The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources

Authors


*Corresponding author. Tel.: +49 (251) 833 9821; Fax: +49 (251) 833 8388, E-mail address: steinbu@uni-muenster.de

Abstract

We recently identified the phaGPp gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaGPa) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaGPp-negative mutant P. putida PhaGN-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaGPa knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaGPa mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty acid de novo synthesis towards PHAMCL synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.

1Introduction

Polyhydroxyalkanoic acids (PHA) consisting of various saturated 3-hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (MCL=medium chain length) as carbon and energy storage compound are mainly produced by fluorescent pseudomonads belonging to rRNA homology group I (for review see [1]). Only few of these pseudomonads, such as Pseudomonas fragi, are not able to accumulate PHAs either from fatty acids or from carbohydrates [2,3]. The composition of PHAs depends on the PHA synthases, the carbon source, and the metabolic routes involved [4,5]. In Pseudomonas aeruginosa two PHA synthase genes, phaC1 and phaC2, which were separated by the PHA depolymerase encoding the phaD gene, were identified and characterized [6]. These PHA synthases are related to type II PHA synthases which prefer 3-hydroxyacyl-CoA with a chain length of 6–14 carbon atoms as substrate. Both PHA synthases have been recently purified to homogeneity, and the purified enzymes were successfully employed for in vitro synthesis of poly-3-hydroxydecanoate [7]. At least three different metabolic routes were found in Pseudomonas putida for the synthesis of 3-hydroxyacyl-CoA thioesters, which are the substrates of the PHA synthase [8,9]. (i) β-Oxidation is the main pathway when fatty acids are used as carbon source. (ii) Fatty acid de novo biosynthesis is the main route during growth on carbon sources which are metabolized to acetyl-CoA, like gluconate, acetate or ethanol. (iii) Chain elongation reaction, in which acetyl-CoA moieties are condensed to 3-hydroxyacyl-CoA, is involved in the PHA synthesis during growth on hexanoate. Recently, recombinant PHAMCL synthesis was also obtained in the β-oxidation mutants LS1298 (fadB) and RS3097 (fadR) of Escherichia coli expressing PHA synthase genes from P. aeruginosa[10–12], indicating that the β-oxidation pathway in E. coli provides precursors for PHA synthesis. Taguchi et al. [13] provided evidence that the overexpression of the E. coli fabG gene, which encodes 3-ketoacyl-ACP reductase, in E. coli HB101 mediated the supply of (R)-3-hydroxyacyl-CoA via fatty acid β-oxidation. Moreover, overexpression of either the E. coli fabH or fabD gene, which encode the 3-ketoacyl-ACP synthase III or the malonyl-CoA-ACP transacylase, respectively, resulted in E. coli in poly(3-hydroxybutyrate) (PHB) synthesis, when the Aeromonas caviae synthase gene was coexpressed, and when cells were cultivated on LB medium plus glucose [14].

The main constituent of PHA from P. putida KT2440 from non-related carbon sources, e.g. gluconate, is (R)-3-hydroxydecanoate [2,7]. Thus, to serve as substrate for PHA synthase, the (R)-3-hydroxyacyl-ACP intermediate of fatty acid de novo synthesis must be converted to the corresponding CoA derivative. Recently, the transacylase PhaG from P. putida has been identified and characterized, which catalyzes the transfer of the (R)-3-hydroxydecanoyl moiety from the ACP to CoA [9], thus directly linking the fatty acid de novo biosynthesis with the PHA biosynthesis. We recently established this PhaG-mediated metabolic pathway in the non-PHA-accumulating bacterium P. fragi, which functionally expressed the phaC1Pa gene (encoding PHA synthase) from P. aeruginosa and the phaGPp gene (encoding transacylase) from P. putida[15]. The aim of this study was the identification and molecular characterization of the phaG homologue from P. aeruginosa and the functional analysis of PhaG with respect of PHAMCL synthesis from non-related carbon sources.

2Materials and methods

2.1Bacterial strains, plasmids and growth of bacteria

Pseudomonads, E. coli strains and plasmids used in this study are listed in Table 1. E. coli was grown at 37°C in Luria–Bertani (LB) medium. Pseudomonads were grown at 30°C either in nutrient broth complex medium (0.8%, w/v) or in a mineral salts medium with 0.05% (w/v) ammonia, unless otherwise indicated.

Table 1.  Bacterial strains and plasmids
Strains and plasmidsRelevant characteristicsSource or reference
Strains  
Pseudomonas aeruginosaprototroph, Alg−ATCC 15692
Pseudomonas aeruginosa KO2phaG::GmThis study
Pseudomonas putida PhaGN-21PhaG-negative mutant of P. putida KT2440 mutants[9]
Pseudomonas oleovoransOCT plasmidATCC 29347
Escherichia coli  
 S17-1recA; harbors the tra genes of plasmid RP4 in the chromosome; proA, thi-1[16]
 JM109recA1 endA1 gyrA96 thi hsdR17 (rk, mk+) supE44 relA1, λ−, lac [F′proAB lac1qZΔM15][17]
Plasmids  
 pBBR1MCS-2Kmr, broad host range, lacPOZ[18]
 pBBR1MCS-3Tetr, broad host range, lacPOZ[18]
 pBBR1MCS-5Gmr, broad host range, lacPOZ[18]
 pEX100TAmpr, oriT, sacB, lacPOZ[19]
 pBHR81pBBR1MCS-2 containing coding region of phaG from P. putida downstream of lac promoter[9]
 pBHR88pBBR1MCS-2 containing coding region of phaG from P. aeruginosa downstream of lac promoterThis study
 pBHR89pBBR1MCS-2 containing coding region plus about 400 bp upstream of phaG from P. aeruginosa downstream of lac promoterThis study
 pBHR88-tetpBHR88, TetrThis study
 pBHR89-tetpBHR89, TetrThis study
 pEXNH2pEX100T, phaGPa::GmThis study

2.2Isolation, analysis and manipulation of DNA

DNA sequences of new plasmid constructs and of phaGPa from P. aeruginosa were obtained by the chain termination method using the automatic sequencer LI-COR model 4000L (MWG-Biotech, Ebersberg, Germany). The DNA sequence and its assignment were deposited in the GenBank database under accession number AF209711. All other genetic techniques were performed as described by Sambrook et al. [17]. The phaGPa gene from P. aeruginosa, i.e. only the coding region (including the native ribosome binding site) or the coding region plus 435 bp upstream, was amplified by tailed PCR employing the following primers: 5′-CGGGGTACCTCGAACGATTCCAGGAGGAA-3′; 5′-CGGGGTACCCCGCGCCGTCCCTTTCCTTC-3′; 5′-TGCTCTAGAGTTTTTCCGCCAGGGGAGCG-3′. The PCR products were inserted into the restriction sites XbaI/KpnI of the vector pBBR1MCS-2 resulting in plasmids pBHR88 and pBHR89, respectively. To enable stable propagation of plasmids in P. aeruginosa a tetracycline resistance cassette was inserted into these plasmids resulting in plasmids pBHR88-tet and pBHR89-tet, respectively.

The isogenic phaGPa knock-out mutant was obtained by replacement of a central 270-bp region of phaGPa with a gentamicin resistance cassette using PCR (primers for the phaG 5′ end: 5′-AAATATGATATCTCGAACGATTCCAGGAGGAAG-3′, 5′-GTTTTCTAGAGGGTGGCGACGCCGCCCCAC-3′; primers for the phaGPa 3′ end: 5′-ATATTCTAGATGAACGCCGATAGCTATACC-3′, 5′-GGGTTTGATATCGTTTTTCCGCCAGGGGAGCG-3′; primers for the gentamicin cassette: 5′-AAATCTAGAGAGAGGCGGTTTGCGTATTGGGCGC-3′, 5-TTTTCTAGAAAGCCGATCTCGGCTTGAACGAATTG-3′) and employing the suicide vector pEX100T as outlined in Fig. 3. The isogenic mutant was verified by PCR using one primer which binds 435 bp upstream of the phaGPa start codon and a second primer binding close to the phaGPa stop codon. The PCR product was analyzed by restriction site mapping and direct DNA sequencing.

Figure 3.

Schematic representation of the strategy to construct an isogenic phaG mutant of P. aeruginosa.

2.3Transfer of plasmids

Plasmids were transferred into E. coli according to the CaCl2 method [16], whereas the transfer of plasmids into P. aeruginosa and P. putida was performed either by electroporation or by conjugation. For electroporation cells were cultivated in LB medium until an OD600 of 0.3 was reached. Cells were harvested and washed twice with 1 vol. 10% (v/v) glycerol in distilled water. Cells were concentrated 100 times by suspension in 10% (v/v) glycerol solution. About 0.5 μg plasmid DNA was mixed with 60 μl electrocompetent cells and subjected to electroporation applying 1215 V mm−1. Conjugation was conducted as described by Simon et al. [16], employing E. coli S17-1 as donor strain.

2.4Gas chromatographic analysis of PHAs in cells

PHA was qualitatively and quantitatively analyzed by gas chromatography (GC). Liquid cultures were centrifuged at 10 000×g for 15 min, then the cells were washed twice in saline and lyophilized overnight. 3–5 mg lyophilized cell material was subjected to methanolysis in the presence of 15% (v/v) sulfuric acid. The resulting methyl esters of the constituent 3-hydroxyalkanoic acids were assayed by GC according to Brandl et al. [20] and as described in detail previously [2]. GC analysis was performed by injecting 3 μl of sample into a Perkin-Elmer 8420 gas chromatograph (Überlingen, Germany) using a 0.5 μm diameter Permphase PEG 25 Mx capillary column 60 m in length.

3Results

3.1Identification and cloning of the phaG gene from P. aeruginosa

Since the P. aeruginosa PAO1 genome DNA sequence has been recently released (http://www.pseudomonas.com), we searched this database for DNA sequences homologous to phaG, encoding a (R)-3-hydroxydecanoyl-CoA:ACP transacylase, from P. putida. One ORF (which will be referred to as phaG) comprising 903 bp was identified in contig 53 of which deduced the amino acid sequence (300 amino acid residues, MW 34 kDa) revealed about 57% identity to PhaG from P. putida as shown in Fig. 1. The conserved HX4D motif, which has been proposed to play an important role in enzymatic catalysis, was also found in the amino acid sequence derived from phaGPa of P. aeruginosa (Fig. 1). The adjacent DNA sequence of phaGPa did not show open reading frames of which the deduced amino acid sequence shows similarity to proteins known to be involved in PHA metabolism. The coding region of the phaGPa and a DNA fragment comprising an additional 435 bp upstream of phaGPa were amplified from chromosomal DNA of P. aeruginosa PAO1 by tailed PCR and subcloned into vector pBBR1MCS-2 directly downstream of the lac promoter leading to plasmids pBHR88 and pBHR89, respectively, as outlined in Fig. 2 and described in detail in Section 2. The amplified DNA regions were sequenced and showed 100% identity with the respective DNA region in the published genome sequence.

Figure 1.

Alignment of the deduced amino acid sequences of PhaG from P. aeruginosa and PhaG from P. putida. Matching amino acid residues are shaded and identical residues are given in bold in the consensus sequence. The HX4D motif is underlined. Numbers indicate the positions of the amino acids in the respective protein.

Figure 2.

Construction of plasmids pBHR88 and pBHR89 and the corresponding restriction site maps.

3.2Functional expression of the phaG gene in various pseudomonads

In order to evaluate the functionality of PhaG from P. aeruginosa, plasmids pBHR88 and pBHR89 were introduced into the phaGPp-negative mutant P. putida PhaGN-21. Since PhaG (transacylase) mediates PHAMCL synthesis from non-related carbon sources in P. putida enzymatically linking fatty acid de novo biosynthesis with PHAMCL synthesis, recombinant pseudomonads were cultivated on gluconate as sole carbon source. In P. putida PhaGN-21, which accumulated PHAMCL contributing to about 1–3% of CDW, PhaG from P. aeruginosa mediated PHAMCL accumulation (Table 2). The PHAMCL contributed to about 13% of CDW and was composed of about 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate, 5 mol% 3-hydroxyhexanoate and 5 mol% 3-hydroxydodecanoate, which was similar to the composition of PHAMCL obtained after expressing phaGPp from P. putida in this mutant. Thus PhaGPa functionally replaced PhaGPp.

Table 2.  PHA accumulation in wild-type and recombinant pseudomonads from various carbon sources
  1. aND, not detectable.

StrainPlasmidCarbon sourcePHA content % (w/w) CDWComposition of PHA (mol%)
    3HHx3HO3HD3HDD
P. putida KT2440NoneGluconate37.61.632.162.73.6
P. putida PhaGN-21NoneGluconate2.9NDa234334
 pBBR1MCS-2Gluconate1.7ND8.16229.9
 pBHR81Gluconate29.71.422.170.36.2
 pBHR88Gluconate12.21.125.365.38.3
 pBHR89Gluconate14.84.728.861.45.1
P. aeruginosa PAO1NoneGluconate16.83.323.763.59.5
P. aeruginosa KO2NoneGluconate7.43.736.250.110
 pBHR89-tetGluconate14.61.847.245.95.1
P. aeruginosa PAO1NoneOctanoate34.57883.91.1
P. aeruginosa KO2NoneOctanoate34.15.989.73.41
Cultivations were performed under PHA storage conditions on mineral salt medium containing either 1.5% (w/v) gluconate or 3×0.1% (w/v) octanoate (0.1 % every 24 h) and 0.05% (w/v) ammonium chloride. Cells on gluconate were grown for 48 h at 30°C; cells on octanoate were grown for 72 h at 30°C. PHA contents of comonomers were analyzed by GC. 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate.

3.3Inactivation of the phaG gene and characterization of the corresponding mutant

To further investigate the functional role of phaGPa particularly with respect to PHAMCL synthesis, we generated an isogenic phaGPa-negative mutant by insertional inactivation of the chromosomal phaGPa. Therefore, we constructed plasmid pEXNH2 which contains phaGPa disrupted by a gentamicin cassette. The gentamicin cassette replaced 270 bp of the internal part (including the presumably essential HX4D motif) of the coding region of phaGPa. Plasmid pEXNH2 was transferred by conjugation into P. aeruginosa PAO1 and gentamicin/sucrose-resistant transformants, which were putative double recombinants carrying an interrupted phaGPa, were selected (Fig. 3). One of the selected transformants was mutant KO2 which was analyzed by PCR employing primers binding 435 bp upstream of the phaGPa gene and its 3′ end. A PCR product of about 2.1 kb from mutant genomic DNA and a PCR product of about 1.4 kb from wild-type genomic DNA indicated that the mutant indeed derived from a double recombination event in which phaGPa was interrupted by the gentamicin cassette (Fig. 3). Further analysis of the 2.1-kb PCR product including restriction site mapping and DNA sequencing showed that the gentamicin cassette was properly inserted in phaGPa (Fig. 3). A tetracycline cassette was amplified by PCR from vector pBBR1MCS-3 as described in Section 2 and subcloned in phaG-containing plasmids in order to enable stable propagation of the respective plasmids in P. aeruginosa. Plasmid pBHR89-tet was transferred into the mutant KO2 of P. aeruginosa. Plasmid pBHR89-tet, which contains phaG from P. aeruginosa, complemented KO2, i.e. the KO2 mutant harboring these plasmids accumulated the same amounts of PHAMCL from gluconate as the wild-type (Table 2).

4Discussion

In this study, we identified the phaG gene in the P. aeruginosa genome, of which the deduced amino acid sequence showed strong homology to PhaG ((R)-3-hydroxydecanoyl-CoA:ACP transacylase) from P. putida (Fig. 1) [9]. The phaG gene from P. aeruginosa was cloned by PCR and functional expression in the phaG-negative mutant P. putida PhaGN-21 strongly enhanced PHAMCL accumulation from gluconate as carbon source (Fig. 2, Table 2). However, the in vivo activity of the P. aeruginosa PhaG was about three-fold less than obtained for PhaG from P. putida. To obtain deeper insight into the biological function of phaG, particularly with respect to PHAMCL synthesis, we generated an isogenic P. aeruginosa PAO1 phaG::Gm mutant (Fig. 3). This mutant KO2 was not affected in growth on mineral medium, but exhibited a strong decrease (only 40% of the wild-type level) in PHAMCL synthesis from gluconate as carbon source, indicating a functional role in this metabolic route (Table 2).

However, the phaG-negative mutant of P. putida PhaGN-21 showed a stronger impact on PHAMCL synthesis from simple carbon sources, exhibiting only 5% of the wild-type level PHAMCL content [9]. These data suggested that in P. aeruginosa alternative pathways might play a more important role for PHAMCL synthesis than in P. putida. However, reintroduction of the intact phaG gene from P. aeruginosa into mutant KO2 led to full recovery of PHAMCL synthesis from gluconate. In conclusion, these data were suggesting that the P. aeruginosa PhaG possesses an important function in PHAMCL synthesis from non-related carbon sources providing a metabolic link between fatty acid de novo biosynthesis and PHAMCL synthesis and that this enzyme exhibits (R)-3-hydroxydecanoyl-CoA:ACP transacylase activity. Interestingly, alternative pathways which lead to PHAMCL synthesis from non-related carbon sources are clearly more emphasized in P. aeruginosa than in P. putida. We are currently investigating transposon mutants of the P. aeruginosa mutant KO2 in which the alternative metabolic routes are strongly impaired.

Acknowledgments

This work was supported by EU-Fair Grant CT96-1780. The authors thankfully acknowledge the provision of plasmid pEX100T by Dr. H.P. Schweizer.

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