Cloning and Characterization of the Pseudomonas sp. 61-3 phaG

are expected to have a potential as biodegradable thermo- plastics. Bacterial PHAs ... in the accumulation of mcl-PHA.13,14 On the other hand,. phaG g...
1 downloads 0 Views 88KB Size
Download PDF
Biomacromolecules 2001, 2, 142-147

142

Cloning and Characterization of the Pseudomonas sp. 61-3 phaG Gene Involved in Polyhydroxyalkanoate Biosynthesis Ken’ichiro Matsumoto,†,‡ Hiromi Matsusaki,†,§ Seiichi Taguchi,† Minoru Seki,‡ and Yoshiharu Doi*,† Polymer Chemistry Laboratory, RIKEN Institute, 2-1, Hirosawa, Wako-shi, Saitama 350-0198, Japan, and Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received September 1, 2000; Revised Manuscript Received October 19, 2000

Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaGPs gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaGPs gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaGPs gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaGPs gene was carried out to investigate the role of a HX4D motif which has been proposed to be related to PhaG activity. Introduction Polyhydroxyalkanoates (PHAs) are accumulated in various microorganisms as intracellular carbon and energy storage material under nutrient-limiting conditions.1-4 These PHAs are expected to have a potential as biodegradable thermoplastics. Bacterial PHAs consist of various monomer units.5 Ralstonia eutropha produces short-chain-length PHAs (sclPHA) with C3-C5 monomer units, whereas Pseudomonas strains belonging to the rRNA homology group I produce medium-chain-length PHAs (mcl-PHA) consisting of C6C14 monomer units from sugars and/or alkanoic acids. We have reported that Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HBco-3HA)] random copolymer consisting of C4-C12 3HA units from sugars and/or alkanoic acids.6,7 In this strain, two gene clusters are involved in PHA biosynthesis, that is, the phb and the pha loci.8 The genes encoding β-ketothiolase (PhbAPs), acetoacetyl-CoA reductase (PhbBPs), and PHB synthase (PhbCPs) that is specific for 3HB-CoA are located in the phb locus. As for the pha locus, an intracellular PHA depolymerase gene (phaZPs) is inserted between two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) specific for both 3HB and 3HA units ranging from C6 to C12. Genes encoding proteins that are involved in providing mcl-3HACoA were not found in these loci.8 When the phbCPs was * Corresponding author. E-mail: [email protected]. Tel.: +8148-467-9404. Fax: +81-48-462-4667. † RIKEN Institute. ‡ The University of Tokyo. § Current address: Study of Food and Health Environment, Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100, Tsukide, Kumamoto 862-8502, Japan.

disrupted, the resulting mutants accumulated only the P(3HBco-3HA) copolymer. Additional copies of β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase genes in the mutant of Pseudomonas sp. 61-3 and R. eutropha PHB-4 resulted in the production of a novel P(3HB-co-3HA) with a very high 3HB fraction.9,10 The physical properties of this copolymer [P(3HB-co-6 mol % 3HA)] were similar to those of low-density polyethylene (LDPE). To further obtain PHA copolymers with various mechanical and physical properties, the compositions of 3HB and mcl-3HA units in the copolymers should be controlled. To achieve this, we have cloned the various genes encoding proteins involved in providing mcl-3HA-CoA as substrates for PHA synthase. We isolated the phaJ genes encoding R-specific enoylCoA hydratases from Aeromonas caViae11,12 and Pseudomonas aeruginosa.13 The enzymes are known to be involved in providing mcl-3HA-CoA from the fatty acid β-oxidation pathway. Heterologous coexpression of the phaJ gene and PHA synthase genes in Escherichia coli successfully resulted in the accumulation of mcl-PHA.13,14 On the other hand, phaG genes encoding 3-hydroxyacyl acyl carrier protein (ACP)-CoA transferase have been cloned from P. putida15 and P. aeruginosa.16 Heterologous coexpression of the phaGPp gene from P. putida and the PHA synthase gene from P. aeruginosa in non-PHA-accumulating bacterium Pseudomonas fragi resulted in the accumulation of mcl-PHA.17 The phaG gene product provides mcl-3HA-CoA from the de novo fatty acid biosynthesis pathway when cells are cultivated on sugars. This suggests that Pseudomonas sp. 61-3 also possesses a phaG gene. In this study, cloning, characterization, and application of the phaG gene of Pseudomonas sp. 61-3 have been carried out.

10.1021/bm005604+ CCC: $20.00 © 2001 American Chemical Society Published on Web 11/29/2000

Pseudomonas sp. 61-3 phaG Gene

Biomacromolecules, Vol. 2, No. 1, 2001 143

Table 1. Bacterial Strains and Plasmids Used in This Study strain or plasmid

Pseudomonas sp. 61-3 Pseudomonas sp. 61-3 (phbC::tet) Pseudomonas sp. G-Gm Pseudomonas sp. BCG-TcGm

Escherichia coli JM109 Escherichia coli S17-1 Escherichia coli S17-1 (λ-pir) pLA2917 pBluescript II KS+ pBBR1MCS-2 pBSL182 pLG3 pBSEE50 pBSXX9F pRKmXS9F pBSSB22R pRKmAS22R pBSEE6

pSLHX6

relevant characteristics Strains wild-type inactivation of chromosomal phbCPs by integration of Tcr; phbCPs-negative mutant inactivation of chromosomal phaGPs by integration of Gmr; phaGPs-negative mutant inactivation of chromosomal phbCPs and phaGPs by integration of Tcr and Gmr, respectively; phbCPs- and phaGPs-negative mutant recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, ∆(lac-proAB)/F′[traD36, proAB+,lacIq, lacZ∆M15] recA and tra genes of plasmid RP4 integrated into chromosome; auxotrophic for proline and thiamine π protein encoded by R6K integrated into chromosome Plasmids Cosmid; Kmr, Tcr, RK2 replicon; Mob+ Apr, lacPOZ,T7 and T3 promoter Kmr, broad host range, lacPOZ′ Apr, Gmr, R6K replicon, suicide, lacIq, tnp(Tn10), Mob+, IS10 pLA2917 derivative containing approximately 20-kb fragment harboring phaGPs pBluescript II KS+ derivative containing the 5.0-kb EcoRI fragment of pLG3 harboring phaGPs with putative promoter pBlusescript II KS+ derivative; phaGPs pBBR1MCS-2 derivative; phaGPs pBluescript II KS+ derivative containing the 2.2-kb StuI-BglII fragment of pBSEE50 harboring phaGPs with putative promoter pBBR1MCS-2 derivative containing the 2.2-kb ApaI-SacI fragment of pBSSB22R harboring phaGPs with putative promoter pBluescript II KS+ derivative containing the 0.6-kb PCR product amplified using pBSXX9 as the template; 5′- and 3′-truncated phaGPs pBSL182 derivative containing the 0.6-kb HindIII-XbaI fragment of pBSEE6; 5′- and 3′-truncated phaGPs

Materials and Methods Bacterial Strains, Plasmids, and Growth Conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Pseudomonas sp. 61-3 was cultivated at 28 °C in a nutrient-rich (NR) medium.8 E. coli strains were grown at 37 °C on a Luria-Bertani (LB) medium.18 When needed, kanamycin (50 mg/l), tetracycline (12.5 mg/l), ampicillin (50 mg/l), and/or gentamicin (10 mg/l) were added to the medium. Production and Analysis of PHA. Cells were cultivated on a reciprocal shaker (130 strokes/min) at 28 °C for 72 h in 500-ml flasks containing 100 mL of nitrogen-limited mineral salt (MS) medium as described previously.6 Filtersterilized carbon sources were added to the medium as indicated in the text. Determination of cellular PHA content and composition by gas chromatography was carried out as described by Kato et al.6 DNA Manipulations. Isolation of total genomic DNA and plasmids, digestion of DNA with restriction endonucleases, agarose gel electrophoresis, and transformation of E. coli were carried out by standard procedures.18 The genomic DNA library of Pseudomonas sp. 61-3 which was constructed previously8 was used in this study. Conjugation of Pseudomo-

source or ref JCM10015, 25 8 this study this study

Stratagene 23 26 27 Stratagene 28 26 this study this study this study this study this study this study this study

this study

nas strains with E. coli S17-1 harboring broad-host-range plasmids was performed as described by Friedrich et al.19 Hybridization Experiments. Hybridization was carried out as described by Southern.20 The phaGPp gene of P. putida kindly provided by Rehm (Westfa¨lische Wilhelms-Universita¨t Mu¨nster) was used as a probe.15 DNA hybridization was carried out using a digoxigenin-labeled probe prepared with a DIG DNA labeling and detection kit (Roche). Nucleotide Sequence Analysis. DNA fragments to be sequenced were subcloned into pBluescript II KS+. DNA was sequenced by the modified dideoxy-chain termination method basically as described by Sanger et al.21 with a 310 Genetic Analyzer (Perkin-Elmer). The sequencing reaction was performed according to the instructions in the Big-dye termination cycle sequencing kit (Perkin-Elmer). The nucleotide sequence was analyzed with SDC-GENETYX genetic information processing software (Software Development Co., Tokyo, Japan). Plasmid Construction. Plasmid pBSEE50 carrying phaGPs, the phaG gene of Pseudomonas sp. 61-3, was constructed by the introduction of a 5.0-kb EcoRI fragment of pLG3 into pBluescript II KS+ as shown in Figure 1. pBSXX9F was constructed by PCR using pBSEE50 as a template. The oligonucleotides for the amplification of the

144

Biomacromolecules, Vol. 2, No. 1, 2001

Matsumoto et al.

Figure 1. Construction strategy of the plasmids used for expression of the phaGPs gene from Pseudomonas sp. 61-3. PPs and TPs are the putative promoter and terminator regions of the phaGPs gene, respectively.

phaGPs structural gene with a Shine-Dalgarno sequence were as follows: N-terminus, 5′-GCTCTAGAGCATACCCGCTTGCCAGGAGTC-3′ and C-terminus, 5′-GCTCTAGAGCTCAAATTGCCAATGC-ATGGT-3′ (XbaI sites are underlined). The PCR product was digested with XbaI in order to produce the insert which was introduced into pBluescript II KS+ to yield pBSXX9F. pRKmXS9F was constructed by the introduction of a 0.9-kb ApaI-SacI fragment of pBSXX9F into pBBR1MCS-2 (Figure 1). pBSSB22R was constructed by the ligation of a T4 DNA polymerase treated 2.2-kb StuIBglII fragment of pBSEE50, including the phaGPs structural gene and its putative promoter and terminator regions, with an EcoRV-digested pBluescript II KS+. A plasmid in which the phaGPs gene was inserted in an opposite direction to the lac promoter was selected. pRKmAS22R was constructed by the introduction of a 2.2-kb ApaI-SacI fragment of pBSSB22R into pBBR1MCS-2 (Figure 1). Construction of phaGPs Mutants. Site-directed mutagenesis of the phaGPs was carried out to generate four mutants ([H177A], [A182D], [E183D], and [E183Q]) by a mutational method using PCR as described by Imai et al.22 Primer locations are given based on the A in the ATG initiation sequence as bp +1. PCRs, using pBSXX9F as the template, were performed with primer pairs 5′-GCGCCGTCAGCACCCTGGCCGAGCAC-3′ (bp 527-552) and 5′-GGTAGTTGAAGCGCTTGAACAACGAC-3′ (bp 526-501) [H177A], 5′-ACGAGCACGAATACGGGCAG-3′ (bp 545-564) and 5′-CCAGGGTGCTGACGTGGCG-3′ {primer G(-)} (bp 544-526) [A182D], 5′-CCGACCACGAATACGGGCAG3′ (bp 545-564) and primer G(-) [E183D], and 5′CCGACCACGAATACGGGCAG-3′ (bp 545-564) and primer G(-) [E183Q]. Kinase treatment and self-ligation were performed on each PCR product to yield pBSXX9F[H177A], pBSXX9F[A182D], pBSXX9F[E183D], and pBSXX9F[E183Q], respectively. The desired mutations were confirmed by sequencing. The ApaI-SacI fragment of these plasmids, carrying site-directed mutations of phaGPs, were introduced into pBBR1MCS-2 to yield pRKmXS9F[H177A],

pRKmXS9F[A182D], pRKmXS9F[E183D], and pRKmXS9F[E183Q], respectively. Disruption of phaGPs. pBSL182 was used as an integration vector to disrupt the chromosomal phaGPs gene of Pseudomonas sp. 61-3. pBSEE6 was constructed by PCR using pBSEE50 as a template. The oligonucleotides to amplify the 0.6-kb 5′- and 3′-region truncated form of the phaGPs gene were as follows: N-terminus, 5′-GGAATTCCTTCACCCGCAATTCAACGTCG-T-3′ and C-terminus, 5′GGAATTCCTGGATGGTGCTGAAGGTGCTGT-3′ (EcoRI sites are underlined). EcoRI digestion was performed on the PCR product in order to produce the insert which was introduced into pBluescript II KS+ to yield pBSEE6. pSLHX6 was constructed by the introduction of a 0.6-kb HindIII-XbaI fragment of pBSEE6 into the multicloning site of pBSL182. Conjugations of Pseudomonas sp. 61-3 or phbCPs-disruptant (recipient cells) with E. coli S17-1 (λ-pir) harboring pSLHX6 (donor cells) were performed as follows. Donor and recipient cells were cultivated overnight at 37 °C in LB medium and at 28 °C in NR medium, respectively. The cells (1.5 mL) were transferred to microcentrifuge tubes and centrifuged at 4000g for 3 min. The supernatant was removed, and the cells were washed with 1 mL of 10 mM MgSO4 solution before resuspending in 1 mL of the same solution. The recipient and donor cells were then mixed at a ratio of 4:1. A portion (500 µL) of this mixture was added to 1.5 mL of LB medium containing 0.05 mM isopropylβ-D(-)-thiogalactopyranoside (IPTG) and was subsequently incubated overnight at 28 °C. Cells were centrifuged at 4000g for 3 min before resuspending the pellet in 400 µL of 10 mM MgSO4. An aliquot (200 µL) of the suspension was plated on Simon’s agar plates23 containing 10 mg/l gentamicin and incubated at 28 °C for 72 h. The chromosomal DNAs from the colonies that appeared were extracted and subjected to Southern hybridization analysis using the phaGPs gene as a probe to confirm the gene disruption. The transconjugants (phaGPs-disruptant and phbCPs- and phaGPs-disruptant) were used for further study.

Pseudomonas sp. 61-3 phaG Gene

Biomacromolecules, Vol. 2, No. 1, 2001 145

Figure 2. Restriction map of the 2.2-kb StuI-BglII region and organization of the phaGPs gene.

Figure 3. Alignment of the deduced amino acid sequence of PhaG from Pseudomonas sp. 61-3 with those of P. putida and P. aeruginosa. Matching amino acid residues are shaded, and identical residues are given in the consensus sequence. The HX4D motif is boxed. The numbers indicate the positions of the amino acids in the respective protein.

Nucleotide Sequence Accession Number. The nucleotide sequence data determined here will appear in the DDBJ databases under accession no. AB047080. Results Cloning and Molecular Analysis of the phaGPs Gene from Pseudomonas sp. 61-3. To clone the phaGPs gene from Pseudomonas sp. 61-3, colony hybridization with the total genomic DNA library of Pseudomonas sp. 61-38 was carried out using the phaGPp gene from P. putida15 as a probe. A positive clone isolated by hybridization screening was further analyzed by Southern hybridization, and a positive 5.0-kb EcoRI fragment was cloned into pBluescript II KS+ (pBSEE50). A 2.2-kb StuI-BglII subfragment of the 5.0-kb EcoRI fragment was sequenced, and its restriction enzyme map was made (Figure 2). In this fragment, one potential open reading frame (ORF) was identified by computer analysis. This ORF encoded a putative protein composed of 294 amino acids with a calculated molecular mass of 33.6 kDa. The deduced amino acid sequence of the ORF revealed high homologies to those of genes encoding (R)-3-hydroxyacyl-ACP-CoA acyltransferases of P. putida (PhaGPp, identity 69%, similarity 81%)15 and P. aeruginosa (PhaGPa, identity 57%, similarity 75%).16 Therefore, the ORF was designated as phaGPs. Figure 3 shows an alignment of the deduced amino acid sequence of PhaGPs with those of PhaGPp and PhaGPa. A similar but not identical sequence to the HX4D motif, which has been proposed to be important for PhaG activity,15 was found at the corresponding location of the phaGPs gene product of Pseudomonas sp. 61-3. Computer analysis revealed the presence of a putative promoter sequence resembling the -35 to -10 sequence of the E. coli σ70-dependent promoter and an inverted repeat sequence plausibly capable of functioning as a F independent termina-

tor in the regions upstream and downstream of the phaGPs gene, respectively. Complementation Studies and Homologous Expression of the phaGPs Gene. To confirm the function of the phaGPs gene, homologous expressions of the phaGPs gene were carried out in the wild-type and mutant strains of Pseudomonas sp. 61-3. Two expression plasmids for the phaGPs gene, pRKmXS9F (controlled by the lac promoter) and pRKmAS22R (controlled by the native promoter), were constructed as shown in Figure 1 and were introduced into the host strains by transconjugation. The transconjugants were cultivated in a MS medium to promote PHA biosynthesis from gluconate as a sole carbon source. Table 2 shows the results of PHA accumulation in recombinant strains of Pseudomonas sp. 61-3 and its mutants. In comparison with the Pseudomonas sp. 61-3 harboring the control plasmid pBBR1MCS-2, Pseudomonas sp. 61-3 harboring pRKmXS9F produced PHA with much higher mcl3HA fractions (C6-C12) (90 mol %). Introduction of pRKmAS22R into Pseudomonas sp. 61-3 also resulted in the accumulation of a higher amount of PHA (42 wt %) with increased mcl-3HA fractions (80 mol %). Thus, additional copies of the phaGPs gene in Pseudomonas sp. 61-3 led to an increase in the mcl-3HA fraction and to a decrease in the 3HB fraction. The phaGPs-disrupted strain Pseudomonas sp. G-Gm harboring a control plasmid produced PHA (19 wt %) with a very high 3HB fraction of 92 mol %. Thus, disruption of the phaGPs gene in Pseudomonas sp. 61-3 caused a remarkable decrease in the mcl-3HA fraction from 53 to 8 mol %. The introduction of pRKmXS9F or pRKmAS22R into the strain G-Gm could complement the decrease in the mcl-3HA fraction in PHA which was caused by the disruption of the phaGPs gene. The phbCPs-negative strain, Pseudomonas sp. 61-3 (phbCPs::tet), harboring the control plasmid pBBR1MCS-2 produced only a copolymer consisting of 25 mol % 3HB

146

Biomacromolecules, Vol. 2, No. 1, 2001

Matsumoto et al.

Table 2. Accumulation of PHA by Wild-Type and Recombinant Strains of Pseudomonas sp. 61-3 Harboring the PhaGPs Genea PHA composition (mol %) strain wild-type

G-Gm

(phbCPs::tet)

BCG-TcGm

plasmid (promoter)

cell dry weight (g/l)

PHA content (wt %)

3HB (C4)

3HHx (C6)

3HO (C8)

3HD (C10)

3HDD (C12)

3H5DD (C12′)

pBBR1MCS-2 pRKmXS9F (lac) pRKmAS22R (native) pBBR1MCS-2 pRKmXS9F (lac) pRKmAS22R (native) pBBR1MCS-2 pRKmXS9F (lac) pRKmAS22R (native) pBBR1MCS-2 pRKmXS9F (lac) pRKmAS22R (native)

1.04 0.93 1.08 0.90 0.93 1.04 1.02 1.24 1.56 1.01 0.89 1.60

23 29 42 19 16 42 20 34 52 6 11 50

47 10 20 92 64 17 25 13 8 71 57 13

3 7 5 0 2 6 4 6 7 0 0 6

12 23 20 1 8 23 16 20 22 5 9 25

20 35 40 3 14 39 27 33 38 11 16 34

8 14 10 2 6 5 14 14 11 10 12 9

10 11 5 2 6 10 14 14 14 3 6 13

a Cells were cultivated at 28 °C for 72 h in a MS medium (100 mL) containing the sodium salt of gluconate (2% wt/vol) as the sole carbon source. All values are averages in triplicated tests. 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecanoate.

Table 3. Accumulation of PHA by the PhaGPs-Negative Strain Pseudomonas sp. G-Gm Carrying the Wild-Type or Mutated PhaGPs Genesa PHA composition (mol %) plasmid

cell dry weight (g/l)

PHA content (wt %)

3HB (C4)

3HHx (C6)

3HO (C8)

3HD (C10)

3HDD (C12)

3H5DD (C12′)

pBBR1MCS-2 pRKmXS9F (lac promoter, phaGPs) pRKmXS9F[H177A] pRKmXS9F[A182D] pRKmXS9F[E183D] pRKmXS9F[E183Q]

0.90 0.93 1.07 0.97 0.94 0.90

19 16 12 17 17 13

92 64 90 68 65 76

0 2 0 1 2 0

1 8 2 7 8 5

3 14 3 12 14 9

2 6 3 7 7 7

2 6 2 5 4 3

a Cells were cultivated at 28 °C for 72 h in a MS medium (100 mL) containing the sodium salt of gluconate (2% wt/vol) as the sole carbon source. All values are averages in triplicated tests. 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecanoate.

and 75 mol % 3HA.8 Additional copies of the phaGPs gene in this strain also led to an increase in both the PHA content and the mcl-3HA fraction (87-92 mol %). The phbCPs- and phaGPs-double negative strain, Pseudomonas sp. BCGTcGm, accumulated a small amount (6 wt %) of P(3HBco-3HA). Introduction of pRKmAS22R into the strain BCGTcGm restored PHA accumulation, and the PHA content and the mcl-3HA fraction were 50 wt % and 87 mol %, respectively. From these results, the phaGPs gene was found to complement the deficiency in monomer supply of mcl3HA units and confer the ability to provide mcl-3HA-CoA. Furthermore, additional copies of the phaGPs gene could increase both the PHA content and the mcl-3HA fraction remarkably. Significance of HX4D Motif in PhaGPs Activity. It has been reported that HX4D configuration is conserved in a variety of glycerolipid acyltransferases.24 Site-directed mutagenesis of glycerolipid acyltransferases revealed that this motif is essential for the acyltransferase activity. This HX4D motif-encoding sequence was also found in the phaG gene products of P. putida and P. aeruginosa.15,16 On the other hand, the phaGPs gene product of Pseudomonas sp. 61-3 had a HX4AE configuration at the corresponding location (Figure 3). This sequence difference prompted us to elucidate the involvement of a HX4AE configuration in PhaGPs activity. First, the role of His at position 177 was investigated by the

substitution by Ala. The [H177A] mutated phaGPs gene was constructed and introduced into Pseudomonas sp. G-Gm (phaGPs-disruptant) as described in Materials and Methods. As shown in Table 3, Pseudomonas sp. G-Gm harboring pRKmXS9F[H177A] produced 12 wt % PHA containing a high 3HB fraction (90 mol %). The plasmid pRKmXS9F[H177A] did not complement the deficiency in the supply of mcl-3HA units, suggesting that the His at this position is indispensable for PhaG activity. Next, the role of Asp in the HX4D motif was investigated. If HX4D were the optimal sequence motif for PhaG catalysis, activity elevation of PhaGPs could be achieved by the substitution of Ala by Asp at position 182. To examine this possibility, the mutant [A182D] was generated and introduced into Pseudomonas sp. G-Gm. The recombinant strain produced 17 wt % PHA containing 32 mol % mcl-3HA (Table 3). The PHA content and the monomer composition were almost the same as those (16 wt % and 36 mol % mcl3HA) harboring pRKmXS9F (wild-type phaGPs gene). The plasmid pRKmXS9F[A182D] did complement the disrupted phaGPs gene. However, the introduction of the HX4D motif into PhaGPs did not lead to a further increase in either the PHA content or the mcl-3HA fraction. In the phaGPs gene product, Glu was located at position 183. To examine the possibility of this amino acid residue being an alternative residue for the Asp residue at position 182 in the phaG gene products of the other two Pseudomo-

Pseudomonas sp. 61-3 phaG Gene

nas, Glu183 was substituted by Asp or Gln. Substitution by an Asp residue was chosen to change the orientation of the acidic carboxyl group, and the substitution by a Gln residue was chosen to eliminate the acidic residue at this position and minimize steric effects. pRKmXS9F[E183D] and pRKmXS9F[E183Q] were constructed and introduced into Pseudomonas sp. G-Gm. These mutations however did not result in significant changes in the PHA content and composition compared to that of the wild-type construct (Table 3). Discussion In this study, the phaGPs gene was cloned from Pseudomonas sp. 61-3, and the effects of PhaGPs activity on the PHA content and composition were investigated. The disruption of the phaGPs gene led to a remarkable decrease in the mcl3HA fraction. Reintroduction of the phaGPs gene into the mutant (strain G-Gm) resulted in its complementation and gave rise to mcl-PHA production. Therefore, it has been concluded that the phaGPs gene product is involved in providing mcl-3HA-CoA from gluconate in Pseudomonas sp. 61-3. Besides that, the results also indicate the presence of other metabolic route(s) providing mcl-3HA-CoA from sugars in Pseudomonas sp. 61-3, as reported for P. aeruginosa.16 The selection of promoters that control the PHA biosynthesis genes is also important for the production of PHA.10 In fact, in this study, the phaGPs gene expressed under the control of the native promoter resulted in both a higher PHA content and a higher mcl-3HA fraction in comparison with those expressed under the control of the lac promoter, indicating that the native promoter is more efficient for phaGPs expression. It has been reported that the HX4D configuration is conserved in a variety of glycerolipid acyltransferases.24 This HX4D motif has been found in (R)-3-hydroxyacyl-ACP-CoA acyltransferases of P. putida (PhaGPp)15 and of P. aeruginosa (PhaGPa).16 However, a slight sequence difference (HX4AE) was found in the phaGPs gene product of Pseudomonas sp. 61-3. Site-directed mutagenesis revealed that PhaGPs was inactivated completely by the substitution of His177 by Ala, suggesting that the His residue at this position plays an important role in PhaG activity. In contrast, the substitution of Ala182 by Asp did not affect PHA production. Although the HX4D motif was constructed in PhaGPs, the mcl-3HA fraction in PHA was at the same level as that of the wildtype gene. In addition, the [E183D] and [E183Q] mutated phaGPs gene products were still active. From these results, it is suggested that acidic residue in the HX4D or HX4AE

Biomacromolecules, Vol. 2, No. 1, 2001 147

configuration is not essential for PhaG activity and that the acyltransfer mechanism of the phaG gene product may be different from that of glycerolipid acyltransferases. Acknowledgment. We are indebted to B. H. A. Rehm (Westfa¨lische Wilhelms-Universita¨t Mu¨nster) for the kind gift of the phaGPp gene from P. putida, to M. E. Kovach (Louisiana State University) for the plasmid pBBR1MCS2, and to the National Institute of Genetics of Japan for the pBSL182 and E. coli S17-1 (λ-pir). This work was supported by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST). References and Notes (1) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (2) Doi, Y. Microbial Polyesters; VHC Publishers: New York, 1990. (3) Mu¨ller, H. M.; Seebach, D. Angew. Chem., Int. Ed. Engl. 1993, 32, 477. (4) Steinbu¨chel, A. In Biomaterials; Byrom, D., Ed.; Macmillan: New York, NY, 1991; p 123. (5) Steinbu¨chel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219. (6) Kato, M.; Bao, H. J.; Kang, C. K.; Fukui, T.; Doi, Y. Appl. Microbiol. Biotechnol. 1996, 45, 363. (7) Kato, M.; Fukui, T.; Doi, Y. Bull. Chem. Soc. Jpn. 1996, 69, 515. (8) Matsusaki, H.; Manji, S.; Taguchi, K.; Kato, M.; Fukui, T.; Doi, Y. J. Bacteriol. 1998, 180, 6459. (9) Matsusaki, H.; Abe, H.; Taguchi, K.; Fukui, T.; Doi, Y. Appl. Microbiol. Biotechnol. 2000, 53, 401. (10) Matsusaki, H.; Abe, H.; Doi, Y. Biomacromolecules 2000, 1, 17. (11) Fukui, T.; Doi, Y. J. Bacteriol. 1997, 179, 4821. (12) Fukui, T.; Shiomi, N.; Doi, Y. J. Bacteriol. 1998, 180, 667. (13) Tsuge, T.; Fukui, T.; Matsusaki, H.; Taguchi, S.; Kobayashi, G.; Ishizaki, A.; Doi, Y. FEMS Microbiol. Lett. 2000, 184, 193. (14) Fukui, T.; Yokomizo, S.; Kobayashi, G.; Doi, Y. FEMS Microbiol. Lett. 1999, 170, 69. (15) Rehm, B. H. A.; Kru¨ger, N.; Steinbu¨chel, A. J. Biol. Chem. 1998, 273, 24044. (16) Hoffmann, N.; Steinbu¨chel, A.; Rehm, B. H. A. FEMS Microbiol. Lett. 2000, 184, 253. (17) Fiedler, S.; Steinbu¨chel, A.; Rehm, B. H. A. Appl. EnViron. Microbiol. 2000, 66, 2117. (18) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (19) Friedrich, B.; Hogrefe, C.; Schlegel, H. G. J. Bacteriol. 1981, 147, 198. (20) Southern, E. M. J. Mol. Biol. 1975, 98, 503. (21) Sanger, F.; Nicklen, S.; Coulson, A. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463. (22) Imai, Y.; Matsushima, Y.; Sugimura, T.; Terada, M. Nucleic Acids Res. 1991, 19, 2785. (23) Simon, R.; Priefer, U.; Pu¨hler, A. Bio/Technology 1983, 1, 784. (24) Heath, R. J.; Rock, C. O. J. Bacteriol. 1998, 180, 1425. (25) Abe, H.; Doi, Y.; Fukushima, T.; Eya, H. Int. J. Biol. Macromol. 1994, 16, 115. (26) Alexeyev, M. F.; Shokolenko, I. N. Gene 1995, 160, 59. (27) Allen, L. N.; Hanson, R. S. J. Bacteriol. 1985, 161, 955. (28) Kovach, M. E.; Elzer, P. H.; Hill, D. S.; Robertson, G. T.; Farris, M. A.; Roop, R. M.; Peterson, K. M. Gene 1995, 166, 175.

BM005604+