chromatography of extracts from the

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 (E) or -ke- tovaleryl-CoA (F) 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.

olism. ORF2 and ORF3 apparently form an operon with over- lapping 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

J. BACTERIOL.

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 condi- tions in these experiments (5). Enhancement of propionate import and activation should yield significantly higher incor- poration of -hydroxyvalerate into polymer (26, 27), poten- tially 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 re- ported 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 ca- talysis of acetoacetyl-CoA (Fig. 6), whereas pure BktB pre- ferred -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, there- fore, 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 avail- able carbon cannot be metabolized directly. This situation oc- curs under anaerobic conditions. R. eutropha is strictly respi- ratory 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 O 2 , indicating that it normally encounters anaerobic conditions (6, 24, 30, 38). Finally, anaerobic conditions induce PHA pro- duction in R. eutropha (33). The ability to form PHA is advan- tageous 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 O 2 becomes avail- able. 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 anaer- obic zone and consuming it in the aerobic zone. In this sce- nario, 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; Ba¨rbel Friedrich, Doug Dennis, and Russ Maurer for providing plasmids; and Barry Goldman for critical reading of the manuscript.

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