FIELD OF INVENTION

The present invention relates to a DNA sequence encoding a lipase, a DNA sequence the product of which affects the pro- duction of the lipase, expression vectors and host cells com¬ prising these sequences, and a method of producing lipase by cultivating the host cells.

BACKGROUND OF THE INVENTION

Lipases are enzymes which catalyze the hydrolysis of ester bonds in triglycerides resulting in the formation of digly- cerides, monoglycerides, glycerine and free fatty acids. Some lipases also catalyze other reactions involving ester bonds such as synthesis of ester bonds or transesterification reac¬ tions. Lipases are produced by a wide variety of different organisms. Microbial lipases in particular are of con¬ siderable practical utility for a variety of purposes where lipolysis of fats is desired, e.g. in the food industry and in detergents.

One particular lipase which has been found to be particularly advantageous for inclusion in a detergent for the removal of fatty stains or soiling from fabrics is a lipase produced by strains of Pseudomonas cepacia. In EP 214 761 (to Novo Indu- stri A/S) , this lipase is disclosed as a lipase which is ac¬ tive at a temperature below 60°C, which is important as most present-day fabrics are washed at temperatures below 60°C.

Another important Pseudomonas cepacia lipase for use as a detergent additive is the one disclosed in WO 89/01032 (to Novo Industri A/S) as a positionally non-specific lipase, i.e. one which is able to react with all three fatty acyl groups of a triglyceride.

In order to facilitate Pseudomonas cepacia lipase production, it may be advantageous to employ recombinant DNA techniques, for instance in order to optimize lipase expression by intro¬ ducing a stronger promoter from which the DNA sequence en- coding the enzyme is expressed or by introducing more effici¬ ent riboso e binding sites or signal peptide coding se¬ quences, or in order to select a host organism for the pro¬ duction of the enzyme which is easier to cultivate (e.g. in terms of its being a standard production organism such as E.coli. B. subtilis or the like) or which results in higher lipase yields. As described below, such approaches will some¬ times fail to yield the expected results, e.g. in cases where one or more genes in addition to the structural gene coding for the protein in question, play some part in the production of the gene product (examples of such genes are the Bacillus sac (Honjo, M. , et al. (1987), Journal of Biotechnology, 6:191-204) and iep (Tanaka, T., Kawata, M. (1988), Journal of Bacteriology, 170:3593-3600) genes, and genes required for the production of Klebsiella pullulanase and E.coli hemoly- sin) .

The cloning of a lipase gene from another Pseudomonas species, Pseudomonas fracri _f is known from, e.g., S. Aoyama et al., FEBS Letters 242(1) . December 1988, pp. 36-40, and W. Kugimiya et al., Biochem. Biophvs. Res. Comm. 141(1) , Novem- ber 26, 1986, pp. 185-190. However, the lipase produced by P.fraαi differs from that of P.cepacia in its amino acid se¬ quence, and in these publications, there is no indication that one or more additional genes may be required in order to achieve a significant lipase production in a host organism.

EP 331 376 discloses a recombinant DNA encoding a Pseudomonas cepacia lipase as well as a protein participating in the pro¬ duction of the lipase. There is, however, no indication that the gene encoding this protein may also be functional in a heterologous host organism.

SUMMARY OF THE INVENTION

The present inventor has isolated and cloned a gene encoding a Pseudomonas cepacia lipase and identified a gene the ex¬ pression of which is important to achieve production of the lipase in significant yields.

Accordingly, in a first aspect, the present invention relates to a DNA construct which comprises a DNA sequence encoding a factor which acts in trans as a modulator of the production of a Pseudomonas cepacia lipase, or encoding a functional analogue of the ______ cepacia lipase modulating factor, the P. cepacia lipase modulating factor or functional analogue thereof being capable of modulating lipase production when the £i cepacia lipase is expressed using expression signals which are heterologous to Pseudomonas and/or when the I ^" \_ cepacia lipase is expressed in a heterologous host cell.

In the course of the research leading to the present inven¬ tion, it was surprisingly found that a region downstream of the DNA sequence coding for the lipase (as present on the chromosome of P___ cepacia) has a pronounced beneficial effect on the production of the lipase. It was experimentally estab¬ lished (among other things, by inserting this region on a another plasmid than that carrying the DNA sequence encoding the lipase) that the region does not merely provide a site on the DNA sequence (such as a promoter, terminator, enhancer, or the like) which is required for lipase expression, but that the region includes a gene encoding a factor which is able to affect the production of the lipase even when the DNA sequences coding for the two products are not located on the same vector. The lipase modulating factor may be an RNA mole- cule or a polypeptide, but is most likely a polypeptide (cf. Example 3 below) .

In- the following, the DNA sequence encoding the lipase modu¬ lating factor will usually be referred to as the "lim" gene,

while the modulating factor will usually be referred to as the "Lim" factor.

It has surprisingly been found that expression of the lim gene exerts a beneficial effect on lipase production even when the lipase is expressed using expression signals which are heterologous to those of Pseudomonas (e.g. a promoter, ribosome binding site and signal peptide-coding region from a Bacillus α-amylase gene) or when the host organism is not Pseudomonas. Therefore, the Lim factor does not act on the initiation of either transcription or translation. However, the precise function of the Lim factor has yet to be eluci¬ dated.

The term "functional analogue" is understood to indicate a factor which has a similar lipase-production modulating func- tion to that of the P.cepacia Lim factor and which is derived from another organism than P.cepacia. or which is a deriva¬ tive of the P.cepacia Lim factor produced by modifying the DNA sequence in a manner resulting in addition, substitution, insertion or deletion of one or more amino acids in the na- tive sequence. There is reason to believe, however, that such modifications of the native sequence should not be too exten¬ sive as the modulating function of the Lim factor may other¬ wise be lost. Apart from this, a functional analogue may be a homologous polypeptide (i.e. one encoded by DNA which hybri- dizes to the same probe as the DNA coding for the Lim factor under certain specified conditions [e.g. presoaking in 5xSSC and prehybridizing for lh at ~40°C in a solution of 20% for- mamide, δxDenhardt's solution, 50mM sodium phosphate, pH 6.8, and 50μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with lOOμM ATP for 18h at -40° ^' C]) , or which is reactive with antibodies raised or directed against the Lim factor. When the term "Lim factor" is used in the following description, this is in¬ tended implicitly to include such functional analogues as well as the native P.cepacia lipase modulating factor.

DETAILED DISCLOSURE OF THE INVENTION

The DNA construct of the invention comprising the DNA se¬ quence encoding the Lim factor may be of genomic or cDNA ori¬ gin, for instance obtained by preparing a genomic or cDNA library of P.cepacia and screening for DNA sequences coding for all or part of the Lim factor by hybridization using syn¬ thetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Clonincr: A Labora¬ tory Manual. Cold Spring Harbor, 1982) .

The DNA sequence encoding the Lim factor may be any one ob¬ tained from a lipase-producing P.cepacia strain. Examples of such strains are the ones deposited in the Deutsche Sammlung von Mikroorganismen in connection with the invention dis¬ closed in EP 214 761, with the deposit numbers DSM 3333-3337 and DSM 3401, as well as the strain deposited in the Deutsche Sammlung von Mikroorganismen in connection with the invention disclosed in WO 89/01032, with the deposit number DSM 3959. It is, however, envisaged that a DNA sequence equivalent to the lim gene may also be derived from another lipase-pro- ducing organism. Such a gene may be screened for by hybridi¬ zation using probes that hybridize to DNA encoding the Lim factor as described above or selected by the reactivity of its gene product with the same antibodies as the Lim factor or identified by screening for the ability to confer lipase production to strains containing the lipase gene but not the lim gene.

The DNA construct of the invention encoding the Lim factor may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22. 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805. According to the phospho¬ amidite method, oligonucleotides are synthesized, e.g. in an

automatic DNA synthesizer, purified, ligated, and cloned in an appropriate vector.

Finally, the DNA construct may be of mixed synthetic and ge¬ nomic, mixed synthetic and cDNA or mixed genomic and cDNA 5 origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire DNA construct, in accordance with standard techniques.

Preferred DNA constructs encoding the Lim factor are those 10 comprising a DNA sequence derived from the chromosome of P.cepacia. in particular those wherein the DNA sequence is located on the P _^ . cepacia chromosome downstream of the se¬ quence encoding a -33 kD lipase. A particularly preferred DNA construct encoding the Lim factor is one which has the DNA 15 sequence shown in Fig. 1 A-C appended hereto or a modifica¬ tion thereof encoding a functional analogue of the ______ cepacia

Lim factor as defined above.

Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another

20 sequence of the Lim factor, but which may correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different sequence and therefore, possibly, a different structure without, however, impairing the properties of

25 either the lipase or the Lim factor. Other examples of pos¬ sible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence and deletion of one or more nucle¬ otides at either end of or within the sequence.

30 For some purposes, it may be convenient to provide a DNA se¬ quence encoding a Pseudomonas cepacia lipase on the same DNA construct as the DNA sequence encoding the Lim factor. Thus, the present invention further relates to a DNA construct

which comprises a first DNA sequence encoding a _9___ cepacia lipase or a derivative thereof and a second DNA sequence en¬ coding the ______ cepacia lipase modulating factor.

In the present context, the term "derivative" is intended to indicate a protein with lipolytic activity which is derived from the native lipase by suitably modifying the DNA sequence coding for the native lipase, resulting in the addition of one or more amino acids to either or both the C- and N-termi- nal end of the the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or more sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence. Such modifications of DNA coding for native proteins are well known and widely practised in the art.

Preferred DNA constructs encoding the P^ cepacia lipase are those comprising a DNA sequence derived from ______ cepacia. DSM

3959, encoding a lipase of -33 kD which in nature is present on the chromosome of ______ cepacia. A particularly preferred DNA construct encoding the lipase is one wherein the DNA sequence encoding the lipase is the one shown in Fig. 2 A-C appended hereto or a modification thereof encoding a derivative of the P. cepacia lipase as defined above.

In one embodiment of the DNA construct of the invention, the lipase DNA sequence may be located upstream of the sequence encoding the Lim factor, and in another embodiment, the DNA sequence encoding the lipase may be located downstream of the sequence encoding the Lim factor. In either case, the DNA sequences may be located relative to each other in such a way that they are transcribed from the same promoter under condi¬ tions permitting expression of the lipase and Lim factor when the DNA construct is present in a host cell. In a particular embodiment, the first DNA sequence encoding the lipase and/or

the second DNA sequence encoding the Lim factor may further comprise expression signals (e.g. a promoter, ribosome bind¬ ing site and/or signal peptide-coding sequence) heterologous to those of Pseudomonas.

In a further aspect, the present invention relates to a re- plicable expression vector which carries an inserted DNA se¬ quence encoding the Lim factor or a functional analogue thereof, as described above.

In a still further embodiment, the invention relates to a replicable expression vector which carries a first inserted DNA sequence encoding the P _^ _ cepacia lipase or a derivative thereof (as described above) and a second DNA sequence en¬ coding the Lim factor (as described above) . In this case, the first and second DNA sequences may be expressed from the same promoter although this need not be required. When the first and second DNA sequences are expressed from the same promo¬ ter, the lipase-encoding sequence may be located downstream or upstream of the Lim-encoding sequence without adversely affecting the modulation of lipase production effected by the Lim factor.

Alternatively, the DNA sequences encoding the lipase and the Lim-polypeptide, respectively, may each be expressed from a separate promoter, even while carried on the same vector.

In a particular embodiment, the first DNA sequence encoding the lipase and/or the second DNA sequence encoding the Lim factor may further comprise expression signals (e.g. a pro¬ moter, ribosOme binding site and/or signal peptide-coding sequence) heterologous to those of Pseudomonas.

The replicable expression vector carrying a DNA sequence en- coding the lipase and/or Lim factor may be any vector which is capable of replicating autonomously in a given host orga¬ nism, typically a plasmid or bacteriophage. In the vector,

the DNA sequence encoding the lipase and/or Lim factor should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes en- coding proteins either homologous or heterologous to the host organism, e.g. a promoter which is heterologous to Pseudomo¬ nas. The promoter is preferably derived from a gene encoding a protein homologous to the host organism. Examples of suit¬ able promoters are lac of E.coli. dagA of Streptomyces coeli- color and amyL of Bacillus licheniformis.

The replicable expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance, such as ampicil- lin, kanamycin, chloramphenicol or tetracyclin resistance, or the dal genes from B.subtilis or B.licheniformis.

The procedures used to ligate the DNA sequences coding for the lipase and/or the Lim factor and the promoter, respec¬ tively, and to insert them into suitable vectors containing the information necessary for replication in the host cell, are well known to persons skilled in the art (cf. , for in- stance, Sambrook et al., op.cit.) . It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence coding for the lipase and for the Lim factor and subsequently inserting this construct into a suitable expression vector, or by preparing separate DNA constructs comprising DNA sequences encoding the lipase or the Lim factor, respectively, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the lipase or Lim factor) followed by ligation after each step. It should further be understood that, in this connection, the DNA sequence en-

coding the lipase may be of genomic origin, while the se¬ quence encoding the Lim factor may be prepared synthetically or vice versa, as described above.

In a still further aspect, the present invention relates to a host cell which contains either two separate DNA constructs comprising a DNA sequence encoding the __ _ cepacia lipase and a DNA sequence encoding the Lim factor as defined above, re¬ spectively, or a DNA construct comprising both DNA sequences on the same fragment; in either case, the DNA constructs may be integrated in the host chromosome which may be an ad¬ vantage as the DNA sequences are more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to con¬ ventional methods, e.g. by homologous recombination. The two DNA sequences may be integrated in the host chromosome in such a way that they are each expressed from a separate pro¬ moter. In a particular embodiment, the DNA sequence encoding the lipase and/or the DNA sequence encoding the Lim factor may further comprise expression signals (e.g. a promoter, ribosome binding site and/or signal peptide-coding sequence) heterologous to those of Pseudomonas.

Alternatively, the host cell may be transformed with a re¬ plicable expression vector which contains both DNA sequences, or with two vectors containing the DNA sequence encoding the lipase and the DNA sequence encoding the Lim factor, respec¬ tively.

As a further alternative, the DNA sequence encoding the li¬ pase may be integrated in the host chromosome, and the DNA sequence encoding the Lim factor may be carried on a replic- able expression vector (as described above) , or vice versa.

The host cell used in the process of the invention may be any suitable bacterium, yeast or filamentous fungus which, on cultivation, produces large amounts of the P___ cepacia lipase.

Examples of suitable bacteria may be grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis, Bacillus stearothermophilus, Bacil¬ lus alkalophilus. Bacillus amylolicruefaciens. Bacillus coagu- lans. Bacillus circulans, or Streptomyces lividans or Strep- tomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favourably be selected from a species of Saccharomyces, e.g. Saccharomvces cerevisiae. The filamen¬ tous fungus may advantageously belong to a species of Asper¬ qillus, e.g. Asperqillus oryzae or Asperqillus ni er. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Asperqillus as a host organism is described in, e.g., EP 238 023.

In a yet further aspect, the present invention relates to a method of producing the _9___ cepacia lipase or a derivative thereof, which method comprises cultivating a host cell as described above under conditions conducive to the production of the lipase or analogue thereof (including conditions en¬ suring the expression of the Lim factor) , and recovering the lipase or analogue from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventio¬ nal medium suitable for growing bacteria. The lipase may be recovered from the medium by conventional procedures includ¬ ing separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells, pre¬ cipitating the proteinaceout components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures,

e.g. ion exchange chromatography, affinity chromatography, or the like.

The invention is further illustrated by the following examples which are not in any way intended to limit the scope of the invention, with reference to the appended drawings, wherein

Fig. 1 A-C shows the DNA sequence of the ______ cepacia lim gene, the derived amino acid sequence being shown below in the con¬ ventional three-letter code,

Fig. 2 A-C shows the DNA sequence encoding the ~33kD ______ _____ epacia lipase, the derived amino acid sequence being shown below in the conventional three-letter code.

Fig. 3 schematically shows the construction of plasmid pSJ518,

Fig. 4 schematically shows the construction of plasmid pSJ910,

Fig. 5 schematically shows the construction of plasmid pSJ485,

Fig. 6 A-B schematically shows the construction of plasmids pSJ622 and pSJ624,

Fig. 7 schematically shows the construction of plasmid PSJ424,

Fig. 8 schematically shows the construction of plasmids pSJ494 and 909,

Fig. 9 schematically shows the construction of plasmid pSJ729,

Fig. 10 schematically shows the construction of plasmid pSJ416,

Fig. 11 schematically shows the construction of plasmid pSJ600,

Fig. 12 shematically shows the construction of plasmid pSJ671, and

Fig. 13 A-B schematically shows the construction of plasmid pSJ669.

MATERIALS AND METHODS

Bacterial strains;

E. coli HW1 is a lacl ^q , z M15 derivative of strain 803 de¬ scribed in Wood, 1966.

E. coli NM539 is described in Frischauf et al., 1983, and was obtained from Promega.

Pseudomonas cepacia SB10, DSM 3959, is described in WO 89/01032.

Bacillus subtilis DN1885 is a a vE. amyR2. Spo ⁺ , Pro ⁺ deriva¬ tive of B. subtilis 168.

Bacillus licheniformis ATCC 9789.

Streptomvces lividans TK24 is described in Hopwood et al., 1983.

Phaqes :

Lambda EMBL4 is described in Frischauf et al., 1983. It was obtained from Promega.

Plasmids;

pUClδ and pUC19 are described in Yanisch-Perron et al., 1985.

pACYC177 is described in Chang and Cohen 1978.

pIJ702 is described in Katz et al., 1983.

pIJ4642 is an E.coli cloning vector allowing positive selec¬ tion for inserts. It resembles pIJ666 (Kieser and Melton, Gene 65. 1988, pp. 83-91) but has more useful sites for clon¬ ing. It was obtained from T. Kieser.

pDN1528 is a derivative of the B. subtilis plasmid pUBHO (Gryczan et al.,1978), carrying a 2.3 kb Hindlll - Sphl frag¬ ment containing the alpha-amylase gene from B. licheniformis strain DN52, an amylase-overproducing derivative of ATCC9789 produced by conventional mutagenesis procedures.

pPL1131 is a pUBHO-derived plasmid containing the amylase promoter and signal peptide-coding region of pDN1528 followed by a synthetic linker.

pIJ2002 is a pUClδ derivative containing the daqA (agarase) gene of S . coelicolor A3(2). It is described in Buttner et al., 1987, and was obtained from M. Bibb.

General Methods;

Standard DNA manipulations were performed essentially as de- scribed in Maniatis et al., 1982.

Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, DNA Polymerase I (Klenow Fragment) , and Exonuclease III were obtained from New England Biolabs and used as recommended by the supplier. Nuclease SI and the restriction enzyme Hindu were obtained from Boehringer Mannheim and used as recom¬ mended by the supplier. Chicken egg white lysozyme was obtained from Sigma.

Preparation of plasmids from all strains was conducted by the method described by Kieser (1984) .

Lambda DNA was packaged in vitro using the Packagene extract from Promega.

DNA sequencing was performed by the dideoxy chain termina¬ tion method (Sanger et al., 1977) using ³⁵ SdATP (DuPont NEN NEG034H, >1000 Ci/mmol) as the radioactive label and using denatured plasmid DNAs as templates. Sequencing was either carried out as described in Hattori and Sakaki, 1986, using the Klenow fragment, or by means of Sequenase™ as described in the booklet from the supplier (United States Biochemical Corporation) . Primers were either the M13 sequencing and re- verse sequencing primers from New England Biolabs or oligo¬ nucleotides (17-21 mers) complementary to regions of the cloned Pseudomonas DNA prepared by the present inventor.

Transformation of E. coli:

Cells of E. coli were made competent and trans- formed as described by Mandel and Higa, 1970.

Transformation of B. subtilis:

Competent cells were prepared and transformed as described by Yasbin et al., 1975.

Transformation of B. licheniformis: Plasmids were introduced into B.licheniformis by polyethylene glycol-mediated protoplast transfor¬ mation as described by Akamatzu (1984) .

Transformation of S. lividans:

Transformation and other procedures for Strepto- myces were as described in Hopwood et al., 1985.

Oligonucleotide synthesis: Synthesis was performed on an automatic DNA synthe¬ sizer using the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22., 19§1, pp. 1859-1869. Crude oligonucleotides were purified by polyacrylamide gel electrophoresis. Preparation of phage DNA:

Phage lambda DNA was prepared from small-scale liquid cultures as described in Maniatis et al., 1982. Exo III deletions: Unidirectional deletions were made using Exonu- clease III as described by Henikoff, 1984.

Lipase analysis

Lipase was measured by a pH-stat method using tributyrine as substrate. 1 LU (Lipase Unit) is the amount of enzyme which liberates 1 umole titratable butyric acid per minute under the following conditions:

Temperature 30.0 °C pH 7.0

Emulsifier Gum Arabic, 1 g/1 Substrate Tributyrine, 50 ml/1

Media;

TY: Trypticase 20 g/1

Yeast extract 5 g/1

FeCl ₂ .4H ₂ 0 6 mg/1 MnCl ₂ .4H ₂ 0 l mg/1

MgS0 ₄ .7H ₂ 0 15 mg/1 pH 7 . 3

BPX: Potato starch 100 g/1

Barley flour 50 g/1

BAN 5000 SKB O.lg/1

Sodium caseinate 10 g/1

Soy Bean Meal 20 g/1

Na ₂ HP0 ₄ , 12 H ₂ 0 9 g/1

Pluronic 0.lg/1

LB agar: Bacto-tryptone 10 g/1

Bacto yeast extract 5 g/1 NaCl 10 g/1

Bacto agar 15 g/1

Adjusted to pH 7.5 with NaOH

LB agar with tributyrine:

To 350 ml of melted LB agar is added 3.5 ml tributyrine and 0.35 g Gum Arabic, the mixture is emulsified using an Ultra Turrax emulsifier and then autoclaved.

Nile Blue indicator plates:

The bottom layer consisted of 15 ml LB agar. The top layer contained a mixture of 3 ml soft agar (LB with 0.6 % agar), 300 μl olive oil emulsion and 20 μl of a 5% Nile Blue solution (made up in water and sterile filtered) .

Olive Oil emulsion:

Olive oil 10 ml Gum Arabic 1 g

Deionised water 90 ml mixed using an Ultra Turrax emulsifier.

Buffers;

TE buffer: 10 mM Tris, pH 8, 1 mM EDTA, pH 8.

Phage buffer: 0.01 M NaCl

0.01 M MgCl ₂ 0.01 M Tris.HCl, pH 7.0

Ligation buffer: 0.066 M Tris.HCl pH 7.5, 0.01 M MgCl ₂ , 25 μg/ml gelatine, 0.001 M ATP, 0.01 M DTT.

EXAMPLE 1

Cloning of the Pseudomonas cepacia lipase gene

A) Preparation of chromosomal DNA from Pseudomonas cepacia SB10.

A pellet of frozen cells from a 3 ml TY culture was resus¬ pended in 1 ml TE buffer containing 1 mg/ml lysozyme. 0.1 ml EDTA (0.5 M, pH 8.0) was added and the mixture was incubated at 37 °C for 15 min. with gentle shaking. 5 μl 20% SDS was added, and the solution was repeatedly extracted with phenol (300 μl portions) until no interface was present. The super¬ natant was then extracted once with CHC1 ₃ (300 μl) . To 900 μl supernatant, 90 μl 3 M Na-acetate and 500 μl isopropanol were added. After 5-10 minutes, the precipitated DNA was recovered on a plastic inoculating loop, washed in 70 and 96 % EtOH and resuspended in 500 μl TE. The solution of chromosomal DNA (chrDNA) was kept at -20 "C.

B) Partial cutting and fractionation of chrDNA from P. cepa¬ cia.

80 μg chrDNA from P.cepacia SB10 (prepared as described in section A above) was digested with 5 units Sau3A at 37 °C for 5 minutes in 400 μl of the buffer recommended by the sup¬ plier, and heated at 70 "C for 10 min, and the total sample was then loaded on a preparative agarose gel. After elec-

trophoresis, gel segments containing DNA fragments of 9-23 kb were cut out. The chromosomal DNA fragments were recovered in the liquid formed after freezing/thawing the gel segments, extracted twice with phenol, once with CHC1 ₃ , ethanol preci- pitated and dissolved in TE buffer.

C) Construction of a P. cepacia DNA library in phage lambda.

2 μg lambda EMBL4 DNA was digested completely with restric¬ tion enzymes BamHI and Sail, extracted twice with phenol, once with CHC1 ₃ , ethanol precipitated and resuspended in 10 μl TE. The suspension was mixed with 2.5 μg size-fractionated chrDNA (in 50 μl TE) , ethanol precipitated, resuspended in 10 μl ligation buffer 100 units of T4 DNA ligase were added and ligation was carried out for 2 h at room temperature followed by 16 hours at 4°C. The ligated DNA was then packaged into lambda phage particles using the Packagene™ extract from Pro- mega, diluted in 500 μl phage buffer and 50 μl CHC1 ₃ were added. This was used for making an amplified phage stock as follows: Packaged phage particles were mixed with an equal volume of a fresh overnight culture of E. coli NM539 grown in TY + 0.4 % maltose + 20 mM MgS0 ₄ , allowed to adsorb for 20 minutes at 37°C and plated on fresh LB plates in 4 ml soft LB agar (0.6 %) containing 20 mM MgS0 ₄ . After incubation at 37°C for 16 hours, the partially lysed top layer containing about 2500 plaques was scraped into a 40 ml centrifuge tube con- taining 5 ml phage buffer, and phage eluted for 2 hours. The supernatant after centrifugation at 5000 rpm for 5 min. in a Sorvall SS34 rotor was kept as an amplified stock and used for screening.

D) Isolation of a recombinant lambda phage carrying a lipase gene.

About 200000 phages from the amplified library were adsorbed to NM539 and plated on 20 LB plates in soft agar containing 1 % glycerol tributyrate emulsion, resulting in total lysis on

each plate. 170 clear plaques (hydrolysing the tributyrine) were identified in the turbid tributyrine emulsion, and a number reisolated. 35 plaques were streaked on Nile Blue in¬ dicator plates and 10 of these gave a positive reaction, i.e. a strong blue colouring. Phage DNA was isolated from three of these phages, which were found to be identical, based on re¬ striction enzyme digests.

E) Subclonin into pUC19

1 μg of the phage DNA prepared in section D above was parti- ally cut with Sail, mixed with 0.3 μg Sail digested pUC19 DNA, ligated, transformed into competent E. coli HW1 and plated on LB plates containing 200 μg/ml ampicillin. These plates were replicated onto LB plates with 200 μg/ml ampicil¬ lin and 1 % glycerol tributyrate emulsion, and colonies sur- rounded by clear halos were isolated. One of these, SJ150, was shown to harbour a plasmid, designated pSJ150, of ap¬ proximately 6 kb, including the lipase-coding gene and the lipase modulating (lim) gene.

F) DNA sequencing

The sequence of most of the cloned DNA contained on pSJ150 was determined by the dideoxy chain terminating method di¬ rectly on double-stranded templates, as described in Materi¬ als and Methods above. The sequences of both strands were determined, using a combination of subcloning of restriction fragments, deletions from either end of the cloned DNA using Exonuclease III, and synthetic oligonucleotide primers. This allowed identification of the lipase coding sequence shown in Fig. 2A-C, and the lipase modulator (lim) coding sequence shown in Fig. 1A-C On pSJ150, the sequence TCG separates the lipase modulator start codon (ATG) from the lipase stop codon (TAA) .

EXAMPLE 2

Expression of the Pseudomonas cepacia lipase

A. Expression of the unmodified lipase in E.coli

(1) lip without lim

Plasmid pSJ518 was constructed by subcloning the 1.5 kb Hindlll-Clal fragment containing the lipase lip) gene from pSJ150 into pUC19, as shown in Fig. 3. Strain SJ518 (E. coli HW1 containing pSJ518) does not produce any lipase as seen on plates containing 1 % glycerol tributyrate (i.e. no halos surround the colonies) .

(2) lip + lim on the same plasmid

On pSJ150, the lipase is expressed from its own as well as from the pUC19 lac promoter. The plasmid pSJ910, containing the same fragment of cloned DNA but in the reverse orienta- tion with respect to the lac promoter, was constructed as outlined in Fig. 4 and introduced into E. coli HW1 to form strain SJ910. When plated on plates containing 1 % glycerol tributyrate, SJ910 colonies were surrounded by smaller halos than colonies of SJ150. This indicates the presence of a pro- moter, active in E. coli. for the lip and lim genes.

The region between the lip and lim genes can be changed with¬ out negatively affecting lipase expression. pSJ150 contains a unique Clal site one basepair after the lipase stop codon. Plasmid pSJ485 was constructed by insertion of an 8 basepair Bglll linker (New England Biolabs) into this Clal site which had been filled-in using the large fragment of DNA polymerase I (Klenow fragment) and dNTP's, thus giving a 10 basepair insertion (Fig. 5) . Strain SJ485 (E. coli HW1 containing pSJ485) produced the same amounts of lipase as SJ150.

(3) lip and lim on separate plasmids

The lim gene was excised on a 1.2 kb Sphl fragment from pSJ150 and inserted into the Sphl site of pUC19. This re¬ sulted in plasmids pSJ377, with the lac promoter reading cor- rectly into the lim gene, and pSJ378, with the lac promoter reading backwards into the lim gene. From pSJ377 and pSJ378, the 1.2 kb insert + the lac promoter could be excised as a 1.5 kb PvuII fragment. This was inserted into the Hindu site of pACYC177 to give pSJ622 (insert from pSJ377) and pSJ624 (insert from pSJ378) . The construction of pSJ622 and pSJ624 is outlined in Fig. 6.

Plasmids pSJ622 and pSJ624 were introduced into competent SJ518 (the strain containing pSJ518 with the lipase gene alone) , selecting for ampicillin and kanamycin resistance, and the transformants were replicated onto plates containing glycerol tributyrate. Large halos were formed around trans¬ formants containing pSJ622, but none around transformants containing pSJ624, indicating lipase production from the for¬ mer but not from the latter.

B. Expression of a modified lipase gene in E.coli

(1) lip without lim

pSJ494 is an expression plasmid for the P.cepacia lipase in Bacillus. It has the promoter, ribosome binding site and sig¬ nal peptide coding region from the B. licheniformis alpha- amylase gene fused in-frame to DNA coding for the mature li¬ pase, but does not contain the lim gene. The construction of pSJ494 is outlined in Figs. 7 and 8. The sequence of the fusion region is as follows:

ATG AAA CAA CAA AAA CGG CTT TAC GCC CGA TTG CTG ACG CTG Met Lys Gin Gin Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu alpha-amylase signal peptide

Pstl TTA TTT GCG CTC ATC TTC TTG CTG CCT CAT TCT GCA GCA GCG GCC Leu Phe Ala Leu lie Phe Leu Leu Pro His Ser Ala Ala Ala Ala

Mlul GCA GCT GGC TAC GCG GCG ACG CGT TAC CCG ATC - - Ala Ala Gly Tyr Ala Ala Thr Arg Tyr Pro lie - - J mature lipase sequence

In order to introduce this alpha-amylase/lipase fusion gene into E. coli. plasmid pSJ909 was constructed (Fig. 8) . Strain SJ909 (E. coli HWl containing pSJ909) does not produce any halos on plates containing glycerol tributyrate.

(2) lip and lim on separate plasmids

Strain SJ909 was made competent and transformed with either pSJ377 (constructed as shown in Fig. 6) or pSJ729, which was derived from pSJ377 by deleting about 600 basepairs from the 5' terminal of the lim gene (Fig. 9). When the double trans¬ formants were streaked on plates containing glycerol tribu¬ tyrate, those containing pSJ377 (the lim ^* plasmid) formed clear halos around the colonies, indicating lipase produc- tion, whereas those containing pSJ729 (li ^"0 did not form any halos.

C. Expression of a modified lipase gene in B.subtilis

(1) lip without lim

Plasmids pSJ493 and pSJ495 are identical to pSJ494, described in section B.l), carrying the amylase-lipase fusion but no lim gene. They were introduced into B. subtilis strain DN1885 and transformants were grown for 4 days at 37 °C in shake flasks containing BPX growth medium, with the addition of oleyl alcohol to 30 g/1 and chloramphenicol to 12 μg/ml.

_L ipase activity in the culture broth was measured by the LU- titrimetric method (described in Materials and Methods) and was 1-2 LU/ml. This is not above the background level found for strain DN1885, which varies between 0-5 LU/ml.

(2) lip and lim on the same plasmid

Plasmid pSJ416 contains the same amylase-lipase fusion as pSJ493-495, but the lipase gene is followed immediately down¬ stream by the lim gene in exactly the same way as on pSJ150 (the construction of pSJ416 is outlined in Fig. 10) .

When this plasmid was introduced into B. subtilis DN1885 and transformants grown in shake flasks as described above, yields reached 40 LU/ml in 4 days.

D. Expression of a modified lipase gene in B.licheniformis

(1) lip without lim

Plasmid pSJ488 contains the same amylase-lipase fusion as pSJ493, but on the Bacillus vector pPL1131 conferring kanamy- cin resistance to its host strain (Fig. 11) . This plasmid was introduced into B. licheniformis strain ATCC 9789 by pro¬ toplast transformation, and the transformants were streaked on plates containing glycerol tributyrate. The halos formed around the transformants were very small, of the same size as those formed around the untransformed strain.

(2) lip and lim on the same plasmid

Plasmid pSJ600 contains the same amylase-lipase fusion as pSJ488, but the lipase gene is followed immediately down¬ stream by the lim gene in exactly the same way as on pSJ150 (the construction of pSJ600 is outlined in Fig. 11) . When this plasmid was introduced into B. licheniformis ATCC 9789 by protoplast transformation, and the transformants were

streaked on plates containing glycerol tributyrate, pro¬ nounced halos were formed around the colonies indicating li¬ pase production.

E. Expression of a modified lipase gene in S.lividans

(1) lip without lim-expression

Plasmid pSJ604 was constructed by isolating a 880 bp Hindlll- Avall fragment from plasmid pIJ2002, carrying the dagA pro¬ moters and part of the signal peptide-encoding sequence (cf. Fig. 2 in Buttner et al., 1987). To the fragment were added two complementary, phosphorylated and annealed oligonucleo¬ tides of the following sequence:

Avail Mlul Xhol

GTCCCGCACCCGCCGCTCATGCCGCAGCTGGCTACGCGGCGACGCGTC

GGCGTGGGCGGCGAGTACGGCGTCGACCGATGCGCCGCTGCGCAGAGCT

This mixture was ligated to pUC19 which had previously been digested with Hindlll and Sail to give plasmid pSJ604.

Plasmid pSJ671 is an E. coli-S. lividans shuttle plasmid carrying an in-frame fusion between the promoter, ribosome binding site and signal peptide coding region of the ;______ coelicolor agarase gene (daqA. Buttner et al., 1987) and the DNA coding for the mature lipase. The lim gene is present on the plasmid, but in such a position that it is not tran¬ scribed by any known promoters. The construction of pSJ671 is outlined in Fig. 12, and the sequence of the fusion region is as follows:

GTG GTC AAC CGA CGT GAT CTC ATC AAG TGG AGT GCC GTC GCA Met Val Asn Arg Arg Asp Leu lie Lys Trp Ser Ala Val Ala agarase signal peptide

Avail CTC GGA GCG GGT GCG GGG CTC GCG GGT CCC GCA CCC GCC GCT CAT Leu Gly Ala Gly Ala Gly Leu Ala Gly Pro Ala Pro Ala Ala His

Mlul GCC GCA GCT GGC TAC GCG GCG ACG CGT TAC CCG ATC - Ala Ala Ala Gly Tyr Ala Ala Thr Arg Tyr Pro lie j mature lipA sequence

When pSJ671 was introduced into S. lividans TK24 by proto¬ plast transformation and the resulting transformants were streaked on plates containing glycerol tributyrate, very small halos were formed around the colonies, indicating little or no lipase production.

(2) lip and lim on the same plasmid

pSJ669 is similar to pSJ671 with the exception that pSJ669 contains the lim gene immediately downstream from the lipase gene in exactly the same way as on pSJ150 (the construction of pSJ669 is outlined in Fig. 13) .

When pSJ669 was introduced as described above into S. livi¬ dans TK24 and transformants were streaked on plates with gly¬ cerol tributyrate, very large halos were formed around the colonies, compared to those around pSJ671.

F. Effect of a modified lim gene

pSJ377, shown in Fig. 6, contains the lim gene expressed from a lacZ promoter. This plasmid was digested with Clal, and deletions around the Clal site were generated using ExoIII. One such deletion plasmid is pSJ721, in which 15 base pairs were deleted, which created a fusion protein derived from the

9 N-terminal codons from the pUC19 lacZ gene plus 8 C-termi- nal codons from the lip gene fused to a lim gene lacking only the first two codons. The sequence of the fusion region is as follows:

LacZ start Sphl lip C-terminal part

ATG ACC ATG ATT ACG CCA AGC TTG CAT GCG AAC CGG CTG AAG CTG Met Thr Met lie Thr Pro Ser Leu His Ala Asn Arg Leu Lys Leu

: lim from 3. codon GCG GGG:GCA CGA GGA GGA CGC GCG CCG CTG GCG CGC CGC GCC GTG Ala Gly Ala Arg Gly Gly Arg Ala Pro Leu Ala Arg Arg Ala Val

GTC Val

The fusion construct plus the lac promoter was excised from pSJ721 as a 1.5 kb PvuII fragment and inserted into the Hindll site of pACYC177 to give pSJ812, in the same manner as in the construction of pSJ622 shown in Fig. 6.

pSJ812 was introduced into competent SJ518 (the strain con¬ taining pSJ518 with the lipase gene alone) , selecting for ampicillin and kanamycin resistance, and the transformants were replicated onto plates containing glycerol tributyrate. The halos formed around the transformants were of approxima¬ tely the same size as those formed around transformants of SJ518 with pSJ622, expressing the non-modified lim gene.

EXAMPLE 3

Expression and identification of the lim encoded polypeptide

The lim gene was excised from pSJ150 as a 1.17 kb Clal - Sphl fragment and inserted into the Ndel site of plasmid pJW2

(Wang et al., 1990) after creating blunt ends with the Kle¬ now fragment of DNA polymerase I. Recombinant plasmids were obtained with the lim gene inserted in either of the two pos¬ sible orientations. pCBE3 and pCBE4 both contain the lim gene 5 in the correct orientation for transcription from the tem- perature-inducible lambda pR and pL promoters on pJW2, where¬ as pCBE2 contain the lim gene in the opposite orientation. The plasmids were introduced into E. coli JA221 (Clarke and Carbon, 1978) , and transcription from the pL and pR promoters

10 was induced by increasing the temperature of the cultures to 42 °C. Proteins produced upon induction were identified by polyacrylamide gel electrophoresis of cells harvested from the induced cultures. This allowed identification of a pro¬ tein of an apparent molecular weight of 34000 which was found

15 in induced cultures of transformants carrying pCBE3 and pCBE4, but not in the induced culture of the transformant carrying pCBE2, or in uninduced cultures.

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