Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis

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Molecular cloning of two (R)-speci¢c enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis Takeharu Tsuge a;b , Toshiaki Fukui b , Hiromi Matsusaki b , Seiichi Taguchi b , Genta Kobayashi a , Ayaaki Ishizaki a , Yoshiharu Doi b; * a

Division of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-858, Japan b Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 4 October 1999; accepted 18 October 1999

Abstract Two Pseudomonas aeruginosa genes, termed phaJ1Pa and phaJ2Pa , homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJAc ) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from L-oxidation cycle. Two expression plasmids for phaJ1Pa and phaJ2Pa were constructed and introduced into Escherichia coli DH5K strain. The recombinants harboring phaJ1Pa or phaJ2Pa showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14^29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1Pa and phaJ2Pa products have the monomer-supplying ability for PHA synthesis from L-oxidation cycle. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Polyhydroxyalkanoate; (R)-speci¢c enoyl-CoA hydratase; Substrate speci¢city; Pseudomonas aeruginosa

1. Introduction Bacterial polyhydroxyalkanoate (PHA) is a novel polyester which has a potential usage as a biodegradable thermoplastic, and a wide variety of bacteria accumulate PHA within cells as an intracellular storage material for energy and carbon source under nutrient-starved conditions [1]. Aeromonas caviae isolated from soil accumulates PHA containing short and medium chain-length (C4 to C7) 3hydroxyalkanoate (3HA) units from alkanoic acids and plant oils [2]. In A. caviae, the L-oxidation intermediates, trans-2-enoyl-CoA, are converted to (R)-3-hydroxyacylCoA via (R)-speci¢c hydration catalyzed by (R)-speci¢c enoyl-CoA hydratase (PhaJAc ), and (R)-3-hydroxyacylCoAs are subsequently polymerized into PHA by the function of PHA synthase (PhaCAc ) [3,4]. The structure gene

* Corresponding author. Tel. : +81 (48) 467-9402; Fax: +81 (48) 462-4667; E-mail: [email protected]

for PHA synthase (phaCAc ) is organized in a pha operon with the gene for (R)-speci¢c enoyl-CoA hydratase (phaJAc ) [3]. On the other hand, £uorescent pseudomonads, such as Pseudomonas oleovorans, Pseudomonas putida, and Pseudomonas aeruginosa, are known to accumulate PHA consisting of medium chain-length (C6 to C14) 3HA units from alkanoic acids or oils [5]. In the production of medium chain-length PHA from fatty acid by £uorescent pseudomonads, L-oxidation pathway is also channeled to PHA biosynthesis [6]; however, the precursor sources for PHA synthesis have not been identi¢ed. As the case of phaJAc , the monomer-supplying related gene for PHA synthesis is not located in the PHA biosynthesis gene locus of £uorescent pseudomonads. Recently, the P. aeruginosa PAO1 genome has been sequenced and the sequence data are available from the Pseudomonas Genome Project web site (http://www.pseudomonas.com). In our search, several genes homologous to the phaJAc gene were found in P. aeruginosa genome, however, their functions were unknown. In this study, we

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 4 6 - X

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focused on two P. aeruginosa genes homologous to the phaJAc , referred to as phaJ1Pa and phaJ2Pa , and cloned these genes by use of PCR. We then investigated whether their gene products supply monomer units for PHA synthesis from the L-oxidation intermediate enoyl-CoA in recombinant Escherichia coli. 2. Materials and methods 2.1. Bacterial strains, plasmids, and genetic techniques Bacterial strains and plasmids used in this study are listed in Table 1. The coding regions for phaJ1Pa (471 bp) and phaJ2Pa (867 bp) were isolated by PCR using chromosomal DNA of P. aeruginosa as template. Oligonucleotide primers for PCR ampli¢cation of phaJ1Pa are as follow : N-terminus: 5P-dGCCGGGATCATTCGACAAAGGAGAG-3P and C-terminus: 5P-dGTAAGACAGCGGACAGGGTAGAGC-3P. A 0.6 kb PCR product including predicted open reading frame (ORF) and ribosome binding site (RBS) was ligated into pT7 Blue(R) vector to which the phaJ1Pa gene was inserted in the opposite direction of lac promoter. The resultant plasmid was digested with EcoRI and HindIII, and a 0.6 kb EcoRI-HindIII fragment was subcloned into the same sites of pUC18 vector to generate expression plasmid for phaJ1Pa , pEH6. Oligonucleotide primers for PCR ampli¢cation of phaJ2Pa are as follow : N-terminus: 5PdCAGGGCGGGGTGGAGTGCTTCATGG-3P and Cterminus: 5P-dGGATGTTGGAGGGCGAGTGGTAGAAC-3P. As the same manner for the pEH6, phaJ2Pa expression plasmid, pBE14, was constructed using pUC19 vector from a 2.3 kb PCR product. The plasmids pAPAC and pPPAC for expression of PHA synthase genes from A. caviae (phaCAc ) [3] and Pseudomonas sp. 61-3 (phaC1Ps ) [10], respectively, were constructed previously [9]. These plasmids were made in common from the broad-hostrange vector, pJRD215 [11], by introducing lacIq gene, trc promoter, and rrnBT1T2 terminator sequences of pTrc99A (Amersham Pharmacia Biotech).

2.2. Enzyme assay Crude extracts were prepared by sonication from the harvested cells of recombinant E. coli DH5K grown in LB medium [12] with IPTG (1 mM) and ampicillin (100 mg l31 ) at 37³C for 24 h, followed by centrifugation (20 000Ug, 20 min, 4³C). The resulting soluble cell extracts were used as an enzyme solution. Enoyl-CoA hydratase activity (including both (S)- and (R)-speci¢c enzymes) were assayed by measuring the reduction in absorbance at 263 nm derived from the decrease of the enoyl-thioester bond, as described by Moskowitz and Merrick [13]. To investigate the con¢guration of 3HA-CoA produced by hydratase, the formation of NADH linked with the oxidation of (S)-3HA-CoA by (S)-speci¢c 3HA-CoA dehydrogenase (Sigma) was observed, as described by Fukui et al. [4]. Crotonyl-CoA substrate used for the hydration assay was purchased from Sigma, and other C6 and C8 trans-2-enoyl-CoA substrates were synthesized from a lithium salt of CoA and the corresponding trans-2-alkanoic acids (Tokyo Kasei) [4]. Protein concentrations were determined by using Bio-Rad assay solution and bovine serum albumin as the standard. 2.3. Cell growth and PHA production E. coli LS5218 (fadR601, atoC2(Con)) strain [7,8], which can grow on fatty acid at a high rate (data not shown), was used for PHA synthesis experiments. The recombinant strains of E. coli LS5218 were inoculated to the M9 medium [12] containing 0.25% sodium dodecanoate as a sole carbon source. When needed, kanamycin (50 mg l31 ), or/and ampicillin (100 mg l31 ) was added into the medium. The cells were cultivated on a reciprocal shaker (130 strokes min31 ) in 500-ml £ask at 37³C for 72 h. 2.4. Analysis of the PHA content and PHA composition The PHA content and PHA composition in dry cells were determined by gas chromatography after methanol-

Table 1 Bacterial strains and plasmids used in this study Bacterial strain and plasmid Strain P. aeruginosa E. coli DH5K E. coli LS5218 Plasmid pT7 Blue(R) pUC18, pUC19 pEH6 pBE14 pAPAC pPPAC a

Genotypea

Source or reference

^ ^ fadR601, atoC2(Con)

DSM1707 Clontech [7,8]

^ ^ pUC18 derivative ; phaJ1Pa pUC19 derivative ; phaJ2Pa pJRD215 derivative ; phaCAc pJRD215 derivative ; phaC1Ps

Novagen Takara This study This study [9] [9]

Only genetic properties concerning the fatty acid and PHA metabolism are shown.

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Fig. 1. Multiple alignment of the deduced amino acid sequence of PhaJ1Pa from P. aeruginosa (P.a. PhaJ1) with those of a hypothetical protein from R. capsulatus (R.c. ORF) [15] and of (R)-speci¢c enoyl-CoA hydratase from A. caviae (A.c. PhaJ) [3] (A). Partial alignment of the deduced amino acid sequence of PhaJ2Pa from P. aeruginosa (P.a. PhaJ2) with those of PauB from P. mendocina 35 (P.m. PauB) [16] and of putative (R)-speci¢c enoyl-CoA hydratase domain of peroxisomal multifunctional protein from S. cerevisiae (S.c. perMFP) [17] (B).

ysis of lyophilized cells in the presence of 15% sulfuric acid, as described previously [14]. 3. Results 3.1. Identi¢cation of the genes homologous to phaJAc through database search A database search revealed that the several genes homologous to the phaJAc exist in P. aeruginosa and other microorganisms. Two of them found in the Pseudomonas Genome Project web site, ORF67288 and ORF63846, were referred to as phaJ1Pa and phaJ2Pa , respectively. As shown in Fig. 1A, the deduced amino acid sequence of phaJ1Pa (PhaJ1Pa ) exhibited high homology to those of

phaJAc (PhaJAc ) (46% identity of 129 amino acids) and of a hypothetical protein of Rhodobacter capsulatus (50% identity of 130 amino acids) [15]. Although the deduced amino acid sequence of phaJ2Pa (PhaJ2Pa ) was partially similar to the PhaJAc (36% identity of 80 amino acids), it showed very high homology to the translated product of pauB (pimelic acid-utilizing B) from Pseudomonas mendocina 35 (80% identity of 198 amino acids) [16], as shown in Fig. 1B. However, P. mendocina pauB is not sequenced completely and the function of its translated product is unknown. The PhaJ2Pa also showed partial homology to a putative (R)-speci¢c enoyl-CoA hydratase (hydratase 2) domain of Saccharomyces cerevisiae L-oxidation multifunctional protein (32% identity of 283 amino acids) [17]. The multifunctional protein from S. cerevisiae is capable of hydrating medium chain-length enoyl-CoA into

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Table 2 Enoyl-CoA hydratase activity in recombinant strains of E. coli DH5K Plasmid (relevant marker)

Speci¢c activity (U mg31 of protein)a Crotonyl-CoA (C4)

2-Hexanoyl-CoA (C6)

2-Octanoyl-CoA (C8)

pUC18 (none) pEH6 (phaJ1Pa ) pBE14 (phaJ2Pa )

4.9U1033 3.7U102 2.4U1031

2.0U1032 3.2U102 5.2U1031

9.8U1033 1.7U1031 1.5

a

Activities in the crude extract from cells grown in LB medium for 24 h at 37³C.

(R)-3-hydroxyacyl-CoA [17], whereas the PhaJAc is speci¢c for short chain-length enoyl-CoA [4]. Therefore, phaJ1Pa and phaJ2Pa are likely to encode di¡erent types of (R)speci¢c enoyl-CoA hydratase on substrate speci¢city. 3.2. Hydratase activity assay of recombinant strains of E. coli DH5K The expression plasmids pEH6 and pBE14 for phaJ1Pa and phaJ2Pa , respectively, were constructed, as described in Section 2. Recombinant E. coli DH5K strains harboring these expression plasmids were cultivated in LB medium in the condition of IPTG induction. The hydratase activity (including both (S)- and (R)-speci¢c enzymes) in the crude extracts of recombinant E. coli strains were assayed by hydration of three trans-2-enoyl-CoA substrates with four, six, and eight carbon atoms, and the results are given in Table 2. The control strain DH5K harboring pUC18 showed low hydratase activities toward all the substrates, indicating the existence of intrinsical hydratase in E. coli. The strain DH5K harboring pEH6 showed very high hydratase activities toward crotonyl-CoA and 2-hexanoyl-CoA, but not toward 2-octanoyl-CoA. This chain length-dependent substrate speci¢city of PhaJ1Pa was in coincidence with that of PhaJAc [4]. Strain DH5K harboring pBE14 also showed a remarkable increase in the hydratase activity concomitantly with an increase in the carbon chain length of 2-enoyl-CoA. For the PhaJ2Pa , the hydratase activity toward 2-octanoyl-CoA was approximately six-fold higher than that for crotonyl-CoA. To determine the stereospeci¢city in the hydration catalyzed by PhaJ1Pa or PhaJ2Pa , NAD‡ and (S)-3HA-CoA dehydrogenases were added to the reaction mixture. How-

ever, no formation of NADH linked with the oxidation of (S)-3HA-CoA was observed. These results suggest that PhaJ1Pa and PhaJ2Pa are (R)-speci¢c enoyl-CoA hydratases with di¡erent substrate speci¢cities, that is, speci¢c for shorter chain-length enoyl-CoAs and longer chainlength enoyl-CoAs, respectively. 3.3. PHA accumulation in recombinant strains of E. coli LS5218 Six recombinant strains of E. coli LS5218 were prepared to verify whether the PhaJ1Pa and PhaJ2Pa are capable of supplying monomer units for PHA synthesis from the Loxidation intermediate enoyl-CoA. Here, we used two PHA synthase genes (phaCAc and phaC1Ps ). The PhaCAc is speci¢c for (R)-3HA-CoAs of C4 to C6 [3], while the PhaC1Ps is capable of polymerizing (R)-3HA-CoAs of C4 to C12 [10]. The strains harboring PHA synthase gene (phaCAc or phaC1Ps ) with or without hydratase gene (phaJ1Pa or phaJ2Pa ) were cultivated in M9 medium containing sodium dodecanoate (0.25% w/v) for 72 h, and then PHA accumulated in the cells were subjected to analysis. The results are given in Table 3. It should be noted that the four strains harboring both PHA synthase gene and hydratase gene accumulated much more PHA (up to 14^29 wt% of cell dry weight) than the two recombinant strains harboring the PHA synthase gene alone, suggesting that PhaJ1Pa and PhaJ2Pa play an important role in supplying the monomer (R)-3HA-CoAs for PHA synthesis in LS5218 strains, as demonstrated for PhaJAc [18]. Strains LS5218 harboring phaCAc with or without the hydratase gene synthesized a copolymer of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6), probably de-

Table 3 PHA accumulation in recombinant E. coli LS5218 strainsa Plasmids (relevant markers)

Cell dry weight (g l31 )

PHA content (wt%)

pAPAC (phaCAc ) pAPAC, pEH6 (phaCAc , phaJ1Pa ) pAPAC, pBE14 (phaCAc , phaJ2Pa ) pPPAC (phaC1Ps ) pPPAC, pEH6 (phaC1Ps , phaJ1Pa ) pPPAC, pBE14 (phaC1Ps , phaJ2Pa )

0.86 0.84 0.84 0.80 0.65 0.79

Trace 28 25 Trace 29 14

PHA composition (mol%) 3HB (C4) 3HHx (C6) 3HO (C8) 3HD (C10) 3HDD (C12) 91 90

9 10

0 0

0 0

0 0

10 8

78 45

7 30

3 11

2 6

3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate; 3HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate. a Cells were cultivated in M9 medium containing sodium dodecanoate (0.25% w/v) for 72 h at 37³C.

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pending on the substrate speci¢city of PHA synthase. On the other hand, by the co-expression with phaC1Ps of a wide substrate-speci¢c PHA synthase gene, recombinant LS5218 strains synthesized PHAs of di¡erent copolymer compositions, with a high 3HHx fraction (78 mol%) for phaJ1Pa and with high fractions of 3HHx (45 mol%) and 3HO (30 mol%) for phaJ2Pa . In spite of lower hydratase activity of PhaJ2Pa toward hexanoyl-CoA than octanoylCoA (Table 2), 3HHx fraction (45 mol%) of PHA synthesized by the strain LS5218 harboring phaC1Ps and phaJ2Pa was higher than 3HO fraction (30 mol%). The di¡erence in these compositions may be due to not only the substrate speci¢cities of PHA synthase and hydratase enzymes but also the enoyl-CoA-supplying capability of the host strain used.

197

Ren et al. [20] attempted to obtain mutants of P. putida lacking PHA accumulation ability under the conditions leading to the expression of the PHA synthase gene. However, they have not obtained PHA-free mutants. This result may suggest that there are various metabolic routes for the synthesis of (R)-3HA-CoA, for example the involvement of 3-ketoacyl-ACP reductase in PHA synthesis as it has been demonstrated in E. coli recombinant system by us [9]. In this context, for analysis of the PHA biosynthesis pathway from fatty acid in P. aeruginosa, we are now investigating the expression of phaJ1Pa and phaJ2Pa during the course of PHA accumulation from fatty acids or plant oils.

References 4. Discussion In a previous paper [4], we have demonstrated that A. caviae (R)-speci¢c enoyl-CoA (PhaJAc ) is a supplier of (R)3HA-CoAs of C4 to C6 for PHA synthesis via L-oxidation pathway from fatty acids or plant oils. Such a (R)-speci¢c hydratase activity toward short chain-length enoyl-CoAs is also found in other PHA-producing bacteria, Rhodospirillum rubrum [13] and Methylobacterium rhodesianum [19], while the activity toward longer enoyl-CoAs (more than C8) has not been found in PHA-producing bacteria. With respect to the distribution or variation of (R)-speci¢c enoyl-CoA hydratase, £uorescent pseudomonads are appropriate gene screening sources because of their capability of producing medium chain-length PHA from fatty acid via L-oxidation pathway. In addition, the precursor sources for PHA synthesis in £uorescent pseudomonads have not been identi¢ed. It has been proposed that (R)-3HACoAs are formed from intermediates of the L-oxidation cycle by (R)-speci¢c enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, or 3-ketoacyl-CoA reductase, although each contribution of their enzymes remains unclear [20]. In this study, based on the database, we identi¢ed and cloned the two genes homologous to the phaJAc , phaJ1Pa and phaJ2Pa , from P. aeruginosa genome. We also demonstrated that these genes encode (R)-speci¢c enoyl-CoA hydratases with di¡erent substrate speci¢cities, and that PhaJ1Pa shows high speci¢cities for shorter chain-length enoyl-CoAs of C4 and C6, while PhaJ2Pa for longer chain-length enoyl-CoAs. Indeed, their translated products were found to exhibit the potential ability to provide monomer units for PHA synthesis from L-oxidation intermediate in E. coli. To date, (R)-speci¢c medium chainlength enoyl-CoA hydratase gene has been cloned as a part of the L-oxidation multifunctional protein gene from S. cerevisiae [17], but not from bacteria. Therefore, this is the ¢rst report on (R)-speci¢c medium chain-length enoyl-CoA hydratase from bacterium. To elucidate PHA biosynthesis pathway from fatty acid,

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