ES2343665A1 - New chitinase of bacterial origin with broad fungicide spectrum - Google Patents

Abstract

The present descriptive specification describes a new DNA sequence having strong promoter activity in bacteria and a method for expressing a gene which determines the synthesis of a bacterial chitinase having a broad fungicide spectrum employing said sequence. It also describes a method for producing said chitinase in bacteria using the sequence described, in particular a method for producing the product of the desired gene in large quantity in the bacterial cell and obtaining it secreted to the cell exterior.

Description

New bacterial origin chitinase with broad fungicidal spectrum

State of the art

Chitin is widely distributed in nature, and very importantly as a polysaccharide structural in the cell wall of fungi, reaching up to 16% of the total dry weight in filamentous fungi and basidiomycetes It is a water-insoluble homopolymer of character structural formed by n repetitions of N-acetyl glucosamine linked by β bonds (1-4). It is also found in the arthropod exoskeleton, covered with crustaceans, etc. It is one of the most abundant polymers in the Nature after cellulose.

Chitin is therefore the compound most important in the maintenance of the fungal wall structure, so it could be used as a target to obtain soluble protein compounds of antifungal action.

The enzyme known as chitinase is present in a wide range of organisms, particularly microorganisms and frequently of bacterial origin, and its action determines the hydrolysis of the chitin polymer preventing the viability of the fungus.

Among the organisms that express chitinase are They find viruses, bacteria, fungi and plants. The function of this Enzyme in each organism is diverse, playing important roles physiological and ecological: in invertebrates it is required for partial degradation of its former exoskeletons, in plants such as a defense mechanism against pathogenic fungi, and in bacteria, for the functions of nutrition and parasitism, being able to apply for the control of phytopathogens in agriculture. Nowadays the agriculture has become more vulnerable to fungal pests due, among other causes, to the limited genetic variation that present large-scale crop plants. Mushrooms proliferate rapidly, stimulated by nutrient-rich soil and wet, exerting a devastating effect on crops. Not only affect agriculture, the human being can also be a host of these opportunistic pathogens, particularly when It is in immunosuppression states.

Bacillus subtilis chitinase (EC 3.2.1.14) is an enzyme of the 18 family of glycosyl hydrolases that catalyzes the degradation of chitin. Bacillus subtilis is one of the microorganisms capable of secreting a wide variety of extracellular enzymes degrading polysaccharides, which makes it an ideal microorganism for the secretion of proteins of commercial interest.

Genetic engineering and biology techniques molecular facilitate the overproduction of enzymes in microorganisms, which can be used in the processes of industrial fermentation to produce large quantities of a particular enzyme From an industrial point of view, produce the greater amount of a given enzyme is necessary to resort to the techniques mentioned above to overproduce the majority of the enzymes used in this industry.

Description

The strain of Bacillus subtilis 168 presents within its genome the sequence SEQ ID 7, which despite being sequenced, did not have up to the date described, nor associated by homology to similar sequences in other genomes, any specific function. In the present invention the function of this particular gene is determined as a coding agent for a new chitinase, the main aspect of the present invention.

The present invention relates to a new chitinase enzyme encoded by Bacillus subtilis 168, to a method for producing said chitinase and its use as a fungicide.

On the other hand, the fungicidal effect, both in vivo and in vitro , of the chitinase of the present invention on different species of fungi is described below, demonstrating the effectiveness thereof.

Therefore, a first aspect of the invention refers to a polypeptide encoded by a polynucleotide that It has at least 70% homology with SEQ ID 1, which presents the at least 80% homology with SEQ ID 1, which has at least 90% of homology with SEQ ID 1 or consisting essentially of SEQ ID 1. From now on we will refer to it as a polypeptide of the invention.

A second aspect of the invention relates to the gene construct comprising a polynucleotide sequence encoding the polypeptide as defined in the claim previous and:

to.

a polynucleotide sequence comprising SEQ ID NO 2,

b.

nucleic acid molecules whose chain complementary hybrid with the polynucleotide sequence of a), or

C.

nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic.

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A third aspect of the plasmid invention recombinant comprising:

-

the gene construct according to the preceding claim,

-

the cat gene,

-

the origin of replication of plasmid pRMIcat from Escherichia coli and plasmid pPCT2 for Bacillus subtilis ,

-

a gene promoter reporter, and

-

a gene Kanamycin resistance.

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Another aspect of the invention relates to method of obtaining the polypeptide of the invention comprising introduce the gene construct according to claim 2 or the recombinant plasmid according to claim 3, in a cell host and incubate the host cell according to a) in a medium of adequate cultivation

A preferred embodiment of said method further comprises purifying the polypeptide obtained. In another preferred embodiment the host cell is selected from the group comprising E. coli and B. subtilis .

Another aspect of the invention relates to the use of the polypeptide of the invention for the preparation of a drug with chitinase activity.

Finally another aspect of the invention is the use of the polypeptide of the invention in the preparation of a drug with fungicidal activity.

Additionally, it is described herein invention a method that allows the overproduction of proteins from biological interest, preferably extracellularly, in this case, overproduction of the enzyme of interest, polypeptide of the invention. For this, a specific promoter is used. This method Expression promotion can also be used for expression of other genes of interest, by inclusion in the Recombinant plasmid described in the present invention.

Specifically, an expression vector is described to overproduce proteins regulated by a B. subtilis promoter or to induce the secretion of the proteins to the outside environment, the cells transformed with this vector and the use thereof for the overproduction of the hydrolytic enzyme. extracellular chitinase.

The use of the promoter used for overexpression is not limited to the gene for the chitinase of B. subtilis , but allows to express other homologous or heterologous proteins since the use of the csn promoter sequence (SEQ ID NO 2) for the regulation of The expression of genes of interest produces increases in them compared to the use of the promoters of these genes.

In the present invention it is described in detail the method of making the pCSN73 recombinant plasmid by amplification of SEQ ID NO 3, using primers specific, preferably primers comprising SEQ ID NO 4 and SEQ ID NO 5 and ligated amplified DNA fragment with the plasmid shuttle pNR2.

A method of making a plasmid is also described, which comprises amplification by specific primers of the nucleotide sequence of B. subtilis, which in turn comprises the coding sequence for the mature protein of the yvbx gene and the factor independent transcription terminator. rho termination; subsequent endonuclease digestion of the amplified fragment and plasmid pCSN73; purification of the fragments obtained in digestion; the defoforilation of the 5 P ends of the plasmid; and ligation of purified fragments. Preferably, the method of making the plasmid of the invention uses primers comprising SEQ ID NO 6 and SEQ ID NO 7.

Such plasmids can be used in the transformation of bacteria, for example but not limited to, the transformed bacteria are selected from the group comprising E. coli and B. subtilis .

The term essentially, as used in the present invention, it refers to the sequences resulting from amino acid elimination from regions N-terminal and / or C-terminal of any of the sequence SEQ ID NO: 1.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The The following examples are provided by way of illustration, and are not It is intended to be limiting of the present invention.

Brief description of the figures

Fig. 1. In vivo assay of chitinase fungicidal activity on Fusarium proliferatum . The dilutions of the fungus culture tested are indicated.

Fig. 2. In vivo assay of the fungicidal activity of chitinase on Aspergillus ochraceus . The dilutions of the fungus culture tested are indicated

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Examples of realization

The bacterial strains used have been: Bacillus subtilis 168 provided by F. Kunst and Escherichia coli MC1061 and the latter has been used for the propagation of the plasmid constructed in this invention.

The methods used for the growth and manipulation of the strains have been performed according to Sambrook et al. [SambrooK, Firtsch and Maniatis, 1989. "Molecular cloning". A laboratory manual . Cold Spring Harbor Laboratory Press].

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Example 1 Preparation of recombinant plasmid pCSN73

The promoter of the present invention, which has a high promoter activity, was identified by sequencing a genomic library of Bacillus subtilis 168 [Anagnostopoulos C, Piggot, PJ and Hoch, JA (1993). "The genetic map of Bacillus subtilis ". In Bacillus subtilis and other Gram-positive Bacteria : Biochemistry, Physiology and Molecular Genetics, pp. 425-461. Edited by AJ Sonenshein, JA Hoch & R. Losick. Washington, DC: American Society for Microbiology] at position 2748.10 kb on the chromosome.

This DNA sequence is located in the promoter region of the csn gene that encodes an extracellular protein with chitosanase activity (accession no .: X92868) and described by Victor Parro and collaborators [Victor Parro, Marta San Román, Immaculate Galindo, Bénédicte Purnelle, Alexei Bolotin, Sorokin and Rafael P. Mellado. (1997). "A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon." Microbiology 143: 1321-1326].

SEQ ID NO 3 shows the 445 nucleotide DNA fragment obtained from the DNA sequence of the Bacillus subtilis 168 chromosomal region comprised between bases 2,748,117 and 2,748,562 of the sequence published by Kunst et al . [Kunst, F. et al ., (1997). "The complete genome sequence of the Gram-positive bacterium Bacillus subtilis ". Nature, vol 390: 249-256]. This DNA sequence includes a promoter DNA sequence that contains a region similar to the TATA box located approximately 10 bp in front of the transcription initiation point and that induces the transcription initiation of RNA polymerase (nucleotides 1-295). The promoter sequence is presented isolated in SEQ ID NO 2.

From this sequence (SEQ ID NO 3), they designed two oligonucleotides that allowed to amplify by PCR a 295 bp DNA fragment containing the promoter region (prcsn1-prcsn2 oligonucleotides).

SEQ ID NO 4: prcsn1: (direct)

SEQ ID NO 5: prcns2: (inverse).

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The 295 bp DNA fragment was amplified by the oligonucleotide pair prcsn1-prcsn2 from Bacillus subtilis 168 genomic DNA. Bacillus subtilis 168 cells obtained by centrifugation from 20 ml of a culture in LB medium, in exponential phase, were lysed following the method of Cutting and Van der Horn [Simón M. Cutting and Peter B. Van der Horn. (1990). "Genetic Analysis." Molecular Biological Methods for Bacillus . Ed. CR Harwood and SM Cutting]. In a PCR tube 10 µl of enzyme buffer, 1 mM dNTPs of final concentration, Cl 2 Mg 1.5 mM of final concentration, 280 ng of each of the oligonucleotides, 500 ng of chromosomal DNA from were added Bacillus subtilis 168 and 1 U of DNA polymerase (Ecogen), completing the final volume at 100 µl. Each PCR cycle consisted of: 1 'at 94 ° C; 5 'at 55 ° C and 2' at 72 ° C for 30 cycles and was performed in a MTC PTC-1000 model thermal cycler. Research, Inc.

The DNA fragment obtained by PCR was ligated to the shuttle plasmid pNR2, which contains the kanamycin resistance gene and the origin of replication of plasmid pPCT2 for Bacillus subtilis , the cat gene, a promoter "reporter" gene encoding the enzyme. chloramphenicol acetyltransferase and the origin of replication of plasmid pRMIcat from E. coli constructed by Parro V. et al. [Victor Parro and Rafael P. Mellado (1993) "Heterologous recognition in vivo of promoter sequences from the Streptomyces coelicolor dagA gene". FEMS Microbiol. Letters 106: 347-356].

Plasmid pNR2 was propagated in a culture of competent E. coli MC1061 cells. The strain was inoculated in 10 ml of LB medium with 100 mg / ml ampicillin and incubated at 37 ° C for 12 h. The culture was centrifuged and the resulting pellet was used to extract the plasmid with the Promega Wizard plus SV minipreps kit.

The plasmid obtained was digested with the Smal restriction enzyme and each fragment obtained by PCR was ligated. The ligaments were transformed into E. coli MC1061 cells and the transformants obtained in LB with 100 µg / ml ampicillin were selected by the filter hybridization technique. The DNA was denatured and immobilized on a nylon membrane (Hymers-N + from Amersham) and hybridized in a solution of SSC 6X, 0.1% SDS, 6 mg / ml Salmon sperm and Denhardt's solution with the fragment obtained by PCR and labeled with the klenow fragment of the DNA polymerase at its 5'OH ends with αdNTP (p32). After hybridization, the non-specific adsorption was washed and the filter was autoradiographed to identify positive clones. The transformants were hybridized at 65 ° C overnight checking the presence and orientation of the insert by sequencing with the prcsn1 or prcsn2 oligonucleotides. The resulting plasmid was named pCSN73 and contains the csn promoter oriented in the direction of the cat gene.

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Example 2 Preparation of the recombinant plasmid of the invention and obtaining cells transformed with said plasmid

Two oligonucleotides were designed from the DNA sequence of the yvbX gene of Bacill? S subtilis 168 published by Kunst et al . [Kunst, F. et al ., (1997). called:

Yvbx2: SEQ ID NO 6

Yvbxt1h: SEQ ID NO 7

The sequence of these oligonucleotides (Boehringer Ingelheim) starts from the 5 'end and ends at the 3' end. To the first one a 5 'end was incorporated with a Bam HI restriction target, and to the second a 5' end with a Hind III target to obtain cohesive ends.

The DNA fragment used for cloning the DNA sequence of the mature protein of the yvbX gene was obtained by the PCR technique (model PTC-1000 of MJ. Research, Inc), using a hybridization temperature of 50 ° C. In a PCR tube 10 µL of enzyme buffer, 1 µL of deoxynucleotides (10 mM dNTPs), 1.5 mM Cl2 Mg of final concentration, 280 ng of each of the oligonucleotides, 500 ng were added of Bacillus subtilis 168 and 1 U DNA polymerase DNA (Ecogen Ecotaq), completing the final volume at 100 µl. The PCR amplification product obtained has a size of 1965 base pairs and comprises a DNA sequence containing the coding sequence for the mature protein and a transcription terminator independent of the rho termination factor.

A recombinant culture of E. coli containing plasmid pCSN73 was grown in LB medium with 100 µg / ml ampicillin. Plasmid DNA was obtained with the Promega Wizard plus SV kit following the manufacturer's instructions: The pellet obtained was resuspended by centrifugation of 5 ml of culture in 250 ml of resuspension buffer to which 250 µl of lysis buffer was added. The two solutions were mixed until a clear supernatant was obtained and 10 µl of alkaline protease was added. The samples were incubated for 5 min at room temperature then adding 350 µl of neutralization solution. The tubes were inverted and centrifuged at 13,000 rpm for 10 min. The resulting supernatant was collected and purified on a Wizard plus SV minipreps spin column, which was washed with 750 µl of wash solution. The columns were then centrifuged and 100 µl of nuclease-free water was added to extract the retained DNA. The columns were centrifuged again at 13,000 rpm for 10 min and the solution was collected with the purified plasmid.

The PCR fragment obtained and the plasmid pCSN73 DNA were digested with the restriction endonucleases Bam HI and Hind III, using 100 ng of DNA in each case, to obtain cohesive ends, releasable from each other. The samples were digested in a final volume of 25 µl, adding 2.5 µl of enzyme buffer, 1 µl of enzyme and sterile distilled water to each tube until the final volume was completed. Both fragments were purified on a 1% low melting agarose gel following the procedure described by Parro, V and Mellado, RP [Parro V. and Pérez Mellado R. (1997). "Procedure for the overproduction, purification and use of Streptomyces coelicolor agarase". National Patent No. 9700090].

The 5'P ends of the plasmid were dephosphorylated with alkaline phosphatase (Amersham) that breaks the phosphate bonds of the 5 'ends of the DNA to avoid recircularization of the plasmid In a final reaction volume of 20 µl they were incubated 100 ng of plasmid, 2 µl of phosphatase buffer and 1 µl of alkaline phosphatase for 1 h at 37 ° C.

The 1965 bp DNA fragment is ligated to the plasmid, using 100 ng of plasmid DNA and 5 times the amount of DNA to be ligated to the plasmid and the necessary milliliters were added to ensure adequate proportion of each DNA fragment in one volume. end of 10 \ mul. 1 µl of enzyme buffer and 1 µl of T4 DNA ligase was added. Each reaction was incubated between 4 and 16 h at 16 ° C. 5 µl of the ligation was transformed into E. coli MC1061 and the cells were plated in LB medium with 100 µg / ml ampicillin to select the transformants.

100 transformants were selected that were replicated in LB agar with 100 µg / ml ampicillin. Clones selected were inoculated in 5 ml of LB medium with ampicillin (100 µg / ml) and incubated at 37 ° C and 250 rpm for 12 h. For check the presence and orientation of the insert were digested plasmids obtained by mini-preparations, as described previously. Plasmids were digested with BamHI and with HindIII and restriction fragments were checked on an agarose gel from 1%.

A clone called pCyvbx was selected.

The plasmid thus obtained was used to transform Bacillus subtilis 168 by selecting the transformants in LB plates with kanamycin (10 µg / ml) as described above for E. coli clones. The cloned DNA fragments were sequenced in an automatic sequencer verifying that they corresponded to the sequence of the cloned fragment and did not contain errors.

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Example 3 Chitinase production assays

Circular Hybond-N + filters (Amersham Biosciences) were used on plates of YPG medium supplemented with the corresponding antibiotic, where the culture of B. subtilis grown to an OD 600nm of 0.8 was seeded. It was incubated overnight at 37 ° C, sufficient period for the microorganism to release the secretion proteins to the medium between which the chitinase is found and which are capable of crossing the filter.

The filter was subsequently removed (with the bacterial biomass) and the mushroom to be tested undiluted (SD) and in serial dilutions (1/5, 1/10, 1/20, 1/50 and 1/100) to observe The effect on growth. When the fungi were tested undiluted sample was used undiluted sample (SD) and dilutions of greater range (10 -1, 10 -2, 10-3, 10-4 and 10-5).

The fungi used in the in vivo activity tests are listed in the following table 1 and the results obtained are indicated in Table 2.

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TABLE 1 List of fungi used

one

TABLE 2 In vivo assay of the fungicidal activity of the overproduced chitinase

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2

\ text {+++} Good growth in the middle solid.

N.D. = not determined.

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These results are completed with those shown in Figures 1 and 2 where the fungicidal activity of chitinase on fungi F? Sarium proliferatum and Aspergillus ochraceus respectively is observed.

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Example 4 Quantification of chitinase overproduction

The chitinase activity produced by the Recombinant bacterium was assessed by supernatant assay of the cultures of the raw producing bacteria using the substrate chromogenic chitin-azure (Sigma) in 0.1 M buffer phosphate at pH 6.9 for 3 h in a final volume of 2 ml and estimation of the release of the chromogen in the spectophotometer a an optical density of 560 nm. A chitinase unit is defined as the 0.01 increase in the measurement of optical density. The Recombinant bacteria produce up to 4 times more active chitinase than the control bacterium that propagates the vector plasmid without the gene inserted.

In addition, the presence of chitinase was checked in the extracellular medium by electrophoresis gels 10% SDS-PAGE observing a majority band of 31 kDa relative molecular mass that was absent in the equivalent fractions of the non-overproductive bacteria and that corresponds to the expected molecular size for chitinase mature extracellular.

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gcgttttaac gtttgattgt cg

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22

Claims (8)

1. Polypeptide encoded by a polynucleotide which has at least 70% or 80% or 90% homology with SEQ ID 1 or consisting essentially of SEQ ID 1. 2. Gene construction comprising a polynucleotide sequence encoding the polypeptide as defined in the preceding claim and:

to.

a polynucleotide sequence comprising SEQ ID NO 2,

b.

nucleic acid molecules whose chain complementary hybrid with the polynucleotide sequence of a), or

C.

nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic.

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3. Recombinant plasmid comprising:

-

a gene construct according to the preceding claim,

-

the Gen cat

-

the origin of replication of plasmid pRMIcat from E. coli and plasmid pPCT2 for B. subtilis ,

-

a gene promoter reporter, and

-

a gene Kanamycin resistance.

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4. Method of obtaining a polypeptide according to claim 1 comprising:

to).

Enter a gene construct according to claim 2 or a recombinant plasmid according to the claim 3, in a host cell e

b).

Incubate the host cell obtained in a) in a suitable culture medium.

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5. Method according to the preceding claim, which It also comprises purifying the polypeptide obtained. 6. A method according to any of claims 4 to 5, wherein the host cell is selected from the group comprising E. coli and B. subtilis . 7. Use of a polypeptide according to claim 1 for the preparation of a drug with chitinase activity. 8. Use of a polypeptide according to claim 1 for the preparation of a drug with fungicidal activity.