HUT63880A - Production of dna sequences encoding polygalacturonase enzyme, vectors and host cells comprising them and process for producing protein with the host cells - Google Patents

Abstract

A DNA sequence encoding the enzyme polygalacturonase is provided. The sequence was isolated from Erwinia carotovora subsp. carotovora and cloned in Escherichia coli. The invention relates to vectors, such as plasmids, comprising the sequences of the present invention, and to host cells transformed with such vectors. By means of the invention polygalacturonase can be synthesized in and secreted from host cells. Use of GRAS-status host cells provides polygalacturonase suitable for use in food processing.

Description

Preparation of DNA sequences encoding polygalacturonase enzyme, vectors and host cells comprising them, and method of producing protein with host cells

OY ALKO AB, Helsinki, Finland

inventors:

PALVA Ilkka,

HEMILA Harri,

PALVA Tapio,

SAARILAHTI Hannu,

HEINO Pekka,

Helsinki,

Helsinki,

Finland,

Uppsala,

Uppsala,

Uppsala,

Sweden

Date of filing: 11/9/91

priority: September 15, 1989 (408,056)

USA

International Application Number: PCT / FI90 / 00212

International Publication Number WO 91/4331

74576-1672-GÁ / KmO • * • · ·

The present invention relates to the field of molecular biology, more particularly to recombinant genetics and genetic engineering. The present invention relates to DNA sequences of Erwinia carotovora origin encoding a polygalacturonase enzyme. The invention relates to vectors, such as plasmids, containing the sequences of the invention and to host cells transformed with these vectors. The invention further relates to a method of producing a protein with the sequences, vectors or transformed host cells of the invention. By using the invention, the polygalacturonase enzyme can be produced in large amounts in pure form.

The use of pectin-degrading enzymes is very important for the food industry. Although mixtures of such enzymes have traditionally been used, such as mixtures of Aspergillus niger or other fungal enzymes, different pectinases catalyze different reactions and are more economical and effective when used in the desired amounts of pure formulations of each enzyme.

Pectin polymers are chains composed of 1,4-linked alpha-D-galacturonic acid and methoxylated derivatives. These polymers are important structural elements of plant central lamellae and primary cell walls.

Pectinase enzymes are of industrial importance in fruit and vegetable juice production, wine making, cooking and baking because of their ability to catalyze the breakdown of skeletal side chain bonds in pectin polymers. Pectin methyl esterase catalyzes the removal of methanol from pectin to leave pectic acid. Polygalacturonase is a galacturonic acid fl • · «·

- Hydrolyses glycoside bonds of 3 chains. Pectate lyases cleave galacturonosyl bonds by B-elimination, while pectin lyases cleave galacturonosyl bonds of highly methylated pectins [Collmer and Keen, Ann. Port. Phytopath. 24, 383409 (1986)]. Effective degradation of naturally occurring pectins can be accomplished using pectin methyl esterase and polygalacturonase or pectate lyase.

For example, pectin methyl esterase, which is naturally present in the cell walls of citrus fruits, destabilizes turbidity in citrus juice. Administration of polygalacturonase can remedy this problem. However, if a colorless, such as orange juice, is to be produced for a prolonged period of time, the polygalacturonase composition may not contain any other enzyme. In order to prepare lemon juice or other juice which does not contain the flesh of the plant, polygalacturonase must be administered in combination with pectin esterase (Rombouts and Fullik, Symbiosis 2: 79-90 (1986)).

The other group of pectinases, bacterial pectate lyases, have no direct practical utility since they have a low activity at the usually acidic pH of fruits and vegetables.

Genes encoding pectate lyases, polygalacturonase and endocellulase have been cloned. However, the nucleotide sequence of only one pectinase, pectin methyl esterase Erwinia chrysanthemi B374, has been described [Plastow, A.S., Mol. Microbiol. 2: 247254 (1988)].

There is a need for commercially available purified pectinases that are active in the pH range of fruit and vegetable processing. These methods require the use of purified and characterized nucleotide sequences encoding these pectinases. However, the nucleotide sequence of most genes encoding pectinases, pectate lyases and polygalacturonases is not yet known.

The state of the art is briefly summarized below.

Plastow et al., Symbiosis 2: 115-122 (1986) describe the molecular cloning of four pectate lyase genes and one Erwinia-derived polygalacturonase gene. Using plasmids, the genes are transferred into E. coli cells and can show some polygalacturonase activity.

Tsuyumu and Miyamoto (Symbiosis 2: 103-110 (1986)) transform E. coli cells with the Erwinia-derived pectate lyase gene and detect the gene product selected from the transformed cells.

Kotoujansky et al., (EMBO, Journal 4, 781-785 (1985)) describe the cloning of four pectate lyase genes and an Erwinia-derived endocellulase gene using the Lambda phage vector to transform E. coli cells.

Ried and Collmer, Appl. and Environ. Micro. 50, 615-622 (1985)] discloses methods for the detection and characterization of pectin-degrading enzymes, which are used to study the pectate lyase isozyme profile of E. coli clones containing Erwinia genes.

Kotoujansky [Ann. Port. Phytopathol. 25: 405-430 (1987)] investigate the dependence of Erwinia-induced pathogenesis on pectinases. See page 410 for a brief summary of polygalacturonase information.

Collmer and Keen, Ann. Port. Phytopathol. 24./ 338-409 · «« «

(1986) provide a general overview of the role of pectin degrading enzymes in plant pathogenesis. They deal with the cloning and expression of a pectate lyase gene into E. coli.

Daniels et al., Ann. Port. Phytopathol 26: 285-312 (1988)] provides an overview of the bacterial pathogenicity of genes.

Rombouts and Fullik (Symbiosis 2: 79-90 (1986)) discuss the role of pectinases in the food industry and mention the possibility that pectinase genes can be transferred to generally recognized as safe microorganisms (GRAS).

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

The present invention relates to the recognition, isolation, cloning and sequencing of a gene encoding the polygalacturonase enzyme of Erwinia carotovora. The gene can be used to produce commercially useful amounts of polygalacturonase using heterologous host cells, including GRAS hosts. In food technology, generally, only enzymes derived from microorganisms that are traditionally involved in the fermentation of known foods can be used. However, transformation of a GRAS microorganism with a polygalacturonase gene, as described herein, and expression of the gene may circumvent this limitation.

The present invention relates to a novel nucleotide sequence encoding the polygalacturonase enzyme. This sequence can be used to generate plasmids and expression vectors. Using these plasmids and vectors, the gene can be found in various host cells, »·» ·

- 6 of which can also be expressed in Gram-negative and Gram-positive bacteria.

An important aspect of the present invention is, inter alia, that it provides a process for the preparation of a commercially useful amount of Erwinia polygalacturonase. Erwinia has a neutral pH η of high activity compared to currently available polygalacturonase from fungi. Thus, the protein of the invention is active in the pH range commonly used in the industrial processing of neutral plants and plant materials, including sugar beet, beet and flax.

The present invention provides the ability to synthesize and excrete bacteria with novel properties, in particular polygalacturonase. Such bacteria are useful in the production of feed.

The foregoing and other aspects of the invention will be readily apparent to one skilled in the art from the following detailed description.

The figures are described below.

Figure 1. The figure shows the location of the pHSK24 region encoding polygalacturonase. The thick line shows a 4.1 kb insert in pHSK24 with restriction sites marked. Deletions were partially generated using the existing restriction enzyme cleavage sites (deletions 24-2-24-6) or exonuclease III (digestion deletions 24-1, 24-7-24-9). The thin lines in each deletion derivative represent segments of the 4.1 kb insert present. The multiple cloning site (mcs) on the left is the · ·· * · ·

- contains 7 HindIII-SphI-PstI-SalI-Xbal sites, while the right side multiple cloning site (mcs) contains the SmaI-KpnI-SacI-EcoRI sites. The pectolytic activity of the deletion derivatives on polygalacturonic acid (PGA) is indicated by the + or symbol on the right side of the figure.

Figure 2 shows the sequencing strategy of the sofA gene. The polygalacturonase coding region is subcloned into M13 using the restriction sites mapped to the insert and the flanking multiple cloning sites (see description in Figure 1). Sequencing reactions were performed starting from the purified single-stranded templates at the indicated starting points and in the indicated sequencing directions according to Sanger [Proc. Natl. Acad. Sci. USA 80, 3963-3965 (1977)] using the dideoxy chain termination method and Sequanase ^R (USB).

Figures 3a, b, c and d show the DNA sequence of the sofA gene. The DNA sequence of the sofA gene and the resulting amino acid sequence of the corresponding protein are shown. The amino acids determined by analysis of the purified NH ₂ -terminal polygalacturonase sequence are underlined. The treatment site is indicated by a vertical arrow. The ribosome binding site (RBS) is designated below the DNA sequence prior to translation. Possible transcription termination loops are indicated by arrows at the 3 'end.

Figure 4 shows the effect of pH on polygalacturonase activity.

Figure 5 shows the effect of temperature on polygalacturonase activity.

Figure 6 shows the result of reverse phase HPLC separation of Erwinia carotovora culture medium. The peak containing polygalacturonase is indicated by an arrow.

Figure 7 shows the nucleotide sequence of the oligonucleotide primers oligo 307 and oligo 308. Also provided is the amino acid sequence deduced from the nucleotide 308 sequence of oligo. The arrows represent the 3 * end of the α-amylase signal sequence and the N-terminus of the mature pectinase.

In the following description, reference is made to various methods known to those skilled in the art of molecular genetics and biology. The publications and other labeled materials to which reference is made herein are incorporated herein by reference in their entirety.

Standard reference work on the general essence of recombinant DNA technology is described in Watson J.D. et al., Molecular Biology of the Gene, I and II. Vol. Benjamin / Cummings Publishing Company, Inc., published by Menlo Park, CA (1987); Darnell, J.E. et al., Molecular Cell Biology, Scientific American Books, Inc., 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 Genetics Engineering, 2nd Edition, University of California Press, Berkeley CA (1981); and Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982), Davis, B.D. et al., Microbiology, 3rd Edition, Harper & Row, Publishers, Philadelphia, PA (1980).

Cloning refers to the use of in vitro recombination techniques to introduce a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to generate DNA fragments by attaching them to vector molecules, inserting the resulting complex DNA molecule into a host cell capable of replication, and selecting a clone containing the target gene from the host host cells. . In a preferred embodiment of the invention, chromosomal DNA from Erwinia carotovora is isolated, digested, ligated into the pUC18 vector and used to transform E. coli cells. The selection of DNA-containing clones encoding polygalacturonase may be accomplished by well-known techniques, including replication (copy) spotting on LGPA Aβ plates.

By vector designation is meant a DNA molecule from a plasmid or bacteriophage into which DNA fragments are inserted or cloned. A vector contains one or more unique restriction sites and is capable of autonomous replication in a particular host or carrier such that the cloned sequence can be reproduced. Thus, the term DNA expression vector refers to any autonomous element that is capable of replication in a host, independently of the host chromosome, after introducing additional DNA sequences into the genome of that autonomous element. Examples of such DNA expression vectors are bacterial plasmids and phages. However, plasmids containing the B. amyloliquefaciens α-amylase gene expression vector are preferred for the present invention, as described in detail in U.S. Patent Application Serial No. 308,861. This application is incorporated herein by reference in its entirety.

By a substantially pure designation is meant any protein or gene encoding a protein of the invention which is substantially free of other proteins or genes or other contaminants that would normally be present in nature, so that this substantially pure protein or gene is not naturally occurring in nature. can be found.

One molecule is referred to as substantially similar to another when the two molecules have substantially the same amino acid sequence. Essentially similar amino acid molecules will exhibit similar biological activity. Thus, provided that two molecules have similar activity, they are considered variants as used in this expression, even if one molecule contains additional amino acid units not found in the other, or if the sequence of the amino acid units is not the same. Thus, an amino acid sequence is substantially similar to the amino acid sequence of a polygalacturonase protein if the polypeptide having the above amino acid sequence has the catalytic properties of the polygalacturonase.

A molecule is considered to be a chemical derivative of another molecule if it contains further chemical units that are not part of the molecule. Such units can improve the solubility, absorption, biological half-life, etc. of the molecule. In addition, these units can reduce the toxicity of the molecule, eliminate or weaken the unwanted side effect of the molecule, etc. Units capable of mediating such effects are, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, Penn. (1980).

Similarly, a functional derivative of a gene of a protein of the invention is understood to mean fragments, variants, or analogs of the gene that are substantially similar in nucleotide sequence and which encode a molecule with similar activity. As used herein, a functional derivative of the polygalacturonase gene encodes a polypeptide having catalytic properties of polygalacturonase.

In a preferred embodiment, the sequence shown is inserted into a plasmid vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. Factors that are important in the selection of a particular plasmid vector include: readily recognizable and distinguishable from host cells that do not contain the vector; the number of copies of the vector required by the particular host and the question whether it is desirable that the vector be shuttle between host cells of different species. Preferred prokaryotic vectors are, for example, plasmids, such as those capable of proliferation in E. coli (e.g., pUC18). Such plasmids are described, for example, in Manatis T. et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982). Particularly preferred vectors for the present invention are those capable of replication in E. coli, B. subtilis, Lactococcus and Lactobacillus hosts.

Once the DNA sequence containing the vector or construct (s) is ready for expression, the vector or DNA construct (s) may be introduced into an appropriate host by any suitable method, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation; and the use of polycations such as diethylaminoethyl (DEAE) dextran; and mechanical means such as electroporation, direct microinjection and micro-bullet (biolistic) bombardment (Johnston et al., Science 240, 1538 (1988)).

In a preferred embodiment of the invention, the polygalacturonase gene of plasmid pHSK24 is combined with a Gram-positive vector and transformed into a Gram-positive bacterium. Preferred Gram-positive hosts include Bacillus, Lactobacillus and Lactococcus strains. In another embodiment, plasmid pHSK24 containing the polygalacturonase DNA sequence is transformed into Gram-negative host cells.

Following the introduction of the vector, the recipient cells are propagated in a selective medium that selects for proliferation of cells containing the vector. Expression of the cloned gene sequence (s) results in the production of the desired heterologous or homologous protein or fragment thereof.

The expressed (produced) protein can be isolated and purified by conventional procedures, for example, extraction, precipitation, chromatography, affinity chromatography or • · ·

- 13 electrophoresis procedures. The cells may be harvested, for example, by centrifugation or appropriate buffers, lysed, and the protein isolated by column chromatography, such as DEAE-cellulose, phosphocellulose, polyribocytidylic acid agarose, hydroxyapatite or immunoassay. In a preferred embodiment, the expressed protein is also secreted from the host cell using a sequence or plasmid of the invention containing a secretion vector, which has the advantage of simplifying the isolation and purification process.

In a further preferred embodiment of the invention, plasmid pHSK24 is modified and combined with plasmid pKTH39, which is an expression vector of Bacillus subtilis based on the α-amylase gene of B. amyloliquefaciens, to obtain a hybrid expression unit. B. subtilis cells are transformed with the plasmid containing the expression vector and the clones are grown and screened. The BRB679 clone expressed the gene and the clone selected the product of the gene as evidenced by its polygalacturonase activity in the supernatant.

In order to further illustrate the invention and the process to those skilled in the art, the following examples are provided by way of non-limiting example.

Example 1

Materials and procedures

the. DNA isolation and modification

E. coli-derived plasmid DNA for rapid clon screening Holmes and Quigley, Anal. Biochem. 134: 193-197 (1980)]. For restriction enzyme digestion and subcloning, DNA from Bimbomim and Doly [Nucl. Acids. Gap. 7, 1513-1523 (1979)] from 200 ml of liquid culture. Isolation of plasmid DNA from B. subtilis by Gryczan et al., J. Med. Bacteriology. 134: 318-329 (1978)].

Chromosomal DNA from Erwinia carotovora was isolated as follows: The cells were first incubated overnight at 28 ° C in 20 ml of L medium, then the cells were harvested and resuspended in 4 ml of 50 mM glucose, 10 mM / L EDTA and 25 mM Tris pH 8.0. 1 mg of lysozyme was added to the suspension and incubated for 20 minutes at room temperature. Then, 0.4 ml of 10% SDS (sodium dodecyl sulfate) was added to the suspension, 200 μg of RNase was added and the incubation was continued under gentle shaking for 40 minutes at 28 ° C. Subsequently, 2.5 mg of proteinase K was added and incubation was continued at the same temperature for 60 minutes. Subsequently, the suspension was extracted twice with an equal volume of phenol, once with a 1: 1 mixture of phenol and chloroform and once with chloroform. 1/10 volumes of 3 M sodium acetate and 2.5 volumes of ethanol are added and the mixture is mixed. The resulting DNA precipitate was transferred to a microcentrifuge tube, the tube was filled with ethanol, and the DNA precipitate was centrifuged. The supernatant was discarded, the precipitate was dried and resuspended in 10 mM Tris-HCl pH 8.0 containing 1 mM EDTA to 1 mg / ml DNA and

- Store 15 zions at 4 ° C.

Restriction enzyme digestion was performed according to the manufacturer's instructions (IBI, Boehringer). The restriction fragments were separated by electrophoresis on a 0.8% agarose gel (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). 1982]. The selected restriction fragments were isolated from a low melting point gel by the procedure described in Bensechni (Biotechniques 2: 66-68 (1984)).

To dephosphorylate the 5'-phosphorylated end of the DNA fragments, calf intestinal phosphatase (CIP) (Boehringer) was used.

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

The ends of the DNA fragments were ligated with T4 DNA ligase (IBI). All DNA modifying enzymes are used according to the manufacturer's instructions for use.

b. DNA transformations

Transformation of E. coli HB101 cells was performed using the CaCl2 method by Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982]. For the transformation of B. subtilis cells, Gryczan et al. Bacteriology. 134, 318-328 (1978)].

c. DNA sequencing

DNA sequencing was performed according to Sanger [Proc. Natl. Acad. Sci. USA, 80, 3963-3965 (1977)] using Sequenase ^R (USB).

• · * · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · |

d. Oligonucleotide synthesis

Synthesis of oligoribonucleotide primers was performed using the phosphoamidite method using a 381A Applied Biosystems synthesizer.

e. Construction of Plasmid pKTH39 Starting from plasmid pKTH 29, the complete disclosure of which is incorporated by reference in U.S. Patent No. 308,861, plasmid pKTH 29 was constructed as follows. Exonuclease-treated, dried pKTH 29 was dissolved in 5 µl of a solution containing a phosphorylated EcoRI-linker molecule, the disclosure of which is disclosed in co-pending U.S. Patent No. 308,861. To the solution was added 0.5 μΐ 10 mM ATP, 0.5 μΐ 1 mM spermidine and 0.5 μΐ T4-DNA ligase (2 Weiss units). The solution is then incubated for 3 hours at 23 ° C and diluted to 20 μΐ with 40 mM Tris containing 100 mM sodium chloride and 10 mM magnesium chloride, pH 7.6. -HCl buffer. To the solution was added 15 units of EcoRI (Biolabs) and incubated for 12 hours at 37 ° C. The reaction was stopped by incubation at 65 ° C for 10 minutes. The Eco RI treated preparation was gel-filtered through a 1 ml column of Sepharose 4B. Elution buffer used for gel filtration was 2 mM Tris-HCl-0.1 mM EDTA pH 7.5. The filtrate was collected in 35 μΐ fractions, and the fractions containing the plasmid were identified, collected and dried for radioactivity. The dried DNA is dissolved in 20 μΐ 66 mM Tris-HCl buffer, pH 8.0, 6.6 mM magnesium chloride and 10 mM dithiothreitol, and • ♦ · ··· · · * ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

17 1.5 μΐ 10 mM ATP and 0.3 μΐ Tΐ DNA ligase were added.

The solution was incubated for 3 hours at 23 ° C and the competent B. subtilis IHO 6064 strain was transformed with the plasmid preparation and the cells were cultured on bacterial plates containing kanamycin.

The plasmids from the transformants were prepared by Gryczan et al., J. Med. Bacteriol 134: 318-329 (1978)], and the plasmids are first characterized by gel electrophoresis and then the DNA base sequence at each end of the EcoRI linker molecule is determined. Thus, plasmids pKTH 38 and pKTH 39 were obtained from pKTH 29.

In plasmid pKTH 38, the EcoRI linker molecule is located 90 nucleotide pairs from the site of the secretory signal in the α-amylase structural gene. In plasmid pKTH 39, the EcoRI molecule is located in the signal sequence region with 16 nucleotide pairs after the methionine initiation of the α-amylase gene.

In order to link the linker molecule at or near the site of the excretion signal, plasmid pKTH 38 is cleaved with EcoRI. Three sections of the cleaved plasmid, each containing 10 Mg, were resuspended in 115 μΐ 20 mM Tris buffer, pH 8.1, containing 600 mM sodium chloride, 12 mM magnesium chloride, 12 mM It also contains 1 copper (II) chloride and 1 mM EDTA. To each plasmid portion was added 10 μΐ BAL-31 (40 U / ml Bethesda Research Laboratories, BRL) and each tube was incubated for 5, 6 and 7 minutes in a water bath at 30 ° C, respectively. The reaction was stopped by the addition of 0.5 M EDTA pH 8.0 to give a final concentration of 12 mM. Part of the BAL-31 Enzyme-Treated DNA

the two were combined, extracted twice with phenol and precipitated with ethanol. The ethanol precipitate was resuspended in 75 µl of 63 mM Tris buffer, pH 8.0, 6.3 mM magnesium chloride, and 5 µl of 1 mM dATP, 1 mM dGTP. 1 mM dCTP and 1 mM dTTP was added and 5 µl of T4 polymerase (PL-Biochemicals, 5 Ε / μl) was added. The solution was incubated for 80 minutes at 11 ° C. The reaction was quenched by the addition of 0.5 M EDTA and the solution was extracted with phenol and the DNA was precipitated with ethanol. The ethanol precipitate was dissolved in 250 µl of a 10 mM Tris buffer containing 10 mM EDTA, pH 8.0. For a 55 µl volume of the solution, a pH of 50 µl of phosphorylated Hind III linker molecule (BRL, 75 pmol), 5 µl of 660 mM Tris, 100 mM magnesium chloride and 50 mM dithiothreitol =

Buffer 7.5 and 10 µl T4 DNA ligase (BRL, 2 U / μΙ) were added. The mixture was incubated at 15 ° C for 15 hours and then at 65 ° C for 10 minutes. The DNA was precipitated by addition of isopropanol, the DNA precipitate was washed with 70% ethanol, dried in vacuo and then 100 µl of 10 mM Tris, 5 mM sodium chloride, 5 mM magnesium chloride and 5 mM / 1 dithiothreitol in pH 8.0 buffer. To the suspension was added 3 µl of Hind III restriction enzyme (BRL, 10 U / μΙ) and incubated for 4 hours at 37 ° C and for 10 minutes at 65 ° C. DNA was purified by electrophoresis on a 0.8% LGT agarose gel (Marina Colloids Inc.) at 30V for 15 hours. The linear plasmid region was excised from the gel and the DNA was extracted with phenol at 65 ° C and precipitated with ethanol. The ethanol precipitate was 35 µl of 66 mM Tris, 10 µl

4

- Dissolve in pH 7.5 buffer containing 19 mM magnesium chloride and 5 mM dithiothreitol and add 1.5 μΐ 10 mM rATP and 1.5 μΐ T4 DNA ligase ( BRL, 2 U / μΙ) was added. The mixture was incubated for 3 hours at 22 ° C and transformed into competent B. subtilis strain IHO 6135 and cultured on nutrient medium plates containing kanamycin. The plasmids were isolated from the transformants as described above and the position of the Hind III linker molecule in the plasmids was determined by DNA sequencing. In this way, a number of plasmids were obtained in which the Hind III linker molecule is located immediately downstream of the secretory signal, or at different positions in the α-amylase structural gene at various positions downstream of the secretory signal.

For plasmid pKTH 39, the procedure for linking the linker molecule at or near the site of the signal sequence is similar to that described above for plasmid pKTH 38. When using plasmid pKTH 39, which contains the deleted signal sequence of the B. amyloliquefaciens α-amylase gene, the desired structural gene can be inserted either at the Eco RI site or by using an in vitro modified site at the same site, e.g. or in any intermediate position between the initiator codon (-met) and the EcoRI site, this operation is well known to those skilled in the art. Establishment of the new linkage at the desired site can be accomplished by methods known to those skilled in the art, including site specific in vitro mutagenesis, polymerase chain reaction (PCR), or the desired site. 4 • · · ····· ···

- In vitro synthesis of 20 promoter fragments, each of which is a routine task for those skilled in the art, in light of the teachings of the present invention.

f. Analysis of proteins encoded by the plasmid

The proteins encoded by the plasmid are described by Sancar et al., J. Med. Bacteriology. 137: 692-693 (1979)]. Alternatively, plasmid-encoded proteins may be produced by a DNA-directed in vitro transcription-translation system following the manufacturer's (Amersham) instructions. The ³⁵ S-labeled protein products were separated by electrophoresis (SDS-PAGE) on sodium dodecyl sulfate polyacrylamide gel (Laureli, Nat. 227: 680-685 (1970)) and the separated fractions were visualized by autoradiography of the stained and dried gels. .

g. Assay for PG activity

Pectolytic activity was assayed on L-agar plates containing 0.7% polygalacturonic acid. Plates were stained with 1M calcium chloride to visualize activity, with a white band around the pectolytic colonies.

Polygalacturonase activity is determined by the release of reducing groups from a reaction mixture of 0.5% w / v polygalacturonic acid, 50 mM sodium acetate (pH 6.0), 100 mM sodium chloride, 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 prepared by the p-hydroxybenzoic acid hydrazide method [Lever, Anal. Biochem. 47, 2763-279 (1972)]. D-galacturonic acid is standard coated in the assay. One unit of polygalacturonase is defined as the enzyme activity that catalyzes the release of 1 μπόι of product under the conditions described above.

h. Purification of polygalacturonase and sequencing of N-terminal amino acid

Polygalacturonase was purified by gel filtration from Erwinia carotovora. 0.5 ml of purified culture was applied to a Bio-Gel P-100 (1.0 x 28 cm) column (Bio-Rad Laboratories, Richmond, CA, USA) and at 50 ml / hr, 50 mM, pH Eluted with 7.5 mM Tris / HCl buffer containing 100 mM sodium chloride. Fractions containing polygalacturonase were identified, collected and concentrated using SDS-PAGE (Laemmli, Natura 227: 680-685 (1970)) and concentrated using a UNISEP Minicent-10 ultrafilter (Bio-Rad). The purified protein is used for polygalacturonase assays. The amount of protein was determined by Bradford [Anal. Biochem. 72, 248 (1976)].

For analysis of the N-terminal sequence, polygalacturonase is purified by reverse phase high performance liquid chromatography (HPLC). 0.5 ml of the purified culture was injected onto a TSK TMS 250 reversed phase column (0.46 x 4 cm) equilibrated with 0.1% trifluoroacetic acid (TFA). Bound proteins were eluted at a flow rate of 1 mL / min using a gradient of 0% to 100% linear acetonitrile in 0.1% TFA over 60 minutes. Proteins are detected at 280 nm. The protein-containing peaks were collected and analyzed by SDS-PAGE.

Purified polygalacturonase (PG) gas pulsed fluid for N-terminal sequence analysis • 4 ···

phase modified sequencer (modified Beckman 890D) [Kalkkinen and Tilgmann, J. Prot. Chem. 1988, 242-243.

About 6 mg of purified PG is applied to a 2 mg glass filter pre-treated with Polybrene. For degradation, the 03CPTH program (Applied Biosystems) is used, and the released amino acids are detected online using a system consisting of a Brownlee MicroGradient LC pump, a Jones chromatography furnace, a Spectra Physics SP 8450 detector, and a Merek Hitachi D-2000 integrator . PHT amino acid separation was performed on a 0.2 x 21 cm reverse phase column (Applied Biosystems) using a gradient of acetonitrile in 25 mM sodium acetate, pH 3.7, 5% tetrahydrofuran, 53 ° C.

Bacterial strains

strains

Erwinia carotovora SCC 3193 subsp. carotovora (Ecc) Escherichia coli K12 HB101

Escherichia coli K12 JM 109

Escherichia coli K12 DH&F '

Bacillus subtilis BRB1

Reference or source

Pirhonen, M., et al

Microbial Pathog. 4, 359-367

Boyer and Roulland-Dussoix, J.

Mole. Biol. 41, 459-472 (1969).

Yanisch-Perron et al

Gene 33, 103-119 (1985).

Hanahan, J. Mol. Biol. 166

557-580 (1983).

Palva, I., Gene 19, 81-87 (1982).

E. coli, B. subtilis and Erwinia carotovora strains were cultured in Lleves or L medium [Miller J., 1972, f ** ** ♦ ♦ · 9

Λ «·

«* · ·

Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] at 37 ° C or 28 ° C (Erwinia). Ampicillin (Ap) and kanamycin (Km) were added to the medium at 150 mg / ml and 10 mg / ml respectively to maintain the plasmid.

Starting plasmids pUC9 pUC18

phages

T4GT7

M13 mpl8 / 19

Example 2

Results

Viera & Messing, Gene 19, 25-268 (1982).

Yanisch-Perron et al.,

1985 Gene 33, 103-119

Wilson G.G. and colleagues,

1979. Natúré 280, 80-82

Yanisch-Perron et al.,

1985 Gene 33, 103-119

the. Cloning of the sofA polygalacturonase gene

Erwinia carotovora subsp. carotovora SCC 3193 genomic DNA library was created in plasmid pUC18: chromosomal DNA was isolated from SCC 3193 and partially digested with Sau3A, and the resulting restriction fragments were separated by gel electrophoresis and the 1.5 kb and 5 kb fragments were isolated from the gel. These fragments were ligated with BamHI-digested and dephosphorylated pUC18. The ligation mixture was transformed into competent E. coli HB101 cells. Transformants were selected by plating the transformation mixture, plating on L-plates containing 150 mg / ml ampicillin

I • 4 • «· * <* ·

I snap. Clones encoding polygalacturonase were screened by replicate plate method using LPGA Aβ plates, and positive clones were isolated for further analysis.

One of these clones, namely clone pHSK24, encodes polygalacturonase as determined by enzyme assay of cell extracts.

b. Characterization of the insert in plasmid pHSK24

The DNA insert in plasmid pHSK24 is characterized by the restriction map (Figure 1). The insert has a size of 4.1 kb. In order to determine the location of the soft gene encoding polygalacturonidase in this fragment, a deletion derivative of pHSK24 was constructed. The most to the right 1.7 kb Aval-Smal fragment can be deleted without affecting PG activity (24-2, the Smal site is located in the pUC18 multiple cloning site (mcs) region). Similarly, approximately 800 bp from the left end of the insert can be removed by exonuclease III digestion without affecting enzyme activity. This supports the hypothesis that the soft gene is the 1.6 kb fragment of the most left EcoRI - Aval fragment (Figure 1).

c. The nucleotide sequence of the sofA gene

The nucleotide sequence of the 1.6 kb fragment containing the sofA gene was determined. The sequencing strategy is shown in Figure 2 and the corresponding sequence, together with the deduced amino acid sequence, is shown in Figure 3. The sequence contains only a long open translational reading frame that starts at the 1-position with Met and is followed by a typical GAGG ribosome binding site at the appropriate position [Shine and Dalgarno, Proc. Natl. Acad. Sci. USA 71: 1342-1346 (1974)]. One

the translational stop codon is located at position 1207. The soft protein has a calculated molecular weight of 42,849.

d. Identification of the PG Protein ³⁵ S-labeled plasmid-encoded proteins were isolated by SDS-PAGE and their molecular weights were determined by comparison with molecular weight markers (Sigma MW-SDS-7L).

Analysis of the proteins encoded by the plasmid led to the conclusion that the soft A gene encodes a polypeptide of apparent molecular weight 42,000.

DNA-dependent transcriptional translation analysis of plasmid-encoded proteins yielded a polypeptide of apparent molecular weight of 45,000, suggesting that the product of the soft A gene is synthesized as a precursor.

e. Isolation of PG and determination of its N-terminal amino acid sequence

To demonstrate that PG is indeed synthesized as a precursor, the N-terminal amino acid sequence of mature PG was determined. To accomplish this, plasmid pHSK24 was transduced into Erwinia carotovora subsp carotovora SCC3193 with bacteriophage T4 and selected for the A? ^R transductants. These transductants produce PG several times higher than SCC3193. This overproduction leads to inhibition of the synthesis or secretion of other exoenzymes normally produced by SCC3193. Approximately 90% of the protein in SCC3193 (pHSK24) culture supernatant was PG.

Polygalacturonase was isolated from culture supernatant from overnight culture of SCC3193 (pHSK24) and purified according to the procedures described in the Methods section.

The purified proteins migrate as a single band in the SDS-PAGE assay with an apparent molecular weight of 42,000. The specific activity of the enzyme is 500 U / mg protein. Optimal pH of the enzyme

5.5 (shown in Figure 4), with an optimum temperature of 35-45 ° C (shown in Figure 5).

Polygalacturonase was purified by reverse phase high performance liquid chromatography (Figure 6) and subjected to amino acid sequence analysis for 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 inferred from the nucleotide sequence (139-180, Figure 3) and defines the N-terminus of the mature PG formed.

f. Expression of polygalacturonase gene in B. subtilus

The PehA gene was synthesized by PCR (Mullis et al., Methods Enzymol. 155, 335-350 (1987)] using plasmid pHSK24 as a template to generate modified 5 'and 3' ends. The 3 'end is modified by four additional restriction enzyme sites (EcoRI, BamHI, Smal and NarI). The oligonucleotide primer (oligo 307) is shown in Figure 7. The 5 'end is synthesized to contain 22 C-terminal amino acid residues of the B. amyloliquefaciens α-amylase gene from the Nterminus of mature PG to the 5' end. The signal sequence structure was slightly modified to include additional cloning sites. The oligonucleotide primer (oligo 308) is shown in Figure 7.

The PCR reaction mixture contained 10 μΐ buffer, 16 μΐ 1.2 mM dNTP • · · ·

containing 0.1 nM oligo 307, 0.1 nM oligo 308, 100 ng pHSK24, 0.5 μΐ enzyme and 100 μΐ water. The PCR reaction was run for 25 cycles in a Perkin-Elmer PCR apparatus with incubation times of 92 'C for 1 minute, 55' C for 1 minute and 72 'C for 7 minutes.

The reaction mixture was extracted twice with phenol, precipitated with ethanol, and the precipitate was dissolved in 30 μΐ TE. A 10 μΐ sample was digested with a total volume of 20 μΐ with EcoRI. Following phenolic extraction, 1 μΐ was ligated with EcoRI-digested and CIP-treated pUC9.

The ligation mixture was transformed into E. coli DH5αF 'cells and plated on LX gel, IPTG, Aβ plates. DNA was isolated from four colonies, one of which (# 5) was found to contain sufficient size fragments. 200 ng EcoRI-digested clone # 5 was ligated with 200 ng EcoRI-digested pKTH39 in a volume of 7 μΐ. PKTH39 is a Bacillus subtilis expression vector based on the amylase gene of B. amyloliquefaciens and contains 8 N-terminal amino acid units of the α-amylase signal sequence outside the promoter SD region. Coupled to the 22 C-terminal amino acid units encoded by oligo 307, a complete functional α-amylase signal sequence is formed. As mentioned above, the full disclosure of plasmid pKTH39 is disclosed in co-pending U.S. Patent No. 308,861. The ligation mixture was transformed into B. subtilis BRB1 and plated on L Km plates.

Clones are screened on an agarose gel for the presence of an insert. One clone, BRB679 (BRB679 = BRB1 [pKTH1892], which • »

• * " · · · • ·· · ····· · • · · the··· · · ·

- 28 contains EcoRI fragment in proper orientation, selected for culture.

Clone BRB679 was cultured in double strength L medium supplemented with 0.1 M potassium phosphate pH 7.0, 5% glucose and Km. The overnight culture was diluted 1: 100 in 20 ml of culture medium. The Klettgg is sampled at 100 + 4 hours. From the sample, the PG activity was determined according to which the supernatant had 10,000 U / ml polygalacturonase activity.

Currently, the most preferred method of achieving polygalacturonase selection in host cells is to combine a full-function signal sequence with the polygalacturonase gene. The most suitable for this purpose are the pKTH50-59 vectors disclosed in U.S. Patent Application Serial No. 308,861. The modified soft gene encoding mature polygalacturonase is introduced into a vector selected from the group pKTH50-59 and used to transform host cells. Expression of the polygalacturonase gene is determined as extracellular polygalacturonase activity after culturing the host in a suitable propagation medium.

G. Polygalacturonase gene expression in Lactobacillus and Lactococcus

Using the vectors described above, the polygalacturonase gene is expressed in Lactobacillus and Lactococcus host cells. A detailed description of a suitable process can be found in Examples V, VI, VII and X of U.S. Patent Application Serial No. 377,450, which is incorporated herein by reference in its entirety.

I will build it.

Briefly, plasmids, exemplified by pKTH1797, pKTH1798, ΡΚΤΗ1799, pKTH1801, pKTH1805, pKTH1806, pKTH1807 and pKTH1809, contain promoter and secretion enhancer signals. The promoter and secretion promoter sequences of plasmids may direct expression of heterologous genes and selection of the gene product in Gram-positive host cells such as Lactobacillus and Lactococcus.

To effect expression of the polygalacturonase gene and selection of the gene product, a host cell, such as Lactobacillus or Lactococcus, is transformed with a plasmid containing the promoter and selection sequences described above and the gene encoding the mature soft protein as described above. The host cell is cultured in an appropriate medium under conditions that allow expression of the protein and the protein is recovered from the medium.

The foregoing description of the invention will make it apparent to those skilled in the art that many variations and modifications thereof may be made within the scope of the invention.

Claims (28)

Claims The nucleotide sequence shown in Figures 3a, b, c and d encoding a polygalacturonase protein or a functional derivative thereof. The nucleotide sequence of claim 1, isolated from Erwinia carotovora or a functional derivative thereof. 3. The substantially pure nucleotide sequence or functional derivative of Figures 3a, b, c and d. 4. A plasmid comprising the nucleotide sequence of any one of claims 1 to 3. 5. A host cell transformed with the plasmid of claim 4. A host cell according to claim 5, characterized in that it is a Bacillus, Lactococcus or Lactobacillus. 7. The host cell of claim 6, wherein said host cell is BRB679. 8. A hybrid expression unit comprising the plasmid of claim 4 and a secretory vector. The hybrid expression unit of claim 8, wherein the secretion vector is plasmid pKTH39. A method for producing a polygalacturonase in a host cell, comprising transforming a host cell with the plasmid of claim 4, culturing the transformed host cell in a suitable medium and conditions for expression of the protein, and recovering the expressed protein from the host cell or medium. 11. Plasmid or a functional derivative of pHSK24. 12. A host cell transformed with a plasmid according to claim 11, wherein the host cell is a Gram-negative bacterium. 13. A hybrid expression unit comprising the plasmid of claim 11 and a secretory vector. 14. The hybrid expression unit of claim 13, wherein said secretion vector is pKTH39. 15. A polypeptide or functional derivative thereof encoded by the substantially pure nucleotide sequence shown in Figures 3a, b, c and d. 16. A polypeptide or functional derivative having the amino acid sequence depicted in Figures 3a, b, c and d. A polypeptide or functional derivative having the amino acid sequence depicted in Figures 3a, b, c and d, which is synthesized by a host cell transformed with the plasmid of claim 4. 18. The polypeptide of claim 17, wherein said host cell is BRB679. A polypeptide encoded by a substantially pure nucleotide sequence shown in Figures 3a, b, c and d, which is synthesized by a host cell transformed with the hybrid expression unit of claim 8. 20. A polypeptide or functional derivative having the amino acid sequence depicted in Figures 3a, b, c and d, which is depicted in Figure 8. • · * - A host cell transformed with a hybrid expression unit according to claim 32. 21. The polypeptide of claim 19, wherein the host cell is BRB679. 22. The polypeptide of claim 20, wherein the host cell is BRB679. 23. A polypeptide or functional derivative encoded by a substantially pure nucleotide sequence shown in Figures 3 a, b, c and d, which synthesizes a host cell transformed with the plasmid of claim 4. 24. The polypeptide of claim 23, wherein the host cell is BRB679. A polypeptide or functional derivative thereof encoded by a substantially pure nucleotide sequence shown in Figures 3a, b, c and d, which is synthesized by a host cell transformed with the hybrid expression unit of claim 13. 26. A polypeptide or functional derivative comprising the amino acid sequence shown in Figures 3a, b, c and d, which is synthesized by a host cell transformed with a hybrid expression vector according to claim 13. 27. The polypeptide of claim 25, wherein the host cell is a Gram-negative bacterium. 28. The polypeptide of claim 26, wherein the host cell is a Gram-negative bacterium.