AU644101B2 - Polygalacturonase gene from erwinia carotovora and its molecular cloning - Google Patents

Description

POLYGALACTURONASE GENE FROM ER INIA CAROTOVORA AND ITS MOLECULAR CLONING

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of molecular biology, and, more particularly, to the fields of recombinant genetics and genetic engineering. The invention further relates to DNA sequences, derived from Er inia carotovora, which code for the enzyme polygalacturonase. The invention relates to vectors, such as plasmids, comprising the sequences of the present invention, and to host cells transformed with such vectors. Additional aspects of the present invention are related to methods for producing proteins employing the sequences, vectors or transformed hosts of the invention. By means of the invention, large quantities of the enzyme polygalacturonase may be produced in pure form.

Description of Related Art

Use of pectin-degrading enzymes is extremely important in the food industry. Although traditionally a mixture of enzymes from, for example, Aspergillus niqer or other fungi, is used, the various pectinases catalyze different reactions and it is more economical and efficient to use pure preparations of individual enzymes in the desired amounts.

Pectin polymers comprise chains of 1,4-1inked alpha-D- galacturonic acid and methoxylated derivatives. These polymers are important structural constituents of plant middle lamellae and primary cell walls.

Pectinase enzymes are of commercial significance in fruit and vegetable juice production, wine making, brewing and baking due to their ability to catalyze the breakdown of the backbone and side-chain bonds of the pectin polymers. Pectin methylesterase catalyzes the removal of methanol from pectin, yielding pectic acid. Polygalacturonase hydrolyzes the glycosidic linkages in the galacturonic chain. Pectate lyases cleave galacturonosyl bonds by ^-elimination, and pectin lyases cleave the gal cturonosyl bonds of highly methylated pectins. (Coll er and Keen, Ann. Rev. Phvtopath. 24:383-409 (1986)). Efficient degradation of naturally occurring pectins can be accomplished by using pectin methylesterase and polygalacturonase or pectate lyase.

For example, pectin methylesterase, naturally present in cell walls of citrus fruits, destabilizes the "cloud" formed in citrus juices. Addition of polygalacturonase can remedy this problem. However, the polygalacturonase preparation should not contain other enzymes, if a cloud-stable pigmented juice such as orange juice is desired. To produce lemon juice and other juices which do not contain pulp, polygalacturonase must be added in conjunction with pectinesterase. (Rombouts and Pilnik, Symbiosis 2:79-90 (1986)).

Another group of pectinases, the bacterial pectate lyases, are not of immediate practical use because they have low activity at the acidic pH commonly encountered in fruit and vegetable processing. Genes encoding pectate lyases, polygalacturonase and endocellulase have been cloned. However, the nucleotide sequence of only one pectinase, pectin methylesterase of Erwinia chrysanthemi B374, has been reported. (Plastow, A.S., Hoi. Microbiol. 2:247-254 (19S8). A need exists for a method of producing commercially useful amounts of purified pectinases that are active at the pH range used in fruit and vegetable processing. Such processing methods require use of purified and characterized nucleotide sequences encoding these pectinases. However, the nucleotide sequences of the genes encoding the major pectin¬ ases, pectate lyase and polygalacturonase have not been reported to date.

INFORMATION DISCLOSURE STATEMENT

In accordance with the requirements of 37 C.F.R. § 1.56, the following are concise explanations of documents known to Applicants or their attorney, submitted in accordance with 37 C.F.R. §§ 1.97 and 1.98.

Applicants will submit hereafter on form PTO-1449 a listing of these documents in accordance with 37 C.F.R. § 1.98, together with copies of the listed documents.

Applicants do not waive any rights to appropriate action to establish patentability over any of the listed documents should they be applied as references against the claims of the present application.

This statement should not be construed as a representa¬ tion that more material information does not exist or that an exhaustive search of the relevant art has been made.

Consideration of these documents, and making the same as record in the prosecution of the present application upon submission of the form PTO-1449 and copies of the documents listed therein, are respectfully requested. Plastow et al . , Symbiosis 2: 115-122 (1986) describes the molecular cloning of four pectate lyase genes and one polygalacturonase gene from Erwinia. Using plasmids, the genes were transferred into E. coli cells and some polygalacturonase activity could be detected. Tsuyu u and Miyamoto, Symbiosis 2: 103-110 (1986) transformed E. coli cells with pectate lyase gene from Erwinia and detected the gene product that was excreted from the transformed cells. Kotoujansky et al .. EMBO Journal 4:781-785 (1985) describe the cloning of four pectate lyase genes and one endocellulose gene from Erwinia, using a Lambda phage vector to transform E. coli cells. Ried and Coll er, Appl . and Environ. Micro. 50:615-622 (1985) describe methods for detecting and characterizing pectic enzymes; the method was used to analyze the pectate lyase isozyme profiles of E. coli clones containing Erwinia genes. Kotoujansky, Ann. Rev. Phvtopathol . , 25:405-430 (1987) discusses the dependence of pathogenesis by Erwinia on pectinases. A brief review of information regarding poly¬ galacturonase is found at page 410.

Collmer and Keen, Ann. Rev. Phvtopathol. £1:383-409 (1986) contains a general review of the role of pectic enzymes in plant pathogenesis. The cloning and expression of a pectate lyase gene in E. coli is discussed.

Daniels et al .. Ann. Rev. Phvtopathol. 26:285-312 (1988) contains a review of bacterial pathogenicity genes. Rombouts and Pilnik, Symbiosis 2:79-90 (1986) discuss the role of pectinases in the food industry, and mention the possibility of transferring pectinase genes into GRAS status microorganisms.

Plastow, Mol. Microbiol. 2:247-254 (1988) describes the cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia. The gene was expressed in E. coli .

SUMMARY OF THE INVENTION

The present inventors have discovered, isolated, cloned and sequenced the gene that encodes the enzyme poly¬ galacturonase in Erwinia carotovora. The gene is used to produce commercially useful amounts of polygalacturonase using heterologous host cells, including GRAS (generally recognized as safe) status hosts. In general, only enzymes derived from microorganisms involved in the fermentation of traditionally known foods can be employed in food technology. However, transformation of GRAS microorganisms with the gene for polygalacturonase disclosed herein and expression of the gene can circumvent this limitation.

In one embodiment, the invention comprises the novel nucleotide sequence encoding the enzyme polygalacturonase. This sequence can be utilized to construct plasmids and expression vectors. Using these plasmids and vectors, the gene can be expressed in a variety of host cells, including both Gram-negative and Gram-positive bacteria.

An important aspect of this invention, inter alia, is that it provides a method for producing commercially useful quantities of Erwinia polygalacturonase. The Erwinia enzyme has high activity at neutral pH, compared to the currently available polygalacturonase from fungi. Thus, the protein of the invention is active in the pH range normally found in industrial processing of neutral plant and vegetable material including sugar beet, carrots and linen.

This invention also provides a means for producing bacteria with new properties, particularly the ability to synthesize and secrete polygalacturonase. Such bacteria are useful in fodder production. These embodiments, as well as additional embodiments of the present invention, will become more apparent and easily understood to those of skill by reference to the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Localization of the polygalacturonase encoding region of pHSK24. The thick line represents the 4.1 kb insert in pHSK24 with the marked restriction sites. Deletions were generated partly by using the existing restriction enzyme cleavage sites (deletions 24-2 -24-6) or by exonuclease III (digestion deletions 24-1, 24-7 - 24-9). The thin lines indicate the segments of the 4.1 kb insert present in each deletion derivative. The multiple cloning site (mcs) on the left contains the sites Hindlll-Sphl-Pstl-Sall-Xbal, and on the right Smal-Kpnl-SacI-EcoRI, respectively. The pectolytic activity (+ or -) of the deletion derivatives on poly- galacturonic acid (PGA) is shown on the right.

Figure 2 Sequencing strategy of the pehA gene. The region encoding the polygalacturonase was subcloned to M13 using the mapped restriction sites in the insert and in the flanking multiple cloning sites (see Figure 1 legend). The sequencing reactions were made from purified single-stranded templates at indicated startpoints and directions of sequencing, employing the dideoxy chain termination method of Sanger (Proc. Natl . Acad. Sci. USA 80:3963-3965 (1977)), and Sequenase™ (USB).

Figures 3a, b and c DNA sequence of the pehA gene. The DNA sequence of the pehA gene and the deduced amino acid sequence of its protein product are shown. The amino acids determined by the NH2~terminal sequence analysis of the purified polygalacturonase are shown underlined. The processing site is shown by a vertical arrow. Riboso e binding site (RBS) before the start of translation is indicated below the DNA sequence. Possible transcription termination loops at the V end are indicated by arrows. Figure 4 The effect of pH on the activity of polygalac¬ turonase.

Figure 5 The effect of temperature on the activity of polygalacturonase.

Figure 6 Reverse-phase HPLC of culture medium of Erwinia carotovora. Polygalacturonase containing peak is indicated by the arrow.

Figure 7 Nucleotide sequence of oligo nucleotide primers oligo 307 and 308. The deduced amino acid sequence of oligo 308 is also shown. The arrows indicate the 3' end of the α- amylase signal sequence and region coding for the N-terminus of the mature pectinase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, reference will be made to various methodologies known to those of skill in the art of molecular genetics and biology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al.. Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al.. Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B.M., Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al .. Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, publisher, Berkeley, CA (1981); and Maniatis, T., et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1982). General principles of microbiology are set forth, for example, in Davis, B.D. et al ., Microbiology, 3d edition, Harper & Row, publishers, Philadelphia, PA (1980).

By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells. In a preferred embodiment, chromosomal DNA from Erwinia carotovora is isolated, digested, ligated into the vector pUC18, and used to transform E. coli cells. Screening of clones containing DNA encoding polygalacturonase may be accomplished by well-known methods, including, preferably, by replica plating on LPGA Ap plates.

By "vector" is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites, and may be capable of autonomous replica- tion in a defined host or vehicle organism such that the cloned sequence is reproducible. Thus, by "DNA expression vector" is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome. Such DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention, however, are plasmids comprising an expression vector based on the α-amylase gene of B. amyloliouefac ens as depicted in Applicants' copending Application Serial No. 308,861, filed February 10, 1989, the specification of which is incorporated herein by reference in its entirety as though set forth in full.

By "substantially pure" is meant any protein of the present invention, or any gene encoding any such protein, which is essentially free of other proteins or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature. A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical. Thus, an amino acid sequence is "substantially similar" to the amino acid sequence of the protein polygalacturonase if the polypeptide consisting of that amino acid sequence possesses the catalytic properties of polygalacturonase.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).

Similarly, a "functional derivative" of a gene of the protein of the present invention is meant to include "frag- ments," "variants," or "analogues" of the gene, which may be "substantially similar" in nucleotide sequence, and which encode a molecule possessing similar activity. As used herein, a "functional derivative" of the polygalacturonse gene will encode a polypeptide having the catalytic properties of polygalacturonase.

In a preferred embodiment, the introduced sequence will be incorporated into a plasmid vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E_ coli (such as, for example, pUC18). Such plasmids are, for example, disclosed by Maniatis, T., et al . (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Particularly preferred vectors according to the invention are those which are able to replicate in E. coli, B. subtilis, Lactococcus and Lactobacillus.

Once the vector or DNA sequence containing the con¬ struct^) has been prepared for expression, the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjuga¬ tion, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al .. Science 240: 1538 (1988)), etc.

In a preferred embodiment of the invention, the poly¬ galacturonase gene from plasmid pHSK24 is combined with a Gram-positive vector to transform Gram-positive bacteria. Preferred Gram-positive hosts include the strains Bacillus, Lactobacillus and Lactococcus. In another embodiment of the invention, the plasmid pHSK24 which contains the DNA sequence for polygalacturonase is transformed into Gram-negative host cells.

After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired heterolo- gous or homologous protein, or in the production of a fragment of this protein.

The expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. For example, the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyriboc- ytidylic acid-agarose, hydroxyapatite or by electrophoresis or i munoprecipitation. In a preferred embodiment, the expressed protein will also be secreted from the host cell when a sequence or plasmid of the invention is employed with a secretion vector, with the advantage that isolation and purification procedures will be simplified. In another preferred embodiment, plasmid pHSK24 was modified and combined with plasmid pKTH39, a Bacillus subtilis expression vector based on the α-amylase gene of B. amylo- liquefaciens, to form a hybrid expression unit. B. subtilis cells were transformed with the plasmid containing the expression vector, and clones were grown and screened. Clone BRB679 expressed the gene and secreted the gene product, as evidenced by polygalacturonase activity in the supernatant. The manner and method of carrying out the present invention may be more fully understood by those of skill by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto.

EXAMPLE I

MATERIALS AND METHODS

a. DNA isolations and modifications Rapid isolation of plasmid DNA from E. coli for screening of clones was done according to Holmes and Quigley (Anal . Biochem. 134:193-197 (1980)). DNA for restriction enzyme digests and subcloning was prepared by the method of Birnboim and Doly (Nucl . Acids. Res. 7:1513-1523 (1979)) from 200 ml liquid cultures. Isolation of plasmid DNA from B. subtilis was carried out according to Gryczan et al . (J. Bacteriol. 134:318-329 (1978)).

Chromosomal DNA from Erwinia carotovora was isolated as follows: Cells were first incubated overnight at 28^βC in 20 ml of L medium, harvested and resuspended in 4 ml of 50 mM glucose - 10 mM EDTA - 25 mM Tris, pH 8.0. 1 mg of lysozyme was added and the suspension was incubated for 20 minutes at room temperature. 0.4 ml of 10% SDS was added and mixed, followed by addition of 200 μg of RNAse and an incubation of 40 minutes at 28'C with gentle shaking. Then, 2.5 mg of proteinase K was added and the incubation was continued for 60 minutes at the same temperature. Next, the suspension was extracted twice with an equal volume of phenol, once with phenol-chloroform (1:1) and once with chloroform. 1/10 volume of 3 M Na-acetate and 2.5 volumes of ethanol was added and mixed. The DNA precipitate was transferred to a icrocentri- fuge tube, the tube was filled with ethanol and the DNA precipitate pelleted. The supernatant was discarded, the pellet was dried and further resuspended in 10 mM Tris-HCl - 1 M EDTA, pH 8.0 to yield a DNA concentration of 1 mg/ml, and stored at 4^βC.

Restriction enzyme digestions were performed according to the instructions provided by the manufacturer (IBI, Boehringer). Restriction fragments were separated by electrophoresis in 0.8% agarose gels (Maniatis et al.. Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982). Selected restriction fragments were isolated from a low melting point gel as described by Benson, Biotechnioues 2:66-68 (1984).

For dephosphorylation of 5'-phosphorylated ends of DNA fragments calf intestinal phosphatase (CIP) (Boehringer) was used.

Exonuclease III (Boehringer) was used to generate deletions in the cloned DNA.

The ends of DNA fragments were joined by T4 DNA ligase (IBI). All the DNA modifying enzymes were used according to instructions provided by the manufacturer.

b. DNA transformations

Transformation of E. coli HB101 cells was done with the CaCl2 method as described in Maniatis et al . , supra. EL subtilis cells were transformed by the method of Gryczan et al. (J. Bacteriol. 134:318-328 (1978)). c. DNA seouencing

DNA sequence determination was based on the method of Sanger (Proc. Natl. Acad. Sci. USA 80:3963-3965 (1977)) using Sequenase™ (USB).

d. Oliqonucleotide synthesis

Synthetic oligo ribonucleotide primers were synthesized by the phosphoamidite method using a model 381A Applied Biosystems synthesizer.

e. Construction of plasmid pKTH39

Beginning with plasmid pKTH 29, which is fully described in Applicants' copending Application Serial No. 308,861, pKTH39 was prepared as follows. The dried pKTH 29 preparation that had been treated with exonuclease, was dissolved into 5 μl of the solution containing phosphorylated EcoRI-linker- molecule described in Applicants' copending Application Serial No. 308,861. 0.5 μl 10 mM ATP, 0.5μl 1 mM spermidine and 0.5 μl T^DNA-ligase (2 Weiss units) were added to the solution. The solution was incubated for 3 hours at 23°C, whereafter it was diluted to 20μl in 40 mM Tris HC1 - 100 mM NaCl - 10 mM MgCl buffer (pH 7.6). Fifteen units of EcoRI enzyme (Biolabs) were added, and the solution was incubated for 12 hours at 37^βC. The reaction was stopped by incubation at 65°C for 10 minutes. The preparate treated with EcoRI was gelfiltered through a 1 ml Sepharose 4B column. Two mM Tris-

HC1 - 0.1 mM EDTA (pH 7.5) was used as elution buffer in the filtering. The filtrate was harvested in 35 μl fractions, and the fractions containing plasmid were identified by their radioactivity, collected and dried. The dried DNA was dissolved into 20 μl 66 mM Tris HC1 - 6.6 mM gCl - 10 mM dithiothreitol buffer (pH 8.0), and 1.5 μl 10 mM ATP and 0.3 μl T_/ DNA-ligase were added. The solution was incubated for 3 hours at 23^βC, whereafter the competent B. subtilis IH0 6064 strain was transformed by the plasmid preparate, and the cells were cultivated on bacterium plates containing kanamycin.

The plasmids were isolated from the transformants by a method described by Gryczan et al., J. Bacteriol. 134:318-329 (1978), and the plasmids were first characterized by gel electrophoresis, whereafter their DNA base sequence at both ends of the EcoRI linker molecule was determined. In this way, plasmids pKTH 38 and pKTH 39 were obtained from the plasmid pKTH 29. In the plasmid pKTH 38, the EcoRI linker molecule is located 90 nucleotide pairs after the cleavage site of the excretion signal in the area of the α-amylase structural gene. In the plasmid pKTH 39, the EcoRI linker molecule is located 16 nucleotide pairs after the initiation methionine of the α- a ylase gene in the area of the signal sequence.

In order to join the linker molecule at the joining site of the excretion signal or in the immediate vicinity thereof, the plasmid pKTH 38 was cleaved with EcoRI. Three portions of 10 μg of the cleaved plasmid were each suspended in 115 μl 20 M Tris, 600 mM NaCl , 12 mM MgCl₂, 12 M CuCl , 1 mM EDTA buffer (pH 8.1). Ten μl BAL-31 enzyme (Bethesda Research Laboratories, BRL, 40 U/ml) were added to each plasmid portion, and the tubes were incubated for 5, 6 and 7 minutes in a water bath of 30^βC. The reaction was stopped by adding 0.5 M EDTA, pH 8.0, so as to obtain a final concentration of 12 M. The DNA portions treated with BAL-31 were combined, extracted twice with phenol and precipitated with ethanol. The ethanol precipitate was suspended in 75 μl 63 M Tris, 6.3 mM MgCl₂ buffer (pH 8.0), and to the solution were added 5 μl 1 mM dATP, 1 mM dGTP, 1 mM dCTP, and 1 mM dTTP, and finally 5 μl T4 polymerase (PL-Biochemicals, 5 U/μl). The solution was incubated for 80 minutes at ll^βC. The reaction was stopped by adding 0.5 EDTA as above, and the solution was extracted with phenol and the DNA was precipitated with ethanol. The ethanol precipitate was dissolved in 250 μl 10 mM Tris, 1 M EDTA buffer (pH 8.0). To 55 μl of this solution were added 50 μl phosphorylated Hind III linker molecule (BRL, 75 pmol), 5 μl 660 mM Tris, 100 mM MgCl₂, 50 mM dithiothreitol buffer (pH 7.5), and 10 μl T4 DNA ligase (BRL, 2 U/μl). The mixture was incubated for 15 hours at 15"C and for 10 minutes at 65'C. The DNA was precipitated by adding isopropanol, the DNA precipitate was washed with 70% ethanol and, after drying jn vacuo, suspended in 100 μl 10 mM Tris, 50 M NaCl , 5 mM MgCl , 5 mM dithiothreitol buffer (pH 8.0). Three μl of Hind III restriction enzyme (BRL, 10 U/μl) were added to the suspen¬ sion, and the solution was incubated for 4 hours at 37^βC and for 10 minutes at 65^βC. The DNA was purified by electro¬ phoresis in 0.8% LGT agarose gel (Marine Colloids Inc.), at 30 V, for 15 hours. The linear plasmid zone was cut off from the gel, and the DNA was extracted at 65'C with phenol and was precipitated with ethanol. The ethanol precipitate was dissolved in 35 μl 66 mM Tris, 10 mM MgCl , 5 mM dithiothreitol buffer (pH 7.5) to which was added 1.5 μl 10 M rATP and 1.5 μl T4 DNA ligase (BRL, 2 U/μl). The mixture was incubated for 3 hours at 22^βC and transformed into the competent B. subtilis IH0 6135 strain, and the cells were cultivated on nutrient medium plates containing kanamycin. The plasmids were isolated from the transfor ants according to a method described earlier, and the location of the Hind III linker molecule in the plasmids was determined by means of DNA sequencing. In this way a series of plasmids was obtained in which the Hind III linker molecule is located immediately after the excretion signal or in different positions after the cleavage site of the excretion signal in the area of the α- amylase structure gene.

With respect to the plasmid pKTH 39, in order to join the linker molecule at the joining site of the signal sequence or in the immediate vicinity thereof, an analogous approach to that described above for the plasmid pKTH 38 was employed. In using the plasmid pKTH 39, which contains the deleted signal sequence of the α-amylase gene of B. amyloliquefaciens, the desired structural gene can thus be inserted either at the EcoRI site, or at the same location using an in vitro modified site as described, for example, in Example 3 of Application Serial No. 308,861, or at any location intermediate between the initiation codon (-met) and the EcoRI site, by well known methods. The creation of a new joint site at the desired location may be accomplished by methods known to those of skill, including but not limited to site directed in vitro mutagenesis, poly erase chain reaction (PCR), or by synthesiz¬ ing in vitro the required promoter fragment, all of which may be accomplished with the exercise of routine skill with an appreciation of the teaching of the present invention.

f. Analysis of plasmid encoded proteins Plasmid-encoded proteins were produced by the maxicell method of Sancar et al . (J. Bacteriol. 137:692-693 (1979)). Alternatively, plasmid encoded proteins were produced by DNA- directed in vitro transcription-translation system following instructions provided by the manufacturer (Amersham). The ^S-labelled protein products were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (Nature 227:680-685 (1970)) and visual¬ ized by autoradiography of stained and dried gels.

g. Assay of PG-activitv

Pectolytic activity was detected on L agar plates containing 0.7% polygalacturonic acid. The activity was developed by staining the plates with 1 M CaCl₂ resulting in the appearance of a white zone around the pectolytic colonies.

Polygalacturonase activity was determined by measuring the release of reducing groups in a reaction mixture contain- ing 0.5% (w/v) polygalacturonic acid, 50 mM sodium acetate (pH 6.0), 100 mM NaCl, 2 mM EDTA, and an appropriate amount of purified polygalacturonase. The reaction mixtures were incubated at 30"C for 1 hour, and the liberated reducing groups were assayed using the p-hydroxybenzoic acid hydrazide method as described elsewhere (Lever, Anal. Biochem. 47:273- 279 (1972)). D-galacturonic acid was used as a standard in the assays. One unit of polygalacturonase was defined as the amount of enzyme activity which catalyzes the liberation of 1 μmol of product under the above conditions.

h. Purification and N-terminal amino acid sequencing of polygalacturonase Polygalacturonase was purified from culture medium of Erwinia carotovora by gel filtration. 0.5 ml of the cleared culture medium was applied onto a Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, CA) column (1.0 x 28 cm) and eluted at 5 ml/hour with 50 mM Tris/HCl, pH 7.5, 100 mM NaCl. Fractions containing polygalacturonase were detected by SDS-PAGE (Laem li, Nature 227:680-685 (1970)), pooled and concentrated using UNISEP Minicent-10 ultrafilters (Bio-Rad). The purified protein was used for polygalacturonase assays. The protein amount was determined by the method of Bradford (Anal . Biochem 72:248 (1976)). For N-terminal sequence analysis, polygalacturonase was purified by reversed-phase high-performance liquid chroma¬ tography (HPLC). 0.5 ml of the cleared culture medium was injected into a TSK TMS 250 reversed-phase column (0.46 x 4 cm) equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA). The bound proteins were eluted at a flow rate of 1 ml/minute with an increasing linear gradient of acetonitrile (from 0 to 100% in 60 minutes) in 0.1% TFA. The proteins were detected at 280 nm. The protein containing peaks were collected and analyzed by SDS-PAGE. ■19-

For N-terminal sequence analysis, the purified PG was degraded in a gas-pulsed-liquid-phase sequencer (Modified Beckman 890D) (Kalkkinen and Tilgmann, J. Prot. Chem. 7:242- 243 (1988)). About 6 μg of the purified PG was loaded on the glass fiber filter pretreated with 2 mg Polybren. For degradation, the 03CPTH program (Applied Biosystems) was used and the released amino acid derivatives were detected on line by using a system consisting of a Brownlee MicroGradient LC pump, Jones chromatography oven, Spectra Physics SP 8450 detector and a Merck Hitachi D-2000 integrator plotter. PHT amino acid separation was performed on a 0.2 x 21 cm reversed phase column (Applied Biosystems) by using a gradient of acetonitrile in 25 mM sodium acetate, pH 3.7, 5% tetrahydro- furan at 53^βC.

Bacteria Strains

Strain Reference or source

Erwinia carotovora SCC 3193 Pirhonen M. et al . , subsp. carotovora (Ecc) Microbial Pathoq.

4.359-367

Escherichia coli K12 HB101 Boyer et Roulland-

Dussoix, J,.

Mol. Biol. 41:459-

472 (1969)

Escherichia coli K12 JM 109 Yanisch-Perron et al ., Gene 3_3:

103-119 (1985) ^■20-

Escheri chia col i K12 DH&F' Hanahan , J . Mol . Biol

166 : 557-580 (1983)

Bacill us subtil i s BRB1 Pal va, I . , Gene 19:81-87 (1982)

The E. col , B. subtilis and Erwinia carotovora strains were propagated in L-broth or L-media (Miller J., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) at +37^βC or at +28^βC (Erwinia), respectively. Ampicillin (Ap) and kanamycin (Km) were added to media at 150 μg/ml and 10 μg/ml, respectively, for plasmid maintenance.

Originating plasmids pUC9 Viera & Messing, Gene 19:259-26

(1982) pUC18 Yanisch-Perron et al . , 1985

Gene 33:103-119

Phages T4GT7 Wilson G.G. et al., 1979. Nature 280:80-82

M13 mpl8/19 Yanisch-Perron, et al . , 1985 Gene 33:103-119

-21-

EXAMPLE II

RESULTS

a. Cloning of the polygalacturonase gene, pehA

A genomic DNA library from Erwinia carotovora subsp. carotovora SCC 3193 was established in the plasmid pUC18: chromosomal DNA from SCC 3193 was isolated and digested partially with Sau3A, and the resulting restriction fragments were separated by electrophoresis and fragments ranging from 1.5 kb to 5 kb were isolated from the gel. These fragments were ligated with pUC18, previously digested with BamHI and dephosphorylated. The ligation mixture was transformed into competent E. coli HB101 cells. Transformants were selected by plating the transformation mixture on L-plates containing 150 μg/ml ampicillin. Clones coding for polygalacturonase were screened for by replica plating onto LPGA Ap plates and positive clones were isolated for further analysis.

One of these clones, named pHSK24, encoded a poly- galacturonase as determined by enzyme assays from cellular extracts.

b. Characterization of the insert in pHSK24

The DNA insert in the plasmid pHSK24 was characterized by restriction mapping (Figure 1). The insert was 4.1 kb in size. To localize the feh gene encoding polygalacturonidase in this fragment deletion derivatives of pHSK24 were con¬ structed. The rightmost 1.7 kb Aval-Smal fragment could be deleted without affecting the PG-activity (24-2, Smal site is located in the multiple cloning site ( cs) region of pUC18). Similarly about 800 bp from the left end of the insert could be removed by exonuclease III digestion without affecting the enzyme activity. This suggests that the pjeh gene is within -22-

the 1.6 kb fragment from the leftmost EcoRI - Aval fragment (Figure 1).

c. Nucleotide sequence of the pehA gene The nucleotide sequence of the 1.6 kb fragment harboring pehA was determined. The sequencing strategy is depicted in Figure 2 and the corresponding sequence together with the deduced amino acid sequence in Figure 3. The sequence contained only one long open translational reading frame starting with a Met at position 1 and preceded by a typical ribosome binding site GAGG at the appropriate position (Shine and Dalgarno, Proc. Natl. Acad. Sci. USA 71:1342-1346 (1974)). A translation stop codon is located at position 1207. The calculated Mr of the Peh protein was 42,849.

d. Identification of the PG protein Plasmid-encoded proteins labelled with ³⁵S were separated by SDS-PAGE and molecular weight determined by comparison with

Mr markers (Sigma MW-SDS-7L). Analysis of plasmid encoded proteins from maxicell harboring pHSK24 suggested that the jDeh A gene encodes a polypeptide of apparent MW 42,000.

When plasmid encoded proteins were analyzed by DNA dependent in vitro transcription translation a polypeptide with apparent Mr of 45,000 was obtained suggesting that rjeh A gene product is synthesized as a precursor.

e. Isolation of PG and determination of its N-terminal amino acid sequence To ensure that the PG was indeed synthesized as a precursor the N terminal amino acid sequence of the mature PG was determined. To accomplish this the plasmid pHSK24 was transduced to Erwinia carotovora subsp carotovora SCC3193 with bacteriophage T4, and selecting for Ap^r transductants. These transductants overproduced PG several-fold as compared to SCC3193. This overproduction led to inhibition of the synthesis or secretion of other exoenzymes normally produced by SCC3193. About 90% of the protein in the culture super¬ natant of SCC3193 (pHSK24) was PG. Culture supernatant from an overnight culture of SCC3193 (pHSK24) was isolated and polygalacturonase was purified as described in the Methods section.

The purified proteins migrated as a single band in SDS- PAGE with an apparent molecular weight of 42,000. The specific activity of the enzyme was 500 U/mg protein. The pH optimum of the enzyme was about 5.5 (Fig. 4) and the tempera¬ ture optimum was in the range of 35-45^βC (Fig. 5).

When polygalacturonase was purified by reversed-phase HPLC (Figure 6) and subjected to amino acid sequence analysis, a single N-terminal amino acid sequence, Ser-Asp-Ser-Arg-Thr- Val-Ser-Glu-Pro-Lys-Thr-Pro-Ser-Ser-, was obtained. This sequence is identical to that deduced from the nucleotide sequence (139-180, Figure 3) and determines the N-terminus of the processed mature PG.

f. Expression of polygalacturonase gene in B. subtilis PehA-gene was synthesized by PCR-reaction (Mullis et al . , Methods Enzvmol . 155:335-350 (1987)) using plasmid pHSK24 as a template, to generate modified 5' and 3' end structures. The 3' end was modified to contain four additional restriction enzyme sites (EcoRI, BamHI, Smal and Narl). The oligonucleo- tide primer (oligo 307) is shown in Figure 7. The 5' end was synthesized to contain, upstream of the N-terminus of the mature PG, 22 C-terminal amino acid residues of the signal sequence of B. amyloliquefaciens α-amylase gene. The signal sequence structure was slightly modified to contain additional cloning sites. The oligo nucleotide primer (oligo 308) is shown in Figure 7. The PCR reaction mix contained 10 μl of buffer, 16 μl of 1.2 M dNTP-mix, 0.1 nmol of oligo 307, 0.1 n ol of oligo 308, 100 ng of pHSK24, 0.5 μl enzyme, 100 μl of water ad. PCR- reaction was continued for 25 cycles in Perkin-Elmer PCR instrument, each cycle consisting of 92^βC 1 minute, 55^βC 1 minute and 72^βC 7 minutes, incubation periods.

The reaction mix was extracted twice with phenol, ethanol precipitated and dissolved into 30 μl TE. 10 μl sample was digested with EcoRI in a total volume of 20 μl . After phenol extraction 1 μl was ligated to EcoRI-digested and CIP-treated plasmid pUC9.

Ligation mix was transformed to E. coli DH5αF' cells and plated on L X-gel, IPTG, Ap-plates. DNA was isolated from four colonies and one (# 5) was found to contain a fragment of right size. 200 ng of EcoRI-digested clone #5 was ligated with 200 ng of EcoRI-digested pKTH39 in 7 μl volume. pKTH39 is a Bacillus subtilis expression vector based on the α- amylase gene of B. amyloliguefaciens and contains in addition to the promoter-SD-region 8 N-terminal amino acid residues of the α-amylase signal sequence. When joined to the 22 C- ter inal amino acid residues coded by oligo 307, a complete functional α-amylase signal sequence is formed. As mentioned above, a full description of the pKTH39 plasmid is found in Applicants' copending application Serial No. 308,861. Ligation mixture was transformed to B. subtilis BRB1 and plated on L Km-plates.

Clones were screened for the presence of an insert using an agarose gel. One clone, designated BRB679 (BRB679 = BRB1 [pKTH1892]), was selected for culture which contained the EcoRI fragment in a correct orientation.

Clone BRB679 was grown in double strength L-broth supplemented with 0.1 M K-phosphate pH 7.0, 5% glucose and Km. Overnight culture was diluted 1:100 to 20 ml of growth medium. A sample was taken at the time point of Klettss 100 + 4 hours. The activity of PG was determined from the sample and the supernatant was shown to contain 10,000 U/ml of poly¬ galacturonase activity.

The most presently preferred method for obtaining secretion of polygalacturonase in host cells is to combine a complete functional signal sequence with the gene for polygalacturonase. The pKTH50-59 vectors disclosed in Applicants' copending Application Serial Number 308,861 are most preferred for this purpose. The modified βeh gene coding for mature polygalacturonase is fused with a vector chosen from the group pKTH50-59 and used to transform the host cells. Expression of the polygalacturonase gene is detected as extracellular polygalacturonase activity following culture in growth medium appropriate for the host.

Expression of polygalacturonase gene in Lactobacil- lus and Lactococcus

Using the vectors described herein the polygalacturonase gene is expressed in Lactobacillus and Lactococcus host cells. The methods are described in detail in Examples V, VI, VII and X of Applicants' copending Application Serial No. 377,450, filed July 10, 1989, the specification of which is incor- porated herein by reference in its entirety as if set forth in full.

Briefly, plasmids as exemplified by pKTH1797, pKTH1798, pKTH1799, pKTH1801, pKTH1805, pKTH1806, pKTH1807 and pKTH1809 contain promoter and secretion promoting signals. The promoter and secretion promoting sequences of these plasmids can direct the expression of heterologous genes and secretion of the gene product in Gram-positive host cells such as Lactobacillus and Lactococcus.

To obtain expression of the polygalacturonase gene and secretion of the gene product, a host cell such as Lac¬ tobacillus or Lactococcus is transformed with a plasmid co prising the promoter and secretion promoting sequences described above and the gene coding for the mature PehA protein described herein. The host cell is cultured in a suitable medium under conditions allowing expression of the protein, and the protein is recovered from the medium.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth below.

Claims (28)

CLAIMS :

1. A nucleotide sequence coding for the protein polygalacturonase, or a functional or chemical derivative thereof.

2. The nucleotide sequence of claim 1 isolated from the bacteria Erwinia carotovora, or a functional or chemical derivative thereof.

3. A substantially pure nucleotide sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof.

4. A plasmid comprising the nucleotide sequence of any one of claims 1-3.

5. A host cell transformed with the plasmid of claim 4.

6. The host cell of claim 5, wherein said cell is selected from the group consisting of Bacillus, Lactococcus and Lactobacil lus.

7. The host cell of claim 6, wherein said cell is BRB679.

8. A hybrid expression unit comprising the plasmid of claim 4 and a secretion vector.

9. The hybrid expression unit of claim 8 in which the secretion vector comprises the plasmid pKTH39.

10. A method for producing polygalacturonase in a host cell, comprising transforming said host cell with the plasmid of claim 4, culturing the transformed host cell in a suitable medium under conditions allowing expression of said protein, and recovering the expressed protein from said host cell or said medium.

11. The plasmid pHSK24, or a functional or chemical derivative thereof.

12. A host cell transformed with the plasmid of claim 11, wherein said host cell is a Gram-negative bacteria.

13. A hybrid expression unit comprising the plasmid of claim 11 and a secretion vector.

14. The hybrid expression unit of claim 13 in which the secretion vector comprises pKTH39.

15. A polypeptide encoded by a substantially pure nucleotide sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof.

16. A polypeptide comprising the amino acid sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof.

17. A polypeptide comprising the amino acid sequence of Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell trans¬ formed by the plasmid of claim 4.

18. The polypeptide of claim 18 wherein said host cell is BRB679.

19. A polypeptide encoded by a substantially pure nucleotide sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell transformed by the hybrid expres- sion unit of claim 8.

20. A polypeptide comprising the amino acid sequence of Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell trans- formed by the hybrid expression unit of claim 8.

21. The polypeptide of claim 19 wherein said host cell is BRB679.

22. The polypeptide of claim 20 wherein said host cell is BRB679.

23. A polypeptide encoded by a substantially pure nucleotide sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell transformed by the plasmid of claim 4.

24. The polypeptide of claim 23 wherein said host cell is BRB679.

25. A polypeptide encoded by a substantially pure nucleotide sequence as shown in Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell transformed by the hybrid expres¬ sion unit of claim 13.

26. A polypeptide comprising the amino acid sequence of Figure 3a, b and c, or a functional or chemical derivative thereof, said polypeptide synthesized by a host cell trans¬ formed by the hybrid expression unit of claim 13.

27. The polypeptide of claim 25 wherein said host cell is a Gram-negative bacteria.

28. The polypeptide of claim 26 wherein said host cell is a Gram-negative bacteria.