EP0652958A1

EP0652958A1 - Cloning and expression of a lipase modulator gene from pseudomonas pseudoalcaligenes

Info

Publication number: EP0652958A1
Application number: EP93917663A
Authority: EP
Inventors: Maria Mathilde Josephina Cox; Gijsbert Gerritse; Wilhelmus Johannes Quax
Original assignee: Gist Brocades NV; Genencor International Inc
Current assignee: Danisco US Inc
Priority date: 1992-07-23
Filing date: 1993-07-23
Publication date: 1995-05-17
Also published as: WO1994002617A2; JPH07509129A; WO1994002617A3

Abstract

The present invention discloses the cloning and expression of a lipase modulator gene obtained from a class I Pseudomonas species. The expression product of the modulator gene is found to give rise to a considerable increase in lipase production especially upon homologous expression. The present invention provides a method for isolating a class I lipase modulator gene, an isolated modulator gene and a class I Pseudomonas transformed with such a gene. Finally the present invention discloses a derivative of plasmid pJRD215 which is segregationally stable in Pseudomonas.

Description

Cloning and expression of a lipase modulator σene from Pseudomonas pseudoalcaliσenes

Technical field The present invention describes the cloning and expression of a lipase gene in combination with a lipase modulator gene, both obtained from a class I Pseudomonas species, in an homologous class I Pseudomonas species.

Background of the invention Lipases are enzymes capable of hydrolyzing lipids they are utilized in a wide range of applications such as fats and oil processing, detergents, diagnostic reagents etc.

Extracellular lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are produced by a wide variety of micro- organisms. Suitable microbial lipases have for example been disclosed in U.S. Patent No. 3,950,277, these lipases were obtained from such diverse microorganisms as Pseudomonas. Asperσillus. Pneumococcus. Staphylococcus. ycobacterium tuberculosis. Mycotorula lipolytica and Sclerotinia. It has turned out that especially Pseudomonas lipases have favourable characteristics for the desired applications. Pseudomonas species have therefore been extensively used for obtaining lipases. To increase lipase yield in fermentation several lipase genes have been cloned and expressed in both homologous and heterologous host strains. Examples of the Pseudomonas species from which lipase gene cloning has been reported are; Pseudomonas cepacia (EP 331376) , Pseudomonas glumae (EP 464922) , Pseudomonas pseudoalcaliσenes (EP 334462), Pseudomonas fraσi (EP 318775). During this work it has been found that a lipase modulator gene was necessary to obtain lipase expression in an heterologous host.

EP 331376 describes the cloning and expression of a lipase gene obtained from Pseudomonas cepacia in P. cepacia. It was found that no expression could be obtained when a second gene located downstream of the lipase gene was deleted. This gene was therefore reported to be essential for lipase production. EP 464922 reports the cloning and expression of a lipase gene together with a gene encoding a protein reported to have a lipase-specific stabilizing/translocation function. The genes are obtained from Pseudomonas qlumae and expression is preferably in heterologous systems. The stabilising protein is reported to differ greatly from the gene described in EP 331376 and therefor assumed to have a different function. WO 91/00908 reports the expression in a heterologous host of the lipase gene and the lipase modulator gene obtained from P. cepacia.

Lipase modulator genes, are reported to be essential for obtaining lipase production, however for an extensively investigated representative of class I Pseudomonas species: Pseudomonas fraqi such a gene was not found. Another class I Pseudomonas lipase gene was described in EP 334462. EP 334462 reports the cloning and expression of the lipase gene from Pseudomonas pseudoalcaliqenes in E. coli it can be concluded that for heterologous lipase production the lipase modulator gene was not essential. The classification of Pseudomonas species is based on DNA-rRNA and DNA-DNA hybridization studies as reported by Palleroni et al. (Palleroni et al. Int.J.Syst.Bacteriol. 2J3. : 333 (1973)). A more extensive overview can be found in Bergey s Manual of Systematic Bacteriology (Vol.l. Section 4 160-161 (1984). Eds N.R. Krieg and J.G.Holt. Williams and Wilkins, Baltimore/ London) . This overview also reports that the classification is supported by morphological data and by 16S ribosomal RNA homology. Generally a lipase modulator gene could not be detected in class I Pseudomonas species on the basis of homology with class 2 lipase modulator genes. Aoyama et al. FEBS Lett. 242 36-40 (1988) report the absence of such a gene in P. fragi. Recently a lipase modulator gene was reported (Ihara et al. J. Ferm. Bioengin. 73. (1992) 337-342) for a Pseudomonas species, which might belong to RNA homology group I, based on DNA homology (shown in table 3 and 4 of the present application) . However, this gene was only used in E. coli and therein shown to be essential for the Pseudomonas lipase gene expression.

Summary of the invention

The present invention discloses a lipase modulator gene and the corresponding protein obtained from a class I Pseudomonas species.

The present invention also discloses Pseudomonas strains which have been transformed with a DNA sequence encoding a lipase and a sequence encoding a lipase modulator gene. These strains are preferably class I Pseudomonas strains and more preferably Pseudomonas pseudoalcaliqenes strains. The present invention further discloses a method for obtaining such transformed strains.

Furthermore the use of these strains for producing high amounts of lipase is disclosed.

The invention further discloses a vector derived from pJRD215 and which is segregationally stable in Pseudomonas. A method for obtaining such a vector is also disclosed.

Brief description of the drawings

Figure l; Restriction map of plasmid P1A. Symbols used are :

Km^r: gene encoding neo ycin resistance of Tn5. lip: gene encoding Ml lipase. Furthermore a number of restriction sites are indicated. - 4 -

Figure 2: Sequence of pJRD215 (derived from Davison et al.

Gene 5_1 275-280 (1987)). The boxes indicate the direct repeat. At this site a recombination event occured, resulting in plasmid P1A5 (shown in figure 3) . The deletion was mapped by sequence analysis. Figure 3: Restriction map of plasmid "BIAS .

Symbols used are :

Km^r: gene encoding neomycin resistance of Tn5. lip: gene encoding Ml lipase. Due to the deletion plasmid PlAδ is about 900 bp smaller than P1A, also several restriction sites are missing. Figure 4: Construction and restriction map of plasmid PIB.

Plasmid pTMPvlδA was described in EP 334462. lip: gene encoding Ml lipase, location indicated by an arrow Tc^r: gene encoding tetracyclin resistance.

Plasmid pLAFR3 and derivatives harbour the replicon of

RP4. Figure 5: Restriction map of plasmid P24A2-5. Symbols used are :

Km^r: gene encoding neomycin resistance of Tn5. lip: gene encoding Ml lipase. lim: gene encoding the Ml lipase modulator protein. Figure 6: Restriction map of plasmid P24B. lip: gene encoding Ml lipase, location indicated by an arrow lim: gene encoding the Ml lipase modulator protein, location also indicated by an arrow.

Tc^r: gene encoding tetracyclin resistance. Figure 7: Construction of plasmid pBRint. amp^r: gene encoding ampicillin resistance. tet^r: gene encoding tetracyclin resistance. Figure 8: Physical map of the integration locus in the chromosome of Pseudomonas pseudoalcaligenes Ml. lip: gene encoding Ml lipase, location indicated by an arrow lim: gene encoding the Ml lipase modulator protein, location also indicated by an arrow. tef: gene encoding tetracyclin resistance. lip^': indicating an inactivated Ml lipase gene. Figure 9: Construction of plasmid pUBint.

Plasmid pBHA-Ml was described in EP 334462 figure 15.

Km^p: gene encoding neomycin resistance of pUBHO.

P_HpaII: Hpall promoter of plasmid pUBHO. flflH - insert derived from Pseudomonas pseudoalcaligenes Ml, containing (part of) the lipase encoding sequence and part of the lipase modulator gene encoding sequence. Figure 10:Physical map of the integration locus in the chromosome of Pseudomonas pseudoalcaljgenes. lip: gene encoding Ml lipase, location indicated by an arrow lim: gene encoding the Ml lipase modulator protein, location also indicated by an arrow. neo^r: gene encoding neomycin resistance of pUBllO. lim^': indicating an inactivated Ml lipase modulator gene.

Detailed description of the invention The recombinant DNA of the present invention is obtained by digestion of chromosomal DNA obtained from a strain of a Pseudomonas class I species. Representatives of class I Pseudomonas species are: Pseudomonas alcaligenes. Pseudomonas pseudoalcaligenes. Pseudomonas stutzeri. Pseudomonas aeruginosa. and Pseudomonas mendocina.

The chromosomal DNA is isolated using standard procedures as disclosed for example in Maniatis et al. Molecular cloning. Cold Spring Harbor Press, 1982 and 1989. I suitable digest is made and the fragments are cloned in a vector which is subsequently used to transform an E. coli. Selection is made on the basis of the presence of the lipase gene this can be performed using hybridization if suitable probes are available. Alternatively it is possible to use an expression vector in which case it becomes possible to select for the presence of the desired genes using a suitable assay such as halo formation when lipase is screened for. Furthermore expression can also be monitored using immunological detection of the protein when suitable antibodies are available.

In the present invention a DNA library was obtained from Pseudomonas pseudoalcaligenes as a representative of class I Pseudomonads. The gene encoding the lipase was localized on a 2.0 kb PvuII fragment. The procedure has been described in EP 334462. Briefly, the lipolytic enzyme from the supernatant of Pseudomonas pseudoalcaligenes M-l (CBS 473.85) was purified. After gel electrophoresis and blotting s on Immobilon transfer membrane the N-terminal sequence was determined. A suitable probe was prepared based on this sequence. This probe was used in Southern hybridization experiments against chromosomal DNA which was ^"isolated from Pseudomonas pseudoalcaligenes and which had been o digested with several restriction enzymes. After size fractionation the fragments were cloned and again hybridized. A 2.0 kb PvuII fragment was found to contain the lipase gene. This fragment was sequenced and was also found to contain at least a part of a putative open reading frame. 5 This 2.0 kb fragment was cloned on an expression vector and the vector transformed to wild type Pseudomonas pseudoalcaligenes the resulting strain showed a 10-20 fold increase in lipase production.

A new expression vector was obtained containing the 0 complete open reading frame which was localized downstream of the lipase gene on a 2.4 kb PvuII/BclI fragment the lipase gene and the open reading frame are found in their natural one operon sequence on this vector. Transformation of P. pseudoalcaligenes with this vector showed a 30 fold increase 5 in lipase production over the expression vector containing only the lipase gene. This indicates that the open reading frame encodes a gene which modulates lipase expression. The open reading frame was sequenced and found not to have a significant identity with the known class 2 lipase modulator genes, the identity was of the order of 30% at the amino acid level. The open reading frame also showed a 25 % identity with the lipase gene itself.

Upon inactivation of this modulator gene in the chromosome no lipase was produced. Cloning of the gene on an expression vector in such a host restored the lipase production. This gene can therefore be considered to be the class I type lipase modulator gene. The cloning and expression of this modulator gene in wild type Pseudomonas pseudoalcaligenes did not have any effect on lipase production. However, when extra copies of the lipase gene were introduced into the cell, lipase production was significantly increased by addition of the modulator gene. From these experiments it is evident that the modulator gene is not required in a 1:1 ratio as compared with the lipase gene. The present invention discloses for the first time that a class I lipase modulator gene increases the lipase productivity in a homologous host cell. Furthermore, it is also conclusively shown that the gene is not required in a 1:1 ratio compared with the lipase gene. The present invention discloses a method for obtaining lipases comprising:

- cloning of a lipase gene and a lipase modulator gene obtained from a class I Pseudomonas species in a strain of an homologous Pseudomonas species, - culturing of the recombinant strain under conditions wherein the lipase is expressed,

- isolating the lipase from the culture.

The host cells of the present invention and the source of the lipase modulator gene are preferably selected from the class I Pseudomonas species, Pseudomonas alcaligenes. Pseudomonas pseudoalcaligenes. Pseudomonas stutzeri. Pseudomonas aeruginosa. and Pseudomonas mendocina. Mutants of these strains can also be employed.

In a most preferred embodiment Pseudomonas pseudoalcaligenes is the host strain. The lipase gene and the lipase modulator gene can be cloned on a replicable expression vector they can however also be inserted in the chromosome of the host strain. Optionally the chromosomal lipase gene and the lipase modulator gene can be inactivated before the cloned genes are introduced. This is especially important when the production of mutated lipase is performed. The presence of the wildtype gene would then give rise to a significant contamination of the product. The copy number of the gene can be regulated by the choice of the vector. The lipase and the lipase modulator genes are found to act both in cis and in trans. They are also found to be in one operon. It is therefore preferable to clone the genes in one operon. It is also possible to clone the genes on two separate vectors thereby enabling independent regulation. The present invention further discloses a method for obtaining a plasmid which is segregationally stable in Pseudomonas. This method comprises:.

- repeated dilution of the transformed Pseudomonas strain in medium without antibiotics,

- followed by incubation periods in the presence of antibiotics.

The invention discloses a segregationally stable derivative of pJRD215 herein called plAδ. Sequence analysis shows that a fragment of 900 basepairs between two direct repeats has been deleted.

The following examples are meant to illustrate but not to limit the present invention. Experimental

Colonies were grown on agar plates by using tributyrin (Merck) or castor oil as a substrate (described by Laurence et al. Nature 191 (1967) 1264-1265) .

Plates were incubated at 30°C during 48-72 hours.

The clear zones that appear indicate lipolytic activity. A linear relationship can be observed when the logarithm of the enzym concentration is plotted against the diameter of zone of intensification.

Colonies were also grown on agar slices containing a defined volume of agar medium. After growth, the full grown colony is placed on a plate containing tributyrin. After an incubation period of 24 hours a zone of intensification can be observed. The diameter of this zone shows a linear relation with the logarithm of the level of enzym produced by the colony. The latter was observed when determining the activity of the supernatant of the strains grown in a lab fermentor as described in EP 334462, example 10. Strains and plasmids described in this patent application are listed in table A.

Table A

Table A (continued)

Examples

Example 1

Cloning of the lipase gene and the lipase modulator gene from Pseudomonas pseudoalcaligenes Ml in Pseudomonas pseudoal¬ caligenes Ml

The lipase gene from Pseudomonas pseudoalcaligenes Ml was cloned in E. coli as described in patent application EP 334462. In order to achieve homologous gene expression the Sstl/Hindlll lipase gene containing fragment of pTMPvlδA (EP 334462: figure 11) was cloned into pJRD215 (Davison et al. Gene 5_1 (1987) 275-280) SstI and Hindlll restriction sites. A restriction map of this plasmid, called P1A, is shown in Figure 1.

A stable derivative of this plasmid P1A was isolated after repeated dilution in medium without antibiotics followed by incubation periods. Characterization of the obtained derivative revealed that a deletion of about 900 basepairs had occured, as shown in Figure 2. Surprisingly this plasmid, named P1A5, was much more stable than plasmid P1A. A restriction map of plasmid P1A6 is shown in Figure 3, The improved segregational stability is shown in Table 1:

Table 1

Cells were grown in a 2xTY culture without anti¬ biotics, at O.D.=l, about 10⁸ cells were inoculated into 2xTY medium and grown for 24 hours (=day 1) , the culture was then diluted a thousand times and incubation was prolonged for 24 hours (=day 2) and so on. Colonies were first grown on plates without antibiotics for 24 hours at 30°C. Subsequently the colonies were replica-plated to plates containing 10 mg/1 neomycin.

The EcoRI/Hindlll lipase gene containing fragment of pTMPvlδA was cloned into pLAFR3 (Staskawicz et al. , J. Bacteriol. 169 (1987) 5789-5794) EcoRI and HinDIII restriction sites. A restriction map of this plasmid, called PIB, is shown in Figure 4.

As described in EP 334462, we also cloned Bell fragments of Pseudomonas pseudoalcaligenes Ml in E. coli. One of these clones, a 1.7 kb Bell fragment, appeared to contain the 3' region of the lipase gene and 1.2 kb of the downstream sequence. In order to investigate whether class I Pseudomonas species also contain lipase modulator genes, both fragments (the 1.7 kb Bell and the Sstl/Hindlll (2.0 kb) lipase gene containing fragment of pTMPvlδA) were combined in one expression cassette, resulting in clone pTZ18B24 (figure not shown) . An internal PvuI/EcoRI fragment was exchanged with Pseudomonas expression cassette P1A«5 resulting in expression cassette P24A2<S. A restriction map of this plasmid is shown in Figure 5. The DNA sequence of the entire insert is shown in the Sequence Listing and contains two open reading frames one encoding the lipase gene and the other encoding a putative lipase modulator gene hereafter described as lipase modulator gene.

The EcoRI/Hindlll lipase and modulator gene containing fragment of pTZ18B2.4 was also cloned into pLAFR3 EcoRI and HinDIII restriction sites, resulting in plasmid P24B2. The restriction map of this plasmid is shown in Figure 6.

All expression cassettes were introduced into Pseu¬ domonas pseudoalcaligenes Ml using electroporation (Wirth et al. Mol. Gen. Gen. 216 (1989) 175-177).

Example 2

Chromosomal inactivation of both lipase and modulator gene of Pseudomonas pseudoalcaligenes

Suicidal integration plasmids, which are unable to replicate in Pseudomonas pseudoalcaligenes but able to replicate in other microorganism, were used to inactivate the lipase gene and the lipase modulator gene in the chromosome of Pseudomonas pseudoalcaligenes Ml.

Inactivation of the lipase gene

An internal PvuI-PstI fragment of the lipase gene was cloned on suicide plasmid pBR322 (Bolivar et al. Gene 2.

(1977) 95-113), able to replicate in E. coli. wherein the 38 N-terminal and the 49 C-terminal aminoacids of the lipase coding sequence are missing (hereafter described as pBRint) .

Detailed information about the construction of pBRint, derived from pBR322 and pTMPvlβA (described EP 334462) , is shown in Figure 7. Pseudomonas pseudoalcaligenes Ml R^'M*

(restriction negative, modification positive) was transformed with pBRint. Several tetracycline resistent (5 mg/1) colonies were selected. They were all lipase negative, demonstrated in lacking a clearing-zone on castor oil (0.5%) agar plates and in a diminished clearing-zone on tributyrin (2%) agar plates. Pseudomonas pseudoalcaligenes Ml R^"M⁺ itself gives rise to a clear halo on both types of agar plates. Southern analysis of two independent lipase negative electroporants revealed that the integration was established through a single cross-over event. As a result of this event the situation in the chromo¬ some of Pseudomonas pseudoalcaligenes undergoes changes, outlined in Figure 8.

Inactivation of the lipase modulator gene The internal EcoRV-PvuII fragment of the lipase modulator gene was cloned on suicide plasmid pUBHO (Gryczan et al. J. Bacteriol. 134 (1978) 318-329), able to replicate in almost all Bacillus species, wherein the 94 N-terminal and 107 C-terminal amino acids of the lipase modulator coding sequence are missing (hereafter described as pUBint) .

Detailed information about the construction of pUBint, derived from pBHA-Ml (described in EP 334462) , is shown in Figure 9.

Pseudomonas pseudoalcaligenes Ml R'M⁺ was transformed with pUBint. Several neomycin resistent (10 mg/1) colonies were selected. They were lipase negative, demonstrated in lacking a clearing-zone on castor oil (0.5%) agar plates and in a diminished clearing-zone on tributyrin (2%) agar plates. Southern analysis of three independent lipase negative elec- troporants revealed that the integration was established through a single cross-over event. As a result of this event the situation in the chromosome of Pseudomonas pseudoalcali¬ genes undergoes changes, outlined in Figure 10.

Complementation Complementation studies were performed with low copy number plasmids PIB, containing only the complete lipase gene and P24B2, containing both the complete lipase gene and modulator gene, and high copy number plasmids, P1A<S containing only the complete lipase gene and P24A25 containing both the complete lipase and modulator gene. The results of the complementation study are shown in Table 2. From these experiments it can be concluded that both the lipase and the lipase modulator gene, need to be intact for the total complementation of the lipase activity of both the lipase gene and lipase modulator gene inactivated strains.

Example 3

Lipase expression of transformed Pseudomonas pseudoalcali¬ genes Ml

The lipase gene was expressed in heterologous host organisms (as described in EP 334462) . However lipase expression levels were extremely low compared with the levels obtained in Pseudomonas pseudoalcaligenes Ml. Therefor homologous gene expression was further developed.

The transformants were tested for their lipase pro¬ duction both on agar plates containing tributyrin and/or castor oil and after fermentation in olive oil based media as described by Odera et al. J. Ferment. Technol. 64. (1986) 363- 371. Results are shown in Table 2.

The improvement achieved by the introduction of multiple copies of the lipase gene expression cassette is 20 fold compared with the level of lipase produced by the parent strain, Pseudomonas pseudoalcaligenes Ml.

However the expression level can be further improved by cloning the lipase modulator gene in the expression cassette as well.

From these data it can be concluded that the lipase modulator gene is not necessary in a 1:1 ratio but does become a limiting factor when lipase expression is increased over 20-fold.

Although the lipase modulator gene is necessary for lipase production the chromosomal copy of the gene is sufficient to allow an increase of 2000%. Only at this point introduction of additional modulation gene copies will result in higher lipase production.

It is unknown yet what the exact function of this gene might be, but our data suggest a chaperone like function, whereas only very low levels of the gene product seem to be sufficient for the secretion of large amounts of lipase.

Table 2

The growth rate of these strains is decreased probably due to a pleiotropic mutation Example 4

Homology comparison of the lipase gene and lipase modulator gene with other lipase and lipase modulator genes Lipase and lipase modulator gene sequences were compared using computer analysis. In order to determine homology with uncharacterized lipase genes, we used a mole¬ cular enzyme screening assay, as described in EP 334462. From this work it was concluded that Pseudomonas species belonging to the same RNA homology group as P. pseudoalcaligenes show a rather strong homology, whereas Pseudomonas species belonging to a different RNA homology group (Palleroni, 1973) and other bacterial species show no hybridization at all. A correlation between both methods was well established, which makes it possible to determine homology with uncharacterized lipase genes.

Sequence comparison of the lipase gene

The sequences of several Pseudomonas lipase genes have been published. Computer analysis of these sequences compared with the Pseudomonas pseudoalcaligenes Ml reveals an identity of 81% for P. aeruginosa (EP 334462), also 81% for a Pseudomonas species (Ihara et al. J. Biol. Chem. 266 (1991) 18135-18140), 56% for P. fragi (Aoya a et al. , FEBS Lett. 242 (1988) 36-40, Kugimiya et al. , Biochem. Biophys. Res. Commun. 141 (1986) 185-190) , all three probably belonging to RNA homology group I, 52% for P. cepacia (Jorgensen et al. , J. Bacteriol. 173. (1991) 559-567) and 59% for P. glu ae (PCT 91/00910) , both belonging to RNA homology group II (Table III) and no homology at all with the lipase gene from S. hvicus (Gόtz et al. , NAR H (1985) 5895-5906).

These data are consistent with the hybridization data, described in EP 334462, where no hybridization can be found for P. fragi , P. cepacia (not shown) , P. glumae (or P. gladioli) , S. hyicus DNA, whereas a proper hybridization signal is obtained from P. aeruginosa DNA. Results are shown in Table 3. Seguence comparison of the lipase modulator gene

Lipase modulator genes have only been described for Pseudomonas species belonging to RNA homology group II. The lipase modulator gene for P. cepacia (both in EP 331376 and by Jorgensen et al. , J. Bacteriol. 173 (1991) 559-567) and the lipase modulator gene of P. glumae (PCT 91/00910) was described. Recently the lipase modulator gene sequence was described (Ihara et al. , J. Fer . Bioeng. 7_3 (1992) 337-342) of a Pseudomonas species, which might belong to Pseudomonas RNA homology group I, based on sequence homology. Results are shown in Table 4.

Table 3

Table 3 percentage identity of different Pseudomonas lipases, The lower part of the table shows a nucleic acid sequence comparison. The upper part shows an amino acid sequence comparison. - 20 -

Table 4

Table 4 percentage identity between different Pseudomonas lipase modulator genes. The lower part of the table shows a nucleic acid sequence comparison. The upper part shows an amino acid sequence comparison. nd => for these species no lipase modulator gene was described.

From these data it can be concluded that the lipase modulator genes seem to be less conserved than the lipase genes itself.

It was established that the degree of homology which was found could even be coincidental. An amino acid homology of 25% was found when the lipase sequence of P. pseudoalcali- genes Ml was compared to the lipase modulator sequence of P. pseudoalcaligenes Ml, indicating that the observed sequence homology between lipase modulator genes belonging to different RNA homology groups is rather low.

Furthermore Ihara et aJL. seem to be the first describing the necessity of a modulator gene for Pseudomonas species derived from RNA homology group I in heterologous organisms. For both P. fragi and P. pseudoalcaligenes Ml lipase expression in 12. coli didn't seem to depend upon the presence of such a modulating gene.

Based on the very low levels of homology it is rather surprising to be dealing with a gene with a comparable function.

Although the modulator gene, derived from P. pseudoalcaligenes Ml, does not seem to be necessary for gene expression in E. coli. it is necessary for levels of lipase gene expression in P. pseudoalcaligenes Ml above 20-fold. This has not been observed before.

SEQUENCE LISTING

(1) INFORMATION FOR SEQ ID NO:_.l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2417 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Pseudomonas pseudoalcaligenes

(B) STRAIN : Ml

(C) INDIVIDUAL ISOLATE : CBS 473 . 85

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 270..1211

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 270..341

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 342..1208

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /codon_Start= 271

/function= "triglyceride hydrolysis" /product= "lipase Ml" /evidence= EXPERIMENTAL /gene*= "lip"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1264..2298

(D) OTHER INFORMATION: /codon_εtart= 1264 /function****- "Lipase modulator" /gene= "lim" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GTACCCCTGG CTGGCAGGCG GCAGCCAGGC CCCACAGGGG GAGTCGAGAA ACGCTCCTGT 60

TCCCCTCGGT AACATCCCCT AGGTAATAGC AGAGCCCTTG CCGGCGCTGG CTTTCGTCAC 120

AAACGCCCGT TTAGAGCCTT TGTTCTAATC CACCCCGTTC CTGGCACAGA TCCTGCCCCA 180

CCGAGCCTGC TGAAGTACCG GCCCGGGAAG CGCCGGATGG CTGGATGCAA GGATGGATCA 240

GTGCCCAACC CTTCGCTCGA GAGCAAAAC ATG AAT AAC AAG AAA ACC CTG CTC 293

Met Asn Asn Lys Lγs T r Leu Leu -24 -20

GCC CTC TGC ATC GGC AGC AGT CTG CTG CTG TCC GGC CCA GCC GAA GCC 341 Ala Leu Cys lie Glγ Ser Ser Leu Leu Leu Ser Gly Pro Ala Glu Ala -15 -10 -5

GGC CTG TTC GGC TCζ,ACC GGC TAC ACC AAG ACC AAG TAC CCG ATC GTC 389 Gly Leu Phe Gly Ser Thr Gly Tyr Thr Lys Thr Lys Tyr Pro He Val 1 5 10 15

CTG ACC CAC GGC ATG CTC GGC TTC GAC AGC ATC CTC GGC GTC GAC TAC 437 Leu Thr His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val Asp Tyr 20 25 30

TGG TAC GGC ATC CCG TCC TCG CTG CGC TCC GAC GGC GCC AGC GTC TAC 485 Trp Tyr Gly He Pro Ser Ser Leu Arg Ser Asp Gly Ala Ser Val Tyr 35 40 45

ATC ACC GAA GTC AGC CAG CTC AAC ACC TCC GAG CTG CGC GGC GAG GAG 533 He Thr Glu Val Ser Gin Leu Asn Thr Ser Glu Leu Arg Gly Glu Glu 50 55 60

CTG CTG GAG CAG GTG GAA GAG ATC GCC GCC ATC AGC GGC AAG GGC AAG 581 Leu Leu Glu Gin Val Glu Glu He Ala Ala He Ser Gly Lys Gly Lys 65 70 75 80

GTC AAC CTG GTC GGC CAC AGC CAT GGC GGC CCG ACC GTC CGC TAC GTG 629 Val Asn Leu Val Gly His Ser His Gly Gly Pro Thr Val Arg Tyr Val 85 90 95

GCC GCC GTA CGC CCG GAC CTG GTG GCC TCG GTG ACC AGC GTC GGC GCC 677 Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala 100 105 110

CCG CAC AAG GGC TCG GAC ACC GCC GAC TTC ATC CGC CAG ATC CCC CCG 725 Pro His Lys Gly Ser Asp Thr Ala Asp Phe He Arg Gin He Pro Pro 115 120 125

GGC TCG GCC GGT GAG GCG ATA GTC GCC GGC ATC GTC AAC GGC CTG GGC 773 Gly Ser Ala Gly Glu Ala He Val Ala Gly He Val Asn Gly Leu Gly 130 135 140 GCG CTG ATC AAC TTC CTC TCC GGC AGC TCC AGC ACC AGC CCG CAG AAC 821 Ala Leu He Asn Phe Leu Ser Gly Ser Ser Ser Thr Ser Pro Gin Asn 145 150 155 160

GCC CTG GGC GCC CTC GAA TCG CTC AAC AGT GAG GGC GCC GCC GCC TTC 869 Ala Leu Gly Ala* Leu Glu Ser Leu Asn Ser Glu Gly Ala Ala Ala Phe 165 170 175

AAC GCC AAG TAT CCG CAG GGC ATT CCG ACC AGT GCC TGC GGC GAA GGC 917 Asn Ala Lys Tyr Pro Gin Gly He Pro Thr Ser Ala Cys Gly Glu Gly 180 185 190

GCC TAC AAG GTC AAT GGC GTC AGC TAC TAC TCC TGG AGC GGC ACC AGC 965 Ala Tyr Lys Val Asn Gly Val Ser Tyr Tyr Ser Trp Ser Gly Thr Ser 195 200 205

CCG CTG ACC AAT GTG CTC GAC GTC AGC GAC CTG CTG CTG GGC GCC AGC 1013 Pro Leu Thr A∑n Val Leu Asp Val Ser Asp Leu Leu Leu Gly Ala Ser 210 " ' 215 220

TCG CTG ACC TTC GAC GAG CCC AAC GAC GGC CTG GTC GGG CGC TGC AGC 1061 Ser Leu Thr Phe Asp Glu Pro Asn Asp Gly Leu Val Gly Arg Cys Ser 225 230 225 240

TCG CAC CTG GGC AAG GTG ATC CGC GAC GAC TAC CGG ATG AAC CAC CTC 1109 Ser His Leu Gly Lys Val He Arg Asp Asp Tyr Arg Met Asn His Leu 245 250 255

GAC GAG GTC AAC CAG ACC TTC GGC CTG ACC AGC CTG TTC GAG ACC GAC 1157 Asp Glu Val Asn Gin Thr Phe Gly Leu Thr Ser Leu Phe Glu Thr Asp 260 265 270

CCG GTC ACC GTG TAC CGC CAG CAG GCC AAC CGC CTC AAA CTG GCC GGC 1205 Pro Val Thr Val Tyr Arg Gin Gin Ala Asn Arg Leu Lys Leu Ala Gly 275 280 285

CTC TGAGCCATGG ATCGGGGCCC ACGGGCCCCG ATGTTTTCCC CCGCCGAGTC TCGCC 1263 Leu

290

GTG AAC AAA GCC CTG CTT CTG GCC GTA CCC CTG CTG ATC GGG GCC GGC 1311 Met Asn Lys Ala Leu Leu Leu Ala Val Pro Leu Leu He Gly Ala Gly 1 5 10 15

ATC GCC GTC ACC CTC GCC CTC AAC CCA CTG ACT CCA GCA CCC AGC CCA 1359 He Ala Val Thr Leu Ala Leu Asn Pro Leu Thr Pro Ala Pro Ser Pro 20 25 30

GCG GCG CTA TCG ACT GCG CCT GGC GTA CCG CTG CCG TCG CCA GCG GTG 1407 Ala Ala Leu Ser Thr Ala Pro Gly Val Pro Leu Pro Ser Pro Ala Val 35 40 45 - 25 -

CAG CGA ACC CTC GAC GAC GCA CCT GCA GCA CCG CCC CTG GCT GCC GAA 1455 Gin Arg Thr Leu Asp Asp Ala Pro Ala Ala Pro Pro Leu Ala Ala Glu 50 55 60

ATC GCG CCC CTG CCA CCC TCC TTC GCC GGA ACC CAG GTG GAT GGC CAG 1503

He Ala Pro Leu Pro Pro Ser Phe Ala Gly Thr Gin Val Asp Gly Gin

65 70 75 80

TTC CGC CTC GAT GCG GCA GGC AAC CTG CTG ATC GAA CGG GAT ATC CGG 1551 Phe Arg Leu Asp Ala Ala Gly Asn Leu Leu He Glu Arg Asp He Arg 85 90 95

CGC ATC TTC GAC TAC TTC CTC AGC GCC TAT GGC GAG GAC AGC CTC AAG 1599

Arg He Phe Asp Tyr Phe Leu Ser Ala Tyr Gly Glu Asp Ser Leu Lys 100 105 110

GCC ACC ATC GAG CGT CTG CAG GCC TAT GTC CGC AGC CAG CTC GAC GAG 1647

Ala Thr He Glu Arg Leu Gin Ala Tyr Val Arg Ser Gin Leu ^f-,**;p Glu 115 -*-"' 120 125

CCG GCC GAA AGC CAG GCC CTG GCG CTG CTG GAG CAG TAC CTG GAG TAC 1695 Pro Ala Glu Ser Gin Ala Leu Ala Leu Leu Glu Gin Tyr Leu Glu Tyr 130 135 140

AAG CGC CAA CTG GTG CAA CTG GAG AAG GAC CTG CCG CAG ATG GCC AGC 1743 Lys Arg Gin Leu Val Gin Leu Glu Lys Asp Leu Pro Gin Met Ala Ser 145 150 155 160

CTG GAT GCC CTG CGT CAG CGC GAG CAG GCG GTG CAG AAC CTG CGT GCC 1791 Leu Asp Ala Leu Arg Gin Arg Glu Gin Ala Val Gin Asn Leu Arg Ala 165 170 175

AGC CTG TTC AGC GTC GAA GCG CAC CAG GCC TTC TTC GCC GAG GAA GAG 1839 Ser Leu Phe Ser Val Glu Ala His Gin Ala Phe Phe Ala Glu Glu Glu 180 185 190

GCC TAC AAC GGC TTC ACC CTG CAG CGC CTG GCG ATC CGT CAC GAC CAG 1887 Ala Tyr Asn Gly Phe Thr Leu Gin Arg Leu Ala He Arg His Asp Gin 195 200 205

ACG CTG GAC GAC CAG CAG AAG GCC GAG GCG CTC GAC CGC CTG CGT GCC 1935 Thr Leu Asp Asp Gin Gin Lys Ala Glu Ala Leu Asp Arg Leu Arg Ala 210 215 220

AGC CTG CCG GAA GAG CTA CAG GCA TTG CTG GCC CCG CAG CTG CAG GCC 1983 Ser Leu Pro Glu Glu Leu Gin Ala Leu Leu Ala Pro Gin Leu Gin Ala 225 230 235 240

GAG CTG CGC CAG CAG ACC GCA GCC CTG CAG GCC CAG GGC GCC AGT GCC 2031 Glu Leu Arg Gin Gin Thr Ala Ala Leu Gin Ala Gin Gly Ala Ser Ala 245 250 255 - 26 -

GCA CAG ATC CAG CAG CTG CGC CTG CAA CTG GTC GGC GCC GAG GCC ACC 2079 Ala Gin He Gin Gin Leu Arg Leu Gin Leu Val Gly Ala Glu Ala Thr 260 265 270

GCA CGC CTG GAA GCG CTG GAC CAG CAG CGC CAG CAG TGG CGC CAG CGC . 2127 Ala Arg Leu Glu Ala Leu Asp Gin Gin Arg Gin Gin Trp Arg Gin Arg 275 280 285

CTC GCC GAC TAC CGT CGG GAA AAG GCC AGG GTG CTG GCC AAC GAC GGC 2175 Leu Ala Asp Tyr Arg Arg Glu Lys Ala Arg Val Leu Ala Asn Asp Gly 290 295 300

CTG AGC GAA AGT GAC AAG CAG GCA GCA ATT GCC GAA CTG GCC GCG CAG 2223 Leu Ser Glu Ser Asp Lys Gin Ala Ala He Ala Glu Leu Ala Ala Gin 305 310 315 320

CGC TTC GAC GAC AAC^SAG CGC CTG CGC CTG GAA GCG GCC GAA CAG CTG 2271 Arg Phe Asp Asp-Ash Glu Arg Leu Arg Leu Glu Ala Ala Glu Gin Leu 325 330 335

GCG CAG AGC CGG GAG GAG AAA CCC TGAATGCAAA AAGGCCCGCT TTCGCGGGCC 2325 Ala Gin Ser Arg Glu Glu Lys Pro

340 345

TTTTCTCGGT GCAACCGTCT CAGCCGGCGA TGCGGCGATC CAGCGACAGC TTGCCGGCGC 2385

CCTCGACCAT CAGCGCCGCG CTGATCCCCG GG 2417

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 313 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Asn Asn Lys Lys Thr Leu Leu Ala Leu Cys lie Gly Ser Ser Leu -24 -20 -15 -10

Leu Leu Ser Gly Pro Ala Glu Ala Gly Leu Phe Gly Ser Thr Gly Tyr -5 1 5

Thr Lys Thr Lys Tyr Pro lie Val Leu Thr His Gly Met Leu Gly Phe 10 15 20

Asp Ser lie Leu Gly Val Asp Tyr Trp Tyr Gly lie Pro Ser Ser Leu 25 30 35 40

Arg Ser Asp Gly Ala Ser Val Tyr lie Thr Glu Val Ser Gin Leu Asn

45 50 55

Thr Ser Glu Leu Arg Gly Glu Glu Leu Leu Glu Gin Val Glu Glu lie 60 65 70

Ala Ala lie Ser Gly Lys Gly Lys Val Asn Leu Val Gly His Ser His 75 80 85

Gly Gly Pro Thr Val Arg Tyr Val Ala Ala Val Arg Pro Asp Leu Val 90 95 100

Ala Ser Val Thr Ser Val Gly Ala Pro His Lys Gly Ser ASD Thr Ala 105 110 115 ^* 120

Asp Phe lie Arg Gin lie Pro Pro Gly Ser Ala Gly Glu Ala lie Val

125 130 135

Ala Gly lie Val Asn Gly Leu Gly Ala Leu lie Asn Phe Leu Ser Gly 140 145 150

Ser Ser Ser Thr Ser Pro Gin Asn Ala Leu Gly Ala Leu Glu Ser Leu 155 160 165

Asn Ser Glu Gly Ala Ala Ala Phe Asn Ala Lys Tyr Pro Gin Gly lie 170 175 180

Pro Thr Ser Ala Cys Gly Glu Gly Ala Tyr Lvs Val Asn Gly Val Ser 185 190 195 200

Tyr Tyr Ser Trp Ser Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Val

205 210 215

Ser Asp Leu Leu Leu Gly Ala Ser Ser Leu Thr Phe Asp Glu Pro Asn 220 225 230 Asp Gly Leu Val Gly Arg Cys Ser Ser His Leu Gly Lys Val lie Arg 235 240 245

Asp Asp Tyr Arg Met Asn His Leu Asp Glu Val Asn Gin Thr Phe Gly 250 255 . 260

Leu Thr Ser Leu Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin Gin 265 270 275 280

Ala Asn Arg Leu Lys Leu Ala Gly Leu

285

INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 344 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Asn Lys Ala Leu Leu Leu Ala Val Pro Leu Leu lie Gly Ala Gly 1 5 10 15 lie Ala Val Thr Leu Ala Leu Asn Pro Leu Thr Pro Ala Pro Ser Pro 20 25 30

Ala Ala Leu Ser Thr Ala Pro Gly Val Pro Leu Pro Ser Pro Ala Val 35 40 45

Gin Arg Thr Leu Asp Asp Ala Pro Ala Ala Pro Pro Leu Ala Ala Glu 50 55 60 lie Ala Pro Leu Pro Pro Ser Phe Ala Gly Thr Gin Val Asp Gly Gin 65 70 75 80

Phe Arg Leu Asp Ala Ala Gly Asn Leu Leu lie Glu Arg Asp lie Arg

85 90 95

Arg lie Phe Asp Tyr Phe Leu Ser Ala Tyr Gly Glu Asp Ser Leu Lys 100 105 110

Ala Thr lie Glu Arg Leu Gin Ala Tyr Val Arg Ser Gin Leu Asp Glu 115 120 125

Pro Ala Glu Ser Gin Ala Leu Ala Leu Leu Glu Gin Tyr Leu Glu Tyr 130 135 140

Lys Arg Gin Leu Val Gin Leu Glu Lys Asp Leu Pro Gin Met Ala Ser 145 150 155 160

Leu Asp Ala Leu Arg Gin Arg Glu Gin Ala Val Gin Asn Leu Arg Ala

165 170 175

Claims

1. A method for obtaining lipases comprising:

- cloning of a lipase gene and a lipase modulator gene obtained from a class I Pseudomonas species in a homologous Pseudomonas species,

- culturing of the recombinant strain under conditions wherein the lipase gene is expressed,

- isolating the lipase from the culture.

2. The method according to claim 1, wherein the class I Pseudomonas species is selected from the group consisting of: Pseudomonas alcaligenes. Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri. Pseudomonas aeruginosa. and Pseudomonas mendocina. or mutants thereof.

3. The method according to claim 1, wherein the class I Pseudomonas species is Pseudomonas pseudoalcaligenes or mutants thereof.

4. The method of any one of the previous claims wherein the class I Pseudomonas species host strain is lipase and/or lipase modulator deficient.

5. A method for increasing the lipase production of class I Pseudomonas strains comprising the cloning and expression of both a class I lipase and lipase modulator gene in the host cell.

6. A class I Pseudomonas strain transformed with a lipase and a lipase modulator gene obtained from the homologous strain.

7. A Pseudomonas strain transformed with a vector containing a lipase gene wherein said strain is characterized in that the amount of lipase produced is at least 15-fold higher than in the untransformed strain.

8. A Pseudomonas strain according to claim 7 wherein the strain is additionally transformed with a lipase modulator gene.

9. A Pseudomonas strain according to claim 7 or 8 selected from the class I Pseudomonas and wherein the strain is transformed with a homologous lipase and/or lipase modulator gene.

10. A derivative of pJRD215 which is segregationally stable in Pseudomonas.

11. A method for obtaining a segregationally stable derivative of pJRD215 comprising:

- repeated dilution of the transformed Pseudomonas strain in medium without antibiotics

- followed by incubation periods in the presence of antibiotics.