Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

Abstract

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro¹¹² residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are hydrolytic enzymes that catalyze the hydrolysis and synthesis of a variety of acylglycerols at the interface of lipid and water (14, 18). Of bacterial extracellular lipases (4), those from the Pseudomonas species have been extensively studied for their industrially applicable properties. Pseudomonas lipases were formerly classified into three classes according to amino acid sequence homology (18). Recently, Pseudomonas lipases were reclassified into six subfamilies among family I of bacterial lipases (4), since a large number of lipases were also isolated from other genera, and some Pseudomonas species have been reclassified as Burkholderia spp. (10, 31, 35). Subfamily I.1 includes the lipases from Pseudomonas aeruginosa (7, 34) and Pseudomonas fragi (3), and subfamily I.2 includes those from Burkholderia cepacia and Burkholderia glumae (11, 20). In the crystal structures of the lipases from B. glumae, B. cepacia, and P. aeruginosa, the calcium-binding site and position of the disulfide bond as well as the catalytic triad are well conserved (23, 28, 29). Most of the lipase genes of subfamilies I.1 and I.2 are clustered with a secondary gene that is located immediately downstream of the lipase genes (1, 12, 15, 16). The protein products of the secondary genes are specifically required for forming active lipases and have been named the lipase modulator, activator, helper protein, and lipase-specific foldase (Lif). Here, the proposed general name Lif (18) was used for the known lipase-specific helper proteins. Lif is directly involved in the folding and secretion of lipase via the two-step type II secretion pathway (33).

In this study, we have cloned, sequenced, and characterized the lipase and lipase modulator genes from Pseudomonas sp. strain KFCC 10818, which produces industrially valuable enzymes, including an alkaline protease, α-amylase, and lipase (this study) (19, 22, 24). When the lipase gene was isolated from Pseudomonas sp. strain KFCC 10818, a secondary gene was also identified immediately downstream of the lipase structural gene. The secondary gene was required for the formation of active lipase, as is true for known Pseudomonas Lifs. However, the deduced amino acid sequence of the secondary protein exhibited very low homology to the known Lifs. The secondary protein was named the lipase modulator since its function has not yet been characterized in detail. In a further study, we have investigated the functional interaction between the lipase and its modulator in lipase folding. Unexpectedly, a lipase variant that does not need the modulator for its correct folding was found. The changed sequences of the variant were identified, and the activity of the lipase variant as expressed without a modulator was assayed in a crude extract.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Pseudomonas sp. strain KFCC 10818 was used as a source of a lipase gene, lipK, and a lipase modulator gene, limK. In order to isolate genomic DNA, Pseudomonas sp. strain KFCC 10818 was grown to the mid-logarithmic phase in Luria-Bertani medium at 30°C. Escherichia coli JM83 and DH5α were used for the transformation of recombinant plasmids. E. coli JM109 and XL1-Blue were used as hosts for expression of the lipK and limK genes and DNA sequencing, respectively. Ampicillin (100 μg/ml), tetracycline (13 μg/ml), and/or chloramphenicol (30 μg/ml) was added to the growth medium when necessary.

Cloning of the lipase and lipase modulator genes.

To construct a genomic DNA library, genomic DNA from Pseudomonas sp. strain KFCC 10818 was isolated by the procedure described by Marmur (27) with a slight modification. Genomic DNA was partially digested with Sau3AI. DNA fragments were fractionated with a sucrose density gradient (10 to 40%, wt/vol) by ultracentrifugation at 25,000 rpm for 24 h at 20°C in a Beckman SW-28 rotor. Fractionated DNAs were ligated into the pUC19 vector at the BamHI site. Screening of the lipase gene(s) from the genomic library was carried out in two steps as previously described (2, 8, 21, 25, 26). First, colonies with halo-forming activity on tributyrin agar plates were isolated. Since both esterases and lipases can hydrolyze tributyrin, the colonies with halo-forming activity were further tested for lipase activity on the agar plates containing olive oil and rhodamine B. A recombinant plasmid carrying a lipase gene was isolated from a lipase-positive colony and sequenced. Nucleotide sequences were determined by the dideoxynucleotide chain termination method (32) with a Sequenase version 2.0 DNA sequencing kit (U.S. Biochemicals).

Construction of expression plasmids.

The fragment containing both lipK and limK was amplified by PCR using the M13/pUC reverse sequencing primer (5′-AGCGGATAACAATTTCACACAGGA-3′) and the Mod-3 primer (5′-GGCATGCATCTTATA CTATCTTATTGACC-3′) from pLIP172, which carries a 2.7-kb fragment containing lipK and limK in the pUC19 vector. In all PCR amplifications for subcloning, Pfu DNA polymerase (Stratagene) was used. The PCR product was digested with EcoRI and NsiI and ligated to the EcoRI and PstI sites of the E. coli expression vector pKK223-3 (Amersham Pharmacia Biotech) containing the strong tac promoter and ampicillin resistance gene, resulting in pKLM11 (lipK plus limK). To express the lipK and limK genes separately, the lipK gene was amplified by PCR with the M13/pUC reverse sequencing primer and Lip-II (5′-GGACAAGCTTACAGTCCAAGTTGTTG-3′). The amplified fragment was inserted into pKK223-3 at the EcoRI and HindIII sites to make pKL11 (lipK). The limK gene was amplified by PCR using primers Mod-1 (5′-CGAGAATTCATGATGCGTTATAAACCCA-3′) and Mod-3. The amplified fragment was digested with EcoRI and NsiI and inserted into the EcoRI and PstI site of pACYC-tac, yielding pACTM12 (limK). Plasmid pACYC-tac was constructed in this study by inserting the tac promoter, ribosomal binding site, and rrnB transcription terminator from pKK223-3 into PvuII- and NcoI-digested pACYC184 (New England Biolabs). Plasmid pACYC184 is a cloning vector compatible with vectors such as pKK223-3 and pUC19 and has tetracycline and chloramphenicol resistance genes. Plasmids pLIP171H and pKLM11H have also been constructed by inserting a 1.4-kb fragment with lipK and truncated limK into pUC19 and pKK223-3, respectively.

Mutagenesis of lipK and limK by PCR.

The high spontaneous error rate (6) of Taq DNA polymerase was utilized to obtain various random mutations. To isolate limK mutants that cannot support the formation of active lipase, the limK gene was amplified from pLIP172 by PCR using primers Mod-1 and Mod-3 under standard conditions. Wild-type limK of pACTM12 was replaced with the amplified products. The resulting plasmids were introduced into E. coli JM109 cells harboring pKL11. The lipase activities of the transformants were tested on tributyrin agar plates supplemented with both ampicillin and tetracycline. From the colonies lacking halo-forming activity, plasmids that contain limK mutant were isolated and put together. Then, lipK mutants that could suppress the putative loss-of-function mutation of limK were screened. The lipK gene was amplified from pLIP172 with the M13/pUC reverse sequencing primer and Lip-II primer under standard conditions. The amplified product was digested with ClaI and HindIII and inserted into the ClaI and HindIII sites of pKL11. The resulting plasmids were introduced into E. coli JM109 cells harboring plasmids containing the limK mutant. The transformants were tested for lipase activity on tributyrin agar plates. From the colonies forming halos on tributyrin agar plates, plasmids derived from pKL11 were isolated and sequenced to identify mutations. Primers Lip438 (5′-CCATTCTAGCCCTAACC-3′) and Lip744 (5′-CACTGGCTTTTAATCGTC-3′) were used for DNA sequencing.

Lipase activity assay.

Tributyrin agar plates (Luria-Bertani medium, 0.5% tributyrin [Sigma], and 1.5% agar [Difco]) were used to detect lipase activity (26). Lipase activity forms halos around colonies. Rhodamine B-olive oil agar plates (nutrient broth, 1% olive oil [Sigma], 0.001% rhodamine B [Sigma], and 1.5% agar [Difco]) were also used for the cloning of true lipase (8, 25), since tributyrin is hydrolyzed by esterases as well as lipases. The interaction of rhodamine B with fatty acids released during hydrolysis of triglycerides causes intense fluorescence around colonies upon UV irradiation. For the liquid assay, p-nitrophenyl ester was used as a substrate (5). The lipase assay in liquid was slightly modified by replacement of p-nitrophenyl butyrate with p-nitrophenyl palmitate. The assay measured the increase in absorbance at 410 nm due to the hydrolytic release of p-nitrophenol. One unit of lipase activity was defined as the activity releasing 1 μmol of p-nitrophenol per min at 25°C.

Nucleotide sequence accession number.

The lipK and limK DNA sequences have been deposited in the GenBank, EMBL, and DDBJ databases under accession no. AF125523.

RESULTS AND DISCUSSION

Cloning of the lipase and lipase modulator genes of Pseudomonas sp. strain KFCC 10818.

The genomic DNA library of Pseudomonas sp. strain KFCC 10818 was constructed and screened to isolate the lipase gene. Out of approximately 10,000 E. coli transformants screened on tributyrin agar plates, 27 halo-forming colonies were detected and further analyzed on rhodamine B-olive oil agar plates to distinguish between lipase and esterase activity. Out of 27 colonies, 1 colony exhibited true lipase activity. Finally, a plasmid named pLIP172, which has a 2.7-kb fragment carrying a lipase gene and a secondary gene, was obtained. The restriction maps of pLIP172 and its derivatives used in this study are represented in Fig. 1. The open reading frame of the lipase gene named lipK begins from a GTG initiation codon and putatively encodes a protein of 311 amino acids. The deduced amino acid sequence of the lipase LipK revealed high homology (36 to 53%) to the subfamily I.1 and I.2 Pseudomonas lipases (11, 20, 34). A secondary gene was located immediately downstream of lipK, similar to what occurs in subfamily I.1 and I.2 Pseudomonas lipase operons. The secondary gene was designated lipase modulator gene limK. The limK gene can encode a hydrophilic protein of 279 amino acids, which is 60 to 74 residues smaller than the previously known Pseudomonas Lifs. An analysis using the TMpred program (European Molecular Biology Network) indicated that the LimK protein is possibly a membrane-associated protein, with amino acids 6 to 25 forming a transmembrane helix. However, the deduced amino acid sequence of LimK shares only 17 to 19% homology with the Lifs, in comparison to the significant homology (28 to 58%) among the known Lifs. These results suggest that Pseudomonas sp. strain KFCC 10818 is distantly related to subfamily I.1 and I.2 Pseudomonas species.

FIG. 1.

Restriction maps of the plasmids used in this study. The thin arrows indicate the direction of transcription. Plac and Ptac represent the lac and tac promoters, respectively. EV, EcoRV; C, ClaI; Nc, NcoI; Hc, HincII; St, StuI; P, PstI; H, HindIII; E, EcoRI; aa, amino acid.

Effect of the limK gene on lipase expression.

The Lifs are known to function as a chaperone for correct folding and efficient secretion of lipase. Therefore, we investigated whether LimK is required for the formation of active LipK. First, a deletion analysis of the limK gene was performed. Plasmid pLIP171H, which contains the lipK gene and a truncated limK gene, was introduced into E. coli JM109 cells and the phenotype was compared to that of E. coli JM109 harboring pLIP172. While E. coli JM109 harboring pLIP172 expressed an active lipase, E. coli JM109 harboring pLIP171H did not show lipase activity, which implies that the limK gene is necessary for the expression of active lipase (Fig. 2A).

FIG. 2.

Expression of the lipK and limK genes in E. coli JM109. (A) Effect of limK on the expression of lipK in cis. The following plasmids were introduced into E. coli JM109 cells: pUC19 (colony row 1), pLIP171H (lipK and truncated limK) (colony row 2), and pLIP172 (lipK and limK) (colony row 3). (B) Effect of limK on the expression of lipK in trans. The following plasmids were introduced into E. coli JM109 cells: pKLM11 (lipK and limK) and pACYC-tac (colony 1), pKK223–3 and pACYC-tac (colony 2), pKL11 (lipK) and pACYC-tac (colony 3), pKLM11H (lipK and truncated limK) and pACYC-tac (colony 4), pKL11 (lipK) and pACTM12 (limK) (colony 5), and pKLM11H and pACTM12 (colony 6). The transformants were grown on a tributyrin agar plate containing 1% tributyrin for 48 h at 37°C.

To examine whether limK also functions in trans, the lipK and limK genes were expressed in different vectors. As shown in Fig. 2B, the lipK gene could encode an active lipase only in the presence of pACTM12 carrying the limK gene, indicating that the limK gene encoded a diffusible protein, LimK. In addition, the addition of the crude extract of E. coli JM109 harboring pACTM12 to the crude extract of E. coli JM109 harboring pKL11 induced the activation of the inactive lipase (data not shown). These results suggest that, despite its low sequence homology with Pseudomonas Lifs, LimK may interact directly with lipase and be required for its correct folding in the same manner that Lifs interact with lipase (9, 13, 17, 30).

Identification of a lipase variant: formation of active enzyme without LimK.

To further investigate the functional interactions of the LipK and LimK proteins, we have attempted to isolate loss-of-function mutations of limK and secondary mutations in lipK that suppress them. As a first step, random mutations were introduced into the limK gene by PCR as described in Materials and Methods. Out of 350 transformants, 27 colonies did not show lipase activity on tributyrin agar plates, indicating they were putative limK mutants that cannot support the formation of active lipase. From the colonies, plasmids containing mutant limK genes were isolated and put together. Next, lipK mutants that could suppress the putative loss-of-function mutation of limK were screened. The plasmids carrying randomly mutated lipK genes were introduced into E. coli JM109 harboring the plasmids containing mutant limK genes. Out of approximately 80,000 transformants, 26 colonies formed halos on tributyrin agar plates. From the colonies, plasmids derived from pKL11 were isolated and sequenced to identify the mutations. All lipK genes from the 26 clones had an identical C→A mutation at codon 112, which converted a Pro residue to a Gln residue, and some had an additional silent T→C mutation at codon 204.

However, a subsequent characterization of the P112Q variant of LipK has resulted in a surprising finding. When expressed in the absence of limK, the LipK^P112Q variant formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK in crude extracts (Fig. 3). Coexpression of LimK did not cause a significant change in enzyme activity. These results indicate that the LipK^P112Q variant does not require LimK for its expression in an active form. Recently, El Khattabi et al. have reported that the denatured lipase of B. glumae refolds into a native-like conformation in the absence of its Lif (10). They demonstrated that the lipase accumulates as a catalytically inactive intermediate of the folding process in the absence of Lif and that Lif helps the lipase overcome an energy barrier in the productive folding pathway. Therefore, it is possible to speculate that the Pro¹¹² residue of LipK participates in the interaction with LimK for overcoming the energy barrier and that the Pro→Gln mutation allows the lipase to spontaneously overcome the energy barrier without the help of LimK.

FIG. 3.

Comparison of the lipase activities of LipK and its mutant LipK^P112Q. (A) Plasmids were introduced into E. coli JM109 cells. E. coli transformants were grown on a tributyrin agar plate containing 1% tributyrin for 72 h at 37°C. Row 1, pKK223–3 and pACYC-tac; row 2, pKL11 (wild-type lipK) and pACYC-tac; row 3, pKL-S1 (encoding LipK^P112Q) and pACYC-tac; row 4, pKL-S1 and pACTM12 (limK); row 5, pKLM11 (wild-type lipK and limK) and pACYC-tac. (B) E. coli JM109 transformants were induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The cells were harvested at 3 h after induction. The lipase activities of the crude extracts were measured.

Pseudomonas lipases belonging to subfamilies I.1 and I.2 are industrially important. However, much difficulty has been encountered in expressing the lipases in E. coli, which is due mainly to their requirements for specific Lifs (13, 17, 35). We expect that the novel finding of this study and further characterization of LipK^P112Q will be useful in studying the folding pathway of Pseudomonas lipases and provide an effective method for the expression of industrially important lipases.