WO1997004106A2 - New saccharomyces cerevisiae pectinase sequences and heterologous expression systems derived therefrom - Google Patents

New saccharomyces cerevisiae pectinase sequences and heterologous expression systems derived therefrom Download PDF

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Publication number
WO1997004106A2
WO1997004106A2 PCT/EP1996/003091 EP9603091W WO9704106A2 WO 1997004106 A2 WO1997004106 A2 WO 1997004106A2 EP 9603091 W EP9603091 W EP 9603091W WO 9704106 A2 WO9704106 A2 WO 9704106A2
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Prior art keywords
polynucleic acid
sequences
sequence
pectinase
acid sequence
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PCT/EP1996/003091
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French (fr)
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WO1997004106A3 (en
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Thierry Scarcez
Annie Van Broekhoven
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Innogenetics N.V.
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Priority to AU66568/96A priority Critical patent/AU6656896A/en
Publication of WO1997004106A2 publication Critical patent/WO1997004106A2/en
Publication of WO1997004106A3 publication Critical patent/WO1997004106A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/231—Pectin; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

Definitions

  • the present invention relates to the field of yeast genes and heterologous yeast expression systems. More particularly the present invention relates to new pectinases, nucleic acids encoding the same, as well as homologous and heterologous expression methods using the regulatory sequences and/or coding sequences comprised in said pectinase nucleic acids.
  • yeast expression systems can be explained by its various advantages it has over both bacterial and mammalian systems. It is a generally regarded as safe (GRAS) organism, having a long historical background in the food indusrty. By contrast, Escherichia coli contains endotoxins, and mammalian cells may contain viral or oncogenic DNA, requiring extensive testing of products derived from these organisms. Yeast is practically as easy to manipulate as E. coli. Its genetics are well understood.
  • High-density cultures can be obtained using relatively simple, substantially protein-free culture media, and large-scale cultures are feasible.
  • S. cerevisiae secretes only 0.5 % of its own proteins, the secretion of heterologous proteins can be achieved, so that a secreted foreign protein is present in the culture medium in a nearly pure form.
  • yeats is capable of higher forms of posttranslational protein processing and can glycosylate secreted proteins - all at the fraction of the cost of a mammalian system. Therefore, it is not surprising that the first commercialized recombinant vaccine, hepatitis B vaccine, was yeast-derived.
  • Pectinases are synthesized by plants and by a variety of microorganisms.
  • Polygalacturonase is one of the best characterized pectinolytic activities. This enzyme cleaves randomly glycosydic bonds of pectic acids or polygalacturonates, producing mono-, di- and oligogalacturonates as final hydrolysis products.
  • the long-standing intrest in pectinases resides in their role in the invasion of plant tissues by phytopathogens, the spoilage of fruits and vegetables and also in their food processing and plant biological applications.
  • the aim of the present invention is to provide polynucleic acid sequences encoding a novel pectinase, more particularly polynucleic acid sequences encoding a yeast pectinase.
  • Another aim of the present invention is to provide a novel pectinase, more particularly from yeast, particularly in a purified and isolated form.
  • Another aim of the present invention is to provide amino acid sequences of these pectinases.
  • Another aim of the present invention is to provide recombinant pectinase, more particularly recombinant yeast pectinase, as well as methods for preparing the same.
  • Another aim of the present invention is to provide recombinant vectors comprising polynucleic acid sequences from the coding and/or non-coding regions of pectinase encoding genes for cloning and/or homologous or heterologous expression purposes.
  • the present invention aims at providing coding or regulatory sequences of new pectinase genes, more particularly of yeast pectinase genes, for heterologous expression purposes.
  • the present invention aims at providing a heterologous yeast expression system wherein said coding or regulatory sequences are used to express heterologous proteins.
  • Another aim of the present invention is to provide probes or primers derived from said new polynucleic acid sequences.
  • Another aim of the present invention is to provide host cells transformed with said recombinant vectors.
  • Another aim of the present invention is to provide methods for producing heterologous or homologous recombinant proteins using said transformed host cells.
  • Another aim of the present invention is to provide fragments or derivatives of said pectinases which have retained the pectinase activity.
  • Another aim of the present invention is to provide for the use of said (recombinant) pectinase for food processing.
  • Another aim of the present invention is to provide monoclonal antibodies which are specifically directed towards said novel pectinase.
  • the present invention relates more particularly to a polynucleic acid sequence in substantially isolated form comprising at least part of a sequence selected from:
  • the Examples section of the present invention teaches the isolation and characterization of a new yeast gene located on yeast chromosome 10. Upon homology comparison with sequences in the databank, limited homology could be found with pectinase sequences from other organisms. Recombinant expression of the open reading frame (ORF) comprised in said new sequence allowed to ascribe a pectinase activity to the new yeast sequence.
  • ORF open reading frame
  • Pectinases which are quivalent or highly homologous to the yeast pectinase as shown in SEQ ID NO 1 of the present invention are likely to exist in other related organisms as well as in other yeast strains. It is to be understood that these pectinases are different from known pectinases.
  • substantially isolated form refers to a purity grade of at least 90%, preferably 95 %, and more particularly 98% as determined by dry weight of the polynucleic acid fraction versus contaminants.
  • polynucleic acid corresponds to either double-stranded or single-stranded cDNA or genomic DNA, or RNA.
  • a polynucleic acid may contain from 10 nucleotides to the complete nucleotide sequence (such as for instance 20, 30, 40, 50, 60, 70, 80 or more nucleotides).
  • a polynucleic acid which is smaller than about 100 nucleotides is often also referred to as an oligonucleotide.
  • a polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes.
  • polynucleic acids of the invention are to be understood as also comprising the complement of the sequence of the top line in Figure 5 or to the nucleic acid as shown in SEQ ID NO 1 .
  • complement refers to a nucleotide sequence which is complementary to an indicated sequence and which is able to hybridize to the indicated sequence.
  • hybridizes refers to conventional hybridization conditions known to the man skilled in the art, preferably to stringent hybridization conditions (see f .i. Maniatis et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982). Under certain conditions hybridization can be allowed between sequences showing a homology of at least 70%, at least 80%, at least 90%, at least 95 % or more.
  • the polynucleic acid according to option (a) can be prepared according to the procedures as set out in the Examples section of the present invention.
  • polynucleic acids may also be derived according to any other procedure obvious from the teaching of the present invention or any other procedure known to the man skilled in the art for preparing polynucleic acid sequences.
  • the polynucleic acids according to options (b) and (c) can be prepared by applying techniques of hybridization, cloning and/or recombinant expression known to the man skilled in the art.
  • the polynucleic acids according to option (c) may be derived by cloning equivalents of the polynucleic acids as depicted in SEQ ID NO 1 from the nucleic acids of other related organisms or of other yeast strains. This may be achieved by screening libraries containing polynucleic acids of other related organisms with probes derived from the polynucleic acid as given in SEQ ID NO 1 . Alternatively, said equivalents may be cloned by performing an amplification reaction, such as PCR, of mRNA, with primers essentially comprising a nucleotide sequence which is part of the polynucleic acid sequence given in SEQ ID NO 1 , to obtain amplified products.
  • the polynucleic acids according to (c) may also be provided for by classical chemical synthesis methods of oligo- or polynucleotides generally known by the person skilled in the art.
  • a polynucleic acid sequence according to the present invention comprises a sequence of preferably 10, 20, 30, 40, 50, 60, 100, 200, 500, 1000 or more nucleotides selected from the sequence as depicted in Figure 5 (SEQ ID NO 1 ), or their complement, or sequences which are degenerate as a result of the genetic code or which hybridize to any of the foregoing sequences.
  • Especially preferred parts of the polynucleic acid sequences of the present invention include regulatory sequences such as promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences, signal sequences, as well as specific parts of the coding sequences (see below).
  • the present invention also relates to a polynucleic acid sequence as defined above comprising at least 10 contiguous nucleotides, for use as a specific hybridization probe for detecting the presence of any target sequence comprising a polynucleic acid according to the present invention, as defined above.
  • probe refers to single-stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to a target sequence to be detected or cloned. Probes may be labelled according to any of the techniques known in the art. Preferably these probes are about 10 to 50 nucleotides long. According to the hybrization solution used, these probes should be hybridized at appropriate temperatures and be of appropriate length to attain sufficient specificity.
  • the present invention relates to polynucleic acid sequences as defined above comprising at least 10 contiguous nucleotides, for use as primer for amplification of any polynucleic acid sequence according to the present invention, as defined above.
  • primer refers to a single-stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied.
  • the length and the sequence of the primer must be such that they allow to prime the synthesis of the extension product.
  • the primer is about 10 to about 50 nucleotides long. Specific length and sequences will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.
  • the amplification method used can be any method known in the art.
  • Probes and primers according to these aspects of the present invention may be used to isolate genes encoding equivalents or proteins which are highly homologous to the polynucleic acid sequence of SEQ ID NO 1 in organisms other than Saccharomyces cerevisiae strain FY1679 from which SEQ ID NO 1 was obtained.
  • Other S. cerevisiae strains include for instance INVSCI, DBY745, AH22, etc.
  • equivalents to the protein of SEQ ID NO 1 may be detected in other organisms than yeast, preferably in a yeast-related organism.
  • the present invention relates more particularly to at least part of any polynucleic acid sequence having a homology of at least 75-85 %, preferably at least 80-90% , more preferably 85-95% with the polynucleic acid sequence as shown in Figure 5 (SEQ ID NO 1 ).
  • the present invention relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising at least part of a polynucleic acid sequence as defined above.
  • polynucleic acid sequence comprised in said recombinant vector of the invention may comprise:
  • vector may comprise a plasmid, a cosmid, a phage or a virus.
  • said vector will be a plasmid.
  • Particularly preferred plasmids (or vectors) for the expression of coding sequences derived from the polynucleic acid sequences according to the present invention in yeast include for instance pYES2 (Invitrogen, CA, USA) including the GAL-1 promoter and CYC1 terminator sequences or the 4,3 kb Pstl fragment of SEQ ID NO 1 cloned in pJDB207.
  • Particularly preferred plamsids (or vectors) for heterologous expression of proteins using the regulatory sequences from the polynucleic acid sequences according to the present invention include for instance derivatives of pPECGAL, pJDB207PECs and pJDB207PECas as disclosed in the Examples section.
  • coding sequence refers to a polynucleic acid sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the ⁇ '-terminus and a translation stop codon at the 3"-terminus.
  • a coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences.
  • the coding sequences of the present invention encode a pectinase.
  • operably linked refers to a juxtaposition wherein the components are figured so as to perform their usual function.
  • control sequences or regulatory sequences operably linked to a coding sequence are capable of effecting the expression of a coding sequence.
  • control sequence refers to those sequences which control the transcription and/or translation of the coding sequences; these may include but are not limited to promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences.
  • control sequences refer to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controling secretion, protease cleavage, and glycosylation of the polypeptide.
  • the signal peptide encoding sequence (and the signal peptide encoded by it) as well as any other of the above-mentioned regulatory sequences of the polynucleotides of the invention form an important aspect of the present invention as such, and it is also contemplated that these regulatory sequences may be inserted upstream and/or downstream of DNA sequences coding for heterologous proteins or peptides so as to obtain expression and/or secretion of the resulting products from yeast host cells.
  • these signal sequences may particularly be used for expression in any recombinant system such as: yeast, or any other bacterial, mammalian or viral host cell system.
  • the present invention relates to a recombinant vector as defined above, more particularly a plasmid, further comprising a nucleotide sequence encoding a heterologous protein or peptide which is desired to be expressed within said vector sequence in such a way that said heterologous protein is expressed under the control of regulatory sequences of the polynucleic acids as defined above.
  • proteins or peptides to be expressed may include, but are not limited to, complete polypeptides or peptides derived therefrom from bacterial, animal, mammalian, human or viral origin.
  • the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor sequence included in any of the polynucleic acid sequences according to the present invention, as defined above.
  • the present invention relates to a recombinant vector (e.g. a plasmid) comprising a yeast pectinase promoter sequence selected from the polynucleotide sequences as defined above.
  • a recombinant vector e.g. a plasmid
  • yeast pectinase promoter sequence selected from the polynucleotide sequences as defined above.
  • yeast pectinase promoter sequences contained in SEQ ID NO 1 More particularly comprising the yeast pectinase promoter sequences contained in SEQ ID NO 1 , more particularly in the region spanning position 1 to 1771 thereof.
  • the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor/secretion signal sequence included in any of the polynucleic acid sequences according to the present invention, as defined above.
  • the present invention relates to a recombinant vector, more particularly a plasmid, comprising a pectinase promotor/secretion signal sequence derived from yeast, selected from the sequences present on the nucleotide sequence as depicted in SEQ ID NO 1 , more particularly in the region spanning position 1 to approximately
  • the present invention further relates to recombinant vectors comprising the signal peptide encoding sequence comprised in any of the polynucleic said sequences of the present invention. More particularly, the signal peptide encoding sequence of a yeast pectinase, selected from the sequences as depicted in SEQ ID NO 1 , more particularly in the region spanning position 1772 to approximately position 1828 of SEQ ID NO 1 .
  • the present invention also relates to recombinant vectors comprising the transcription/translation stop regulatory sequences comprised in any of the polynucleic eaid sequences of the present invention. More particularly a transcription/translation stop regulatory sequence of a yeast pectinase as depicted in SEQ ID NO 1 , more particularly in the region spanning positions 2855 to position 4300 of SEQ ID NO 1 .
  • the present invention also relates to recombinant vectors comprising a polynucleic acid as defined above with said polynucleic acid comprising a deletion of at least part of the coding region for heterologous expression of foreign proteins or peptides in the place of said deletion and in the same reading frame as the original coding sequence. More particularly, a deletion in the coding region of a yeast pectinase gene as shown in SEQ ID NO 1 . For instance a deletion of the whole region or part of the region comprised between spproximately nucleotide position 1828 and 2855 of SEQ ID NO 1 .
  • the present invention also relates to a recombinant vector, more particularly a plasmid, comprising polynucleic acid sequences of the invention for homologous recombinant expression of at least part of the coding sequence of a polypeptide of the invention as defined below. More particularly for homologous recombinant expression in yeast of the complete pectinase. More particularly for homologous recombinant expression of at least part of a yeast pectinase encoded by SEQ ID NO 1 . Examples of such a homologous recombinant expression vector are pJDB207PECas and pJDB207PECs as disclosed in the Examples section.
  • the present invention also relates to a recombinant vector, more particularly a plasmid, comprising at least part of the coding region of a polynucleic acid sequences of the invention operably linked to heterologous regulatory sequences for heterologous recombinant expression of at least part of a polypeptide of the invention as defined above in a certain host which recognizes these regulatory sequenecs. More particularly, for heterologous expression of at least part of the coding sequence of a yeast pectinase as depicted in SEQ ID NO 1 .
  • the present invention also relates to a recombinant vector, more particularly a plasmid, comprising at least the regulatory and at least part of the coding sequences of a polynucleic acid of the present invention operably linked to at least part of a coding sequence of a heterologous protein, for expression of a fusion protein. More particularly, for expression of a heterolgous fusion protein wherein said at least part of the coding region is from a pectinase gene encoded in SEQ ID NO 1 .
  • yeast pectinases of the invention in: bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out:
  • a recombinant vector heterologous or homologous expression vectors
  • a nucleotide sequence coding for a pectinase of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence
  • heterologous proteins or peptides from recombinant vectors as defined above which comprise regulatory (heterologous protein or peptide expression) and possibly also part of coding sequences (in case of fusion proteins) of the pectinases of this invention in S. cerevisiae the following steps are carried out:
  • the present invention relates to a host cell transformed with a recombinant vector as defined above.
  • the present invention contemplates a Saccharomyces cerevisiae host cell transformed with a recombinant vector as defined above, with said vector comprising yeast regulatory sequences for homologous pectinase or heterologous protein/peptide expression.
  • yeast host cell strains are GRF18, INVSCI, DBY745, AH22, etc.
  • the present invention contemplates bacterial, yeast, fungal or animal host cells transformed with a recombinant vector as defined above, with said vector comprising at least part of a coding sequence of a pectinase as defined above as well as regulatory sequences suitable for heterologous expression of said pectinase in said specific host.
  • the present invention also contemplates bacterial, yeast, fungal or animal host cells transformed with a recombinant vector as defined above, with said vector comprising at least part of a coding sequence of a yeast pectinase according to the present invention as well as regulatory sequences suitable for heterologous expression of said yeast pectinase in said specific host.
  • the present invention relates also to a polypeptide in substantially pure form comprising at least part of an amino acid sequence substantially corresponding to any of the amino acid sequences encoded by a polynucleic acid sequence according to the present invention, as defined above, or derivatives or fragments thereof having pectinase activity.
  • pectinase activity refers to a pectinase-like activity as can be measured by any of the techniques known in the art for determining pectinase activity, such as for instance the techniques disclosed in the Examples section.
  • the pectinase activity of the polypeptides of the present invention may involve more particularly a polygalacturonase activity which may be measured as in the Examples section or by any other technique known in the art.
  • Derivatives or fragments are considered to have pectinase activity if they have at least between 50-70%, more preferably between 60-90%, and even more preferably 70- 95% of the activity of the complete pectinase.
  • Fragments of the pectinase of SEQ ID NO 1 which are tested for their pectinase activity include fragments spanning approximately amino acid positions 1-20, 21-40, 41-60, 61-80, 81-100, 101 -120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241 -260, 261-280, 281-300, 301 -320, 321 -340 or 341 -360.
  • the present invention relates also to a polypeptide in substantially pure form comprising an amino acid sequence substantially corresponding to any of the amino acid sequences encoded by a polynucleic acid sequence as set out in SEQ ID 1 , or derivatives and fragments thereof having pectinase activity.
  • the present invention relates to a polypeptide comprising at least part of the amino acid sequence as depicted in SEQ ID NO 1 in its amino acid sequence.
  • a polypeptide comprising at least 10, 20, 30, 40, 50, 60 or more contiguous amino acids of the amino acid sequence as depicted in SEQ ID NO 1 /2 in its amino acid sequence.
  • Said polypeptides according to the present invention is preferably a recombinant polypeptide.
  • Said recombinant polypeptides are produced by a method as defined below.
  • substantially pure form corresponds to a purity grade of at least 90%, preferably 95 %, more preferably 98% or more expressed as dry weight of proteins versus contaminants determined by any known technique.
  • the contaminating proteins should be undectectable by conventional methods such as SDS-PAGE and silver staining, or 2D-PAGE and silver staining.
  • an amino acid sequence substiantially corresponding to refers to an amino acid sequence which is at least 75-90%, preferably at least 80-95 %, more preferably at least 85-98 % or more homologous to any of the amino acid sequences of the invention as set out in SEQ ID NO 1 .
  • polypeptide of SEQ ID NO 1 shows a homology of at the highest 54% to known polygalacturonases as shown in Figure 6.
  • homologous or “homology” used within the present invention is to be understood as meaning identical and identity. This means that for instance 80% homology is the same as 80 on 100 identical base pairs or amino acids in the same relevant positions upon alignment of the sequences.
  • derivatives corresponds to muteins or homologoues of the amino acid sequences which (i) are derived from other yeast strains (purified naturally occuring derivatives) or other organisms, or (ii) which have in their amino acid sequence insertions, substitutions or deletions in comparison to the naturally occuring amino acid sequences which do not influence the pectinase activity of said polypeptides or fragments, or (iii) which are encoded by polynucleic acid sequences according to the present invention which are degenerate as a result of the genetic code to any of the polynucleic acid sequences of the invention. It is to be understood that the derivatives within the scope of the present invention have retained the pectinase activity, more particularly they have retained the galacturonidase activity.
  • mutant corresponds to polypeptides which are derived from the polypeptides of the invention and which include amino acid substitutions, deletions or insertions compared to the naturally occuring polypeptides of the invention.
  • Table 1 gives an overview of the amino acid substitutions which could be the basis of some of the muteins as defined above.
  • fragments refers to smaller portions of about 5 to about 50 amino acids in length of any of the above-mentioned polypeptides or their derivatives
  • the latter polypeptides and derivatives according to the present invention may be produced by recombinant DNA technology.
  • the derivatives and particularly the fragments according to the present invention may also be prepared by classical chemical synthesis methods.
  • the synthesis can be carried out in homogenous solution or in solid phase.
  • the synthesis technique in homogenous solution which can be used is the one described by Houbenweyl in the book entitled “Methode der organischen Chemie” (Method of organic chemistry) edited by
  • polypeptides of the invention can also be prepared in solid pahse according to the methods described by Atherton and
  • the present invention particularly relates to a method for producing a desired heterologous or homologous protein or peptide in a host cell, comprising at the least the following steps: - transforming said host cell with a recombinant vector as defined above,
  • the present invention relates to a composition
  • a composition comprising as an active ingredient a polypeptide or peptide having pectinase activity as defined above together with at least one of a carrier, and/or diluent, or excipient.
  • Said polypeptide is preferably a recombinant polypeptide.
  • Suitable diluents or carriers include any diluent or carrier known in the art of pectinase activity.
  • the present invention relates to the use of a pectinase polypeptide of the invention or a composition comprising the same as defined above for use in any type of food processing, such as the preparation of fruit juices. It is to be understood that the pectinases of the present invention may also be used for any other known use for pectinases.
  • a further embodiment of the present invention relates to an antibody, more particularly a monoclonal antibody, characterized in that it is specifically directed against an antigenic determinant of a pectinase polypeptide of the present invention as defined above.
  • the present invention also relates to an antigen-binding fragment of the antibody, said fragment being of the F(ab') 2 , Fab or single chain Fv type, or any type of recombinant antibody derived from said specific antibodies or monoclonal antibodies.
  • antigenic determinant or “epitope” refer to that portion of a molecule that is specifically bound by an antibody combining site. Antigenic determinants may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art.
  • the expression "specifically directed against” means that binding between the antigen as a ligand and a molecule containing an antibody combining site, such as a Fab portion of a whole antibody, is specific, signifying that no cross-reaction occurs.
  • Antibodies according to this preferred embodiment of the invention include specific polyclonal antisera prepared against the pectinase polypeptides of the invention, more particularly the yeast pectinases of the invention, and having no cross-reactivity to others proteins, or monoclonal antibodies prepared against the pectinase (particularly the yeast pectinase) polypeptides of the invention.
  • the monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the polypeptides according to the invention, defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which have been initially used for the immunization of the animals.
  • the monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
  • fragments derived from these monoclonal antibodies such as Fab, F(ab)' 2 and ssFv (“single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention.
  • Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses.
  • the antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • FIGURE LEGENDS Figure 1 Physical map of cosmid 109 showing the position and name of the subclones. restriction sites Apa I, BamHI, Kpnl, Pstl and Smal are respectively noted by A, B, K, P and S.
  • Figure 7 Amino acid sequence homology between deposited pectinase sequences and the sequence of PEC encoded by SEQ ID NO 1 .
  • CHIEF-purified chromosome 10 was digested by Sau3AI. Fragments were ligated to the BamHI linearized pWE15 cosmid vector (Huang et al., 1994, DNA-Seq., 4:293-300).
  • One of the obtained cosmid clones is cosmid 109.
  • FIG. 1 A physical map of cosmid 109 (p109) constructed by single and multiple restriction digestion analysis is presented in Figure 1 . Smaller fragments of cosmid 109 were subcloned in pSK + . These subclones are indicated on Figure 1 . To clone the small areas of the cosmid for which no subclones were available, PCR was carried out using primers flanking these gaps. The PCR products were cloned in pGEMT (Promega, Madison, Wl, USA) and the sequence was verified. The complete sequence of the insert of cosmid 109 could be generated by combining the sequence data obtained from all the subclones of cosmid 109.
  • Sequencing was performed according to routine sequencing techniques including PCR, primer walking, use of the Exo-lll-SI unidirectional deletion technique, shotgun library technology, etc in combination with the dideoxy chain termination method (Sanger, 1977, PNAS, 74:5463- 5467).
  • a range of 166 primers was used for different purposes: (a) cosmid walking for fragment assembling, (b) primer walking for gap filling, indetermination solving, or double- strand sequencing, (c) PCR in combination with another primer for gap cloning, gap size evaluation or generation of templates for sequencing.
  • Table 2 summarizes the position and orientation of each designed primer referring to each consensus sequence.
  • the full DNA sequence of the insert of cosmid 109 is 40,876 bases long and is shown in Figure 2 (SEQ ID NO 3).
  • the ORF spanning positions 3461-4546 of cosmid 109 was chosen for further analysis.
  • the protein encoded by the gene carrying this ORF is predicted to have a pectinase activity and more precisely a polygalacturonase activity: random hydrolysis of 1 ,4- ⁇ -D-galactosiduronic linkages in pectate and other galacturonans.
  • Pectinases belong to a family of 28 glycosyl hydrolases and are classified in 3 groups regarding their specific enzymatic activity : polygalacturonase (EC 3.2.1 .15), pectin esterase (EC 3.1 .1 .1 1 ), and pectin lyase (EC 4.2.2.10).
  • Figure 6 summarizes some other polygalacturonase (EC 3.2.1.15) data as well as the percentage of amino acid sequence homology with our pectinase.
  • the homology strongly decreases with more distantly related organisms. Even with only 50% homology, the homology is significantly important in predicting that the protein coded by this gene has pectinase activity.
  • the homology comparison analysis reveals that 4 cysteines are completely conserved. Within the first part of the sequence which corresponds to the signal sequence and the propeptide, less homology is observed.
  • Figure 7 shows the homology between our pectinase sequence (PEC) and 4 deposited pectinase amino acid sequences : 2 sequences from Aspergillus niger (S 1 7980 and S12895), Aspergillus tubigensis (S1 1391 ), and Achliobolus carbonum (S28771 ).
  • PEC pectinase sequence
  • S1 7980 and S12895 Aspergillus niger
  • S1 1391 Aspergillus tubigensis
  • Achliobolus carbonum S28771
  • Cosmid 109 has been cut with Pstl and the 4.3 kb Pstl fragment containing the PEC gene, flanked by its 5' and 3' regulatory sequence, has been purified from an agarose gel. This fragment was ligated into plasmid pJDB207 (Beggs, Multiple -copy yeast plasmid vectors. Molecular Genetics Yeast. Alfred Benzon Symposium 16, Ed. D. Von Wettstein et al., Copenhagen, 1981 , p. 383-389), which has been treated with Pstl and Calf Intestinal Phosphatase. The resulting plasmids pJDB207PECs and pJDB207PECas are shown in Figures 8 and 9. These two plasmids contain the same 4.3 kb fragment but in opposite direction.
  • Leucine prototrophs were grown in minimal medium lacking leucine and containing 3% glucose for 2 days at 28 °C. They were then used to inoculate cultures of complex medium at a ratio of 1 : 150 and these cultures were grown at 28 °C at 180 rpm. After 7 days of culture, aliqouts of 10 ml were removed, centrifuged at 2500 g for 10 minutes and pectinase activity determined in the cell and in the culture supernatant.
  • the proteins were extracted from the medium by TCA precipitation at 4°C and centrifuged for 10 min. at 10000g.
  • the protein pellet was washed with 80% ethanol solution, and redissolved in a 1 ml of 50 mM phosphate buffer, pH 5.5. 100 //I of this protein solution was added to 200 ⁇ l of 1 % polygalacturonic acid, 50 mM phosphate buffer, pH 5.5, incubated at 30°C and the galacturonase activity was determined according to the method of Nelson (J. Biol. Chem., 1944, 153: 375), modified by Milner and Avigad (Carbohyd.
  • the cell pellets were vortexed in a Braun homogenizer for 5 min. with glass beads in 1 ml 50 mM phosphate buffer, pH 5.5. After separation of the cellular debris by centrifugation at 12500 g, the galacturonase activity was determined as described above.
  • Total pectinase activity was 1 U/ml of culture (one unit releases 1 .0 /vmole of reducing sugar from polygalacturonic acid per hour at pH 5.5 at 30°C) for both constructs. Between 50 and 70% of total activity is secreted. No significant pectinase activity was found in GRF18 transformed with pJDB207. These results demonstrate that the 4.3 kb fragment contains all information for the expression and secretion of a yeast enzyme having a pectinase activity.
  • PCR The PCR was done with the Taq polymerase enzyme using cosmid 109 as a template and the oligonucleotides 4053 and 4054 as primers.
  • the sequences of the oligonucleotides used were the following: 4053 5'- AGGGAAGCTTATGATTTCTGCTAATTCATTAC -3' (SEQ ID NO 4) 4054 5'- TCCTTCTAGATTAACAGCTTGCACCAG -3 * , (SEQ ID NO 5) where primer 4053 contain the ATG codon and primer 4054 the STOP codon.
  • a tail was designed at the 5' end of each oligo, which allows excision with resp. Hindlll and Xbal at the 5' end the 3' end.
  • PCR was performed using the following conditions: one ng of cosmid 109 was used as template in 50 ⁇ PCRs, with 1 unit of Taq polymerase (Stratagene, wesburg, NL), supplied with standard Taq DNA polymerase buffer (10 mM Tris-HCl, pH8.3, 1 .5 mM MgCI 2 , 50 mM KCl), 200 nM of each primer, and 0.5 mM dNTP (Pharmacia, Brussels, Belgium).
  • a Perkin-Elmer 480 thermocycler (Perkin-Elmer, Brussels, belgium) was used under the following conditions: 95°C, 1 min; 55°C, 1 min; and 72°C, 1 min. twenty amplification cycles were performed.
  • the 1.2 kb PCR product was cloned into the pGEM-T vector (Promega), the sequence verified, and subcloned between the Hindlll and Xbal site of pYES2 vector (Invitrogen, CA, USA).
  • the resulting PEC expression vector pYES2PEC contains the expression cassette of the regulated GAL 1 promoter, the PEC signal sequence, the mature PEC coding region and the CYC1 terminator. This vector is shown in Figure 10.
  • Yeast strain INVSCI was transformed, and transformants were obtained on uracil selective plates.
  • Total pectinase activity was 0.5 U/ml of culture. In this example, more than 60% of the activity is found in the medium.
  • Example 6 Expression of a heterologous protein under the control of the PEC promoter
  • the promoter region of the PEC gene has been isolated by PCR.
  • the PCR was done with Taq polymerase using cosmid 109 as a template and the oligonucleotides IGA1 and IGA2 as primers.
  • sequences of the oligonucleotides used were the following: IGA1 5'-AGGGAAGCTTAACTTGGCAGGACCACA-3' (SEQ ID NO 6)
  • a tail was designed at the 5' end of each oligo, allowing excision with respectively Hindlll and BamHI at the 5' and 3' end.
  • the 1 ,8 kD PCR product was cloned into the pGEM-T vector (Promega), the sequence verified, and subcloned in the Hindlll and BamHI site of the yeast vector YepZ36 (Oberto and Davison, 1985, Gene, 40: 57-65).
  • This vector contains the LEU2 marker gene and the 2 ⁇ yeast plasmid replication origin with the LacZ gene coding for the E. coli ⁇ - galactosidase.
  • the resulting vector pPECGAL contains the LacZ gene under the control of the PEC promoter.
  • Yeast strain GRF18, transformed with pPECGAL was grown in minimal medium at
  • the clone produced ⁇ -galactosidase to a level of at least 0.5 % of total protein.
  • Val (V) Val Met, lie, Tyr, Phe, Leu
  • Gly Gly, Ala, Thr, Pro, Ser lie (I) lie. Met, Leu, Phe, Val, Ile, Tyr
  • Gin (Q) Gin, Glu, His, Lys, Asn, Thr, Arg

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Abstract

The present invention relates to new pectinases, more particularly to the nucleic acids encoding the same as well as homologous and heterologous expression systems using the regulatory and/or coding sequences comprised in said pectinase nucleic acids. More particularly, the present invention relates to new yeast pectinase polynucleic acid and amino acid sequences, and yeast (heterologous) expression systems based thereon. The present invention also relates to new pectinases or fragments or derivatives thereof for use in food processing.

Description

NEW SACCHAROMYCES CEREVISIAE PECTINASE SEQUENCES AND HETEROLOGOUS EXPRESSION SYSTEMS DERIVED THEREFROM
The present invention relates to the field of yeast genes and heterologous yeast expression systems. More particularly the present invention relates to new pectinases, nucleic acids encoding the same, as well as homologous and heterologous expression methods using the regulatory sequences and/or coding sequences comprised in said pectinase nucleic acids.
Over the past few years, Saccharomyces cerevisiae and alternative yeasts have proved their ability to express heterologous proteins. The exhaustive list of examples in the literature and the commercialization of yeast-derived products clearly proves the importance and versatility of the yeast family in the field of biotechnology. The success of yeast expression systems can be explained by its various advantages it has over both bacterial and mammalian systems. It is a generally regarded as safe (GRAS) organism, having a long historical background in the food indusrty. By contrast, Escherichia coli contains endotoxins, and mammalian cells may contain viral or oncogenic DNA, requiring extensive testing of products derived from these organisms. Yeast is practically as easy to manipulate as E. coli. Its genetics are well understood. High-density cultures can be obtained using relatively simple, substantially protein-free culture media, and large-scale cultures are feasible. Although S. cerevisiae secretes only 0.5 % of its own proteins, the secretion of heterologous proteins can be achieved, so that a secreted foreign protein is present in the culture medium in a nearly pure form. Moreover, as an eukaryotic organism, yeats is capable of higher forms of posttranslational protein processing and can glycosylate secreted proteins - all at the fraction of the cost of a mammalian system. Therefore, it is not surprising that the first commercialized recombinant vaccine, hepatitis B vaccine, was yeast-derived.
Pectinases are synthesized by plants and by a variety of microorganisms. Polygalacturonase is one of the best characterized pectinolytic activities. This enzyme cleaves randomly glycosydic bonds of pectic acids or polygalacturonates, producing mono-, di- and oligogalacturonates as final hydrolysis products. The long-standing intrest in pectinases resides in their role in the invasion of plant tissues by phytopathogens, the spoilage of fruits and vegetables and also in their food processing and plant biological applications.
Despite a major role for S. cerevisiae in the food industry, few studies have been undertaken concerning its pectinolytic enzymes.
The aim of the present invention is to provide polynucleic acid sequences encoding a novel pectinase, more particularly polynucleic acid sequences encoding a yeast pectinase. Another aim of the present invention is to provide a novel pectinase, more particularly from yeast, particularly in a purified and isolated form.
Another aim of the present invention is to provide amino acid sequences of these pectinases.
Another aim of the present invention is to provide recombinant pectinase, more particularly recombinant yeast pectinase, as well as methods for preparing the same.
Another aim of the present invention is to provide recombinant vectors comprising polynucleic acid sequences from the coding and/or non-coding regions of pectinase encoding genes for cloning and/or homologous or heterologous expression purposes.
Particularly the present invention aims at providing coding or regulatory sequences of new pectinase genes, more particularly of yeast pectinase genes, for heterologous expression purposes.
Particularly, the present invention aims at providing a heterologous yeast expression system wherein said coding or regulatory sequences are used to express heterologous proteins. Another aim of the present invention is to provide probes or primers derived from said new polynucleic acid sequences.
Another aim of the present invention is to provide host cells transformed with said recombinant vectors.
Another aim of the present invention is to provide methods for producing heterologous or homologous recombinant proteins using said transformed host cells.
Another aim of the present invention is to provide fragments or derivatives of said pectinases which have retained the pectinase activity.
Another aim of the present invention is to provide for the use of said (recombinant) pectinase for food processing. Another aim of the present invention is to provide monoclonal antibodies which are specifically directed towards said novel pectinase.
All the aims of the present invention have been met by the following embodiments of the invention.
The present invention relates more particularly to a polynucleic acid sequence in substantially isolated form comprising at least part of a sequence selected from:
(a) a polynucleic acid sequence as shown in Figure 5 (SEQ ID NO 1 ),
(b) a polynucleic acid sequence which hybridizes to the polynucleic acid as shown in Figure 5 (SEQ ID NO 1 ), (c) a polynucleic acid sequences which is degenerate as a result of the genetic code to the polynucleic acid sequences as defined in (a) or (b) and which either codes for a polypeptide as defined below, or which hybridizes to a polynucleic acid as defined in (a) or (b).
The Examples section of the present invention teaches the isolation and characterization of a new yeast gene located on yeast chromosome 10. Upon homology comparison with sequences in the databank, limited homology could be found with pectinase sequences from other organisms. Recombinant expression of the open reading frame (ORF) comprised in said new sequence allowed to ascribe a pectinase activity to the new yeast sequence. Pectinases which are quivalent or highly homologous to the yeast pectinase as shown in SEQ ID NO 1 of the present invention are likely to exist in other related organisms as well as in other yeast strains. It is to be understood that these pectinases are different from known pectinases.
The expression "in substantially isolated form" refers to a purity grade of at least 90%, preferably 95 %, and more particularly 98% as determined by dry weight of the polynucleic acid fraction versus contaminants.
The term "polynucleic acid" corresponds to either double-stranded or single-stranded cDNA or genomic DNA, or RNA. A polynucleic acid may contain from 10 nucleotides to the complete nucleotide sequence (such as for instance 20, 30, 40, 50, 60, 70, 80 or more nucleotides). A polynucleic acid which is smaller than about 100 nucleotides is often also referred to as an oligonucleotide. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for therapeutic purposes.
The polynucleic acids of the invention are to be understood as also comprising the complement of the sequence of the top line in Figure 5 or to the nucleic acid as shown in SEQ ID NO 1 . The term "complement" refers to a nucleotide sequence which is complementary to an indicated sequence and which is able to hybridize to the indicated sequence.
The term "hybridizes" refers to conventional hybridization conditions known to the man skilled in the art, preferably to stringent hybridization conditions (see f .i. Maniatis et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, 1982). Under certain conditions hybridization can be allowed between sequences showing a homology of at least 70%, at least 80%, at least 90%, at least 95 % or more. The polynucleic acid according to option (a) can be prepared according to the procedures as set out in the Examples section of the present invention. Alternatively, said polynucleic acids may also be derived according to any other procedure obvious from the teaching of the present invention or any other procedure known to the man skilled in the art for preparing polynucleic acid sequences. The polynucleic acids according to options (b) and (c) can be prepared by applying techniques of hybridization, cloning and/or recombinant expression known to the man skilled in the art.
The polynucleic acids according to option (c) may be derived by cloning equivalents of the polynucleic acids as depicted in SEQ ID NO 1 from the nucleic acids of other related organisms or of other yeast strains. This may be achieved by screening libraries containing polynucleic acids of other related organisms with probes derived from the polynucleic acid as given in SEQ ID NO 1 . Alternatively, said equivalents may be cloned by performing an amplification reaction, such as PCR, of mRNA, with primers essentially comprising a nucleotide sequence which is part of the polynucleic acid sequence given in SEQ ID NO 1 , to obtain amplified products. The polynucleic acids according to (c) may also be provided for by classical chemical synthesis methods of oligo- or polynucleotides generally known by the person skilled in the art.
The expression "comprising at least part" refers to the fact that a polynucleic acid sequence according to the present invention comprises a sequence of preferably 10, 20, 30, 40, 50, 60, 100, 200, 500, 1000 or more nucleotides selected from the sequence as depicted in Figure 5 (SEQ ID NO 1 ), or their complement, or sequences which are degenerate as a result of the genetic code or which hybridize to any of the foregoing sequences.
Especially preferred parts of the polynucleic acid sequences of the present invention include regulatory sequences such as promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences, signal sequences, as well as specific parts of the coding sequences (see below).
According to another embodiment, the present invention also relates to a polynucleic acid sequence as defined above comprising at least 10 contiguous nucleotides, for use as a specific hybridization probe for detecting the presence of any target sequence comprising a polynucleic acid according to the present invention, as defined above.
The term "probe" refers to single-stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to a target sequence to be detected or cloned. Probes may be labelled according to any of the techniques known in the art. Preferably these probes are about 10 to 50 nucleotides long. According to the hybrization solution used, these probes should be hybridized at appropriate temperatures and be of appropriate length to attain sufficient specificity.
According to another embodiment, the present invention relates to polynucleic acid sequences as defined above comprising at least 10 contiguous nucleotides, for use as primer for amplification of any polynucleic acid sequence according to the present invention, as defined above.
The term "primer" refers to a single-stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension product. Preferably, the primer is about 10 to about 50 nucleotides long. Specific length and sequences will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. The amplification method used can be any method known in the art.
The fact that amplification-primers do not have to match exactly with the corresponding template sequence to warrant proper amplification, providing that an exact match at the last three nucleotides at the 3' end of the primer is maintained, is amply documented in the literature (Kwok et al., Nucleic Acids Research 18:999-1005, 1990; Sommer and Tautz, Nucleic Acids Research 17, 6749, 1989).
Probes and primers according to these aspects of the present invention may be used to isolate genes encoding equivalents or proteins which are highly homologous to the polynucleic acid sequence of SEQ ID NO 1 in organisms other than Saccharomyces cerevisiae strain FY1679 from which SEQ ID NO 1 was obtained. Other S. cerevisiae strains include for instance INVSCI, DBY745, AH22, etc. In addition, equivalents to the protein of SEQ ID NO 1 may be detected in other organisms than yeast, preferably in a yeast-related organism.
The present invention relates more particularly to at least part of any polynucleic acid sequence having a homology of at least 75-85 %, preferably at least 80-90% , more preferably 85-95% with the polynucleic acid sequence as shown in Figure 5 (SEQ ID NO 1 ).
According to another embodiment, the present invention relates to a recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising at least part of a polynucleic acid sequence as defined above.
It is to be understood that said polynucleic acid sequence comprised in said recombinant vector of the invention may comprise:
(a) only regulatory elements/sequences of said polynucleic acid sequences operably linked to a heterologous coding sequence and capable of providing for the expression of this heterologous coding sequence by specific host cells, or,
(b) only a coding sequence (parts or complete) of said polynucleic acid sequence which is operably linked to heterologous regulatory sequences present in the vector sequence for expressing at least part of the coding sequence in a heterolgous system, or, (c) both regulatory and at least part of the coding sequence of said polynucleic acid sequence capable for providing for the expression of at least part of the products encoded by said polynucleic acids (homologous yeast expression), or, (d) regulatory sequences and parts of the coding sequence (f.i. the N-terminal part of the coding sequence) of said polynucleic acid sequences operably linked to a heterlogous protein coding sequence and capable of providing for the expression of fusion proteins consisting of at least part of the products encoded by said polynucleic acids (f.i. N-terminus) and heterologous proteins or parts thereof. The term "vector" may comprise a plasmid, a cosmid, a phage or a virus. Preferably said vector will be a plasmid. Particularly preferred plasmids (or vectors) for the expression of coding sequences derived from the polynucleic acid sequences according to the present invention in yeast include for instance pYES2 (Invitrogen, CA, USA) including the GAL-1 promoter and CYC1 terminator sequences or the 4,3 kb Pstl fragment of SEQ ID NO 1 cloned in pJDB207. Particularly preferred plamsids (or vectors) for heterologous expression of proteins using the regulatory sequences from the polynucleic acid sequences according to the present invention include for instance derivatives of pPECGAL, pJDB207PECs and pJDB207PECas as disclosed in the Examples section.
The term "coding sequence" refers to a polynucleic acid sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the δ'-terminus and a translation stop codon at the 3"-terminus. A coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences. The coding sequences of the present invention encode a pectinase.
The term "recombinant" refers to the fact that a fusion is being made between polynucleic acids from different origins.
The term "operably linked" refers to a juxtaposition wherein the components are figured so as to perform their usual function. Thus, control sequences or regulatory sequences operably linked to a coding sequence are capable of effecting the expression of a coding sequence.
The term "regulatory sequence" or control sequence refers to those sequences which control the transcription and/or translation of the coding sequences; these may include but are not limited to promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences. In addition, control sequences refer to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controling secretion, protease cleavage, and glycosylation of the polypeptide. It should be understood that the signal peptide encoding sequence (and the signal peptide encoded by it) as well as any other of the above-mentioned regulatory sequences of the polynucleotides of the invention form an important aspect of the present invention as such, and it is also contemplated that these regulatory sequences may be inserted upstream and/or downstream of DNA sequences coding for heterologous proteins or peptides so as to obtain expression and/or secretion of the resulting products from yeast host cells.
More particularly, these signal sequences may particularly be used for expression in any recombinant system such as: yeast, or any other bacterial, mammalian or viral host cell system. According to a particularly preferred embodiment, the present invention relates to a recombinant vector as defined above, more particularly a plasmid, further comprising a nucleotide sequence encoding a heterologous protein or peptide which is desired to be expressed within said vector sequence in such a way that said heterologous protein is expressed under the control of regulatory sequences of the polynucleic acids as defined above.
Examples of such proteins or peptides to be expressed may include, but are not limited to, complete polypeptides or peptides derived therefrom from bacterial, animal, mammalian, human or viral origin. According to another embodiment, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor sequence included in any of the polynucleic acid sequences according to the present invention, as defined above.
More particularly, the present invention relates to a recombinant vector (e.g. a plasmid) comprising a yeast pectinase promoter sequence selected from the polynucleotide sequences as defined above.
More particularly comprising the yeast pectinase promoter sequences contained in SEQ ID NO 1 , more particularly in the region spanning position 1 to 1771 thereof.
According to another embodiment, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a promotor/secretion signal sequence included in any of the polynucleic acid sequences according to the present invention, as defined above.
More particularly, the present invention relates to a recombinant vector, more particularly a plasmid, comprising a pectinase promotor/secretion signal sequence derived from yeast, selected from the sequences present on the nucleotide sequence as depicted in SEQ ID NO 1 , more particularly in the region spanning position 1 to approximately
1828 of SEQ ID NO 1 .
The present invention further relates to recombinant vectors comprising the signal peptide encoding sequence comprised in any of the polynucleic said sequences of the present invention. More particularly, the signal peptide encoding sequence of a yeast pectinase, selected from the sequences as depicted in SEQ ID NO 1 , more particularly in the region spanning position 1772 to approximately position 1828 of SEQ ID NO 1 .
The present invention also relates to recombinant vectors comprising the transcription/translation stop regulatory sequences comprised in any of the polynucleic eaid sequences of the present invention. More particularly a transcription/translation stop regulatory sequence of a yeast pectinase as depicted in SEQ ID NO 1 , more particularly in the region spanning positions 2855 to position 4300 of SEQ ID NO 1 .
The present invention also relates to recombinant vectors comprising a polynucleic acid as defined above with said polynucleic acid comprising a deletion of at least part of the coding region for heterologous expression of foreign proteins or peptides in the place of said deletion and in the same reading frame as the original coding sequence. More particularly, a deletion in the coding region of a yeast pectinase gene as shown in SEQ ID NO 1 . For instance a deletion of the whole region or part of the region comprised between spproximately nucleotide position 1828 and 2855 of SEQ ID NO 1 . The present invention also relates to a recombinant vector, more particularly a plasmid, comprising polynucleic acid sequences of the invention for homologous recombinant expression of at least part of the coding sequence of a polypeptide of the invention as defined below. More particularly for homologous recombinant expression in yeast of the complete pectinase. More particularly for homologous recombinant expression of at least part of a yeast pectinase encoded by SEQ ID NO 1 . Examples of such a homologous recombinant expression vector are pJDB207PECas and pJDB207PECs as disclosed in the Examples section.
The present invention also relates to a recombinant vector, more particularly a plasmid, comprising at least part of the coding region of a polynucleic acid sequences of the invention operably linked to heterologous regulatory sequences for heterologous recombinant expression of at least part of a polypeptide of the invention as defined above in a certain host which recognizes these regulatory sequenecs. More particularly, for heterologous expression of at least part of the coding sequence of a yeast pectinase as depicted in SEQ ID NO 1 . The present invention also relates to a recombinant vector, more particularly a plasmid, comprising at least the regulatory and at least part of the coding sequences of a polynucleic acid of the present invention operably linked to at least part of a coding sequence of a heterologous protein, for expression of a fusion protein. More particularly, for expression of a heterolgous fusion protein wherein said at least part of the coding region is from a pectinase gene encoded in SEQ ID NO 1 .
In order to carry out the expression of the pectinases of the invention, more particularly the yeast pectinases of the invention in: bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out:
- transformation of an appropriate cellular host with a recombinant vector (heterologous or homologous expression vectors), in which a nucleotide sequence coding for a pectinase of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence,
- culture of said transformed cellular host under conditions enabling the expression of said pectinase insert.
In order to carry out the expression of heterologous proteins or peptides from recombinant vectors as defined above which comprise regulatory (heterologous protein or peptide expression) and possibly also part of coding sequences (in case of fusion proteins) of the pectinases of this invention in S. cerevisiae the following steps are carried out:
- transformation of an appropriate S. cerevisiae host with a recombinant heterologous expression vector, in which a nucleotide sequence coding for said heterologous peptide or polypeptide has been inserted under the control of regulatory sequences comprised in the polynucleic acids of the invention, and possibly also part of the coding sequence of said S. cerevisiae pectinase, and enabling the expression in said cellular host of said nucleotide sequence,
- culture of said transformed cellular host under conditions enabling the expression of said polypeptide, peptide or pectinase fusion protein insert.
According to yet another aspect, the present invention relates to a host cell transformed with a recombinant vector as defined above.
More particularly, the present invention contemplates a Saccharomyces cerevisiae host cell transformed with a recombinant vector as defined above, with said vector comprising yeast regulatory sequences for homologous pectinase or heterologous protein/peptide expression. Particularly preferred yeast host cell strains are GRF18, INVSCI, DBY745, AH22, etc.
Also, the present invention contemplates bacterial, yeast, fungal or animal host cells transformed with a recombinant vector as defined above, with said vector comprising at least part of a coding sequence of a pectinase as defined above as well as regulatory sequences suitable for heterologous expression of said pectinase in said specific host. Alternatively, the present invention also contemplates bacterial, yeast, fungal or animal host cells transformed with a recombinant vector as defined above, with said vector comprising at least part of a coding sequence of a yeast pectinase according to the present invention as well as regulatory sequences suitable for heterologous expression of said yeast pectinase in said specific host. According to a preferred embodiment, the present invention relates also to a polypeptide in substantially pure form comprising at least part of an amino acid sequence substantially corresponding to any of the amino acid sequences encoded by a polynucleic acid sequence according to the present invention, as defined above, or derivatives or fragments thereof having pectinase activity.
The term "pectinase activity" refers to a pectinase-like activity as can be measured by any of the techniques known in the art for determining pectinase activity, such as for instance the techniques disclosed in the Examples section. The pectinase activity of the polypeptides of the present invention may involve more particularly a polygalacturonase activity which may be measured as in the Examples section or by any other technique known in the art.
Derivatives or fragments are considered to have pectinase activity if they have at least between 50-70%, more preferably between 60-90%, and even more preferably 70- 95% of the activity of the complete pectinase. Fragments of the pectinase of SEQ ID NO 1 which are tested for their pectinase activity include fragments spanning approximately amino acid positions 1-20, 21-40, 41-60, 61-80, 81-100, 101 -120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241 -260, 261-280, 281-300, 301 -320, 321 -340 or 341 -360.
More particularly the present invention relates also to a polypeptide in substantially pure form comprising an amino acid sequence substantially corresponding to any of the amino acid sequences encoded by a polynucleic acid sequence as set out in SEQ ID 1 , or derivatives and fragments thereof having pectinase activity.
More particularly, the present invention relates to a polypeptide comprising at least part of the amino acid sequence as depicted in SEQ ID NO 1 in its amino acid sequence. Preferably a polypeptide comprising at least 10, 20, 30, 40, 50, 60 or more contiguous amino acids of the amino acid sequence as depicted in SEQ ID NO 1 /2 in its amino acid sequence.
Said polypeptides according to the present invention is preferably a recombinant polypeptide. Said recombinant polypeptides are produced by a method as defined below.
The expression "substantially pure form" corresponds to a purity grade of at least 90%, preferably 95 %, more preferably 98% or more expressed as dry weight of proteins versus contaminants determined by any known technique. Essentially, the contaminating proteins should be undectectable by conventional methods such as SDS-PAGE and silver staining, or 2D-PAGE and silver staining.
The expression "an amino acid sequence substiantially corresponding to" refers to an amino acid sequence which is at least 75-90%, preferably at least 80-95 %, more preferably at least 85-98 % or more homologous to any of the amino acid sequences of the invention as set out in SEQ ID NO 1 .
The polypeptide of SEQ ID NO 1 shows a homology of at the highest 54% to known polygalacturonases as shown in Figure 6.
The term "homologous" or "homology" used within the present invention is to be understood as meaning identical and identity. This means that for instance 80% homology is the same as 80 on 100 identical base pairs or amino acids in the same relevant positions upon alignment of the sequences.
The term "derivatives" corresponds to muteins or homologoues of the amino acid sequences which (i) are derived from other yeast strains (purified naturally occuring derivatives) or other organisms, or (ii) which have in their amino acid sequence insertions, substitutions or deletions in comparison to the naturally occuring amino acid sequences which do not influence the pectinase activity of said polypeptides or fragments, or (iii) which are encoded by polynucleic acid sequences according to the present invention which are degenerate as a result of the genetic code to any of the polynucleic acid sequences of the invention. It is to be understood that the derivatives within the scope of the present invention have retained the pectinase activity, more particularly they have retained the galacturonidase activity.
It should be understood that also the polynucleic acid sequences encoding such "derivatives" are within the scope of the present invention.
The term "mutein" referred to above corresponds to polypeptides which are derived from the polypeptides of the invention and which include amino acid substitutions, deletions or insertions compared to the naturally occuring polypeptides of the invention.
Table 1 gives an overview of the amino acid substitutions which could be the basis of some of the muteins as defined above.
The term "fragments" or peptides refers to smaller portions of about 5 to about 50 amino acids in length of any of the above-mentioned polypeptides or their derivatives The latter polypeptides and derivatives according to the present invention may be produced by recombinant DNA technology.
The derivatives and particularly the fragments according to the present invention may also be prepared by classical chemical synthesis methods. The synthesis can be carried out in homogenous solution or in solid phase. For instance, the synthesis technique in homogenous solution which can be used is the one described by Houbenweyl in the book entitled "Methode der organischen Chemie" (Method of organic chemistry) edited by
Wunsh, vol. 1 5-1 et II. THIEME, Stuttgart, 1974. The polypeptides of the invention can also be prepared in solid pahse according to the methods described by Atherton and
Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).
The present invention particularly relates to a method for producing a desired heterologous or homologous protein or peptide in a host cell, comprising at the least the following steps: - transforming said host cell with a recombinant vector as defined above,
- culturing the transformed host cell in a suitable medium under conditions allowing expression of said protein or peptide, and,
- recovering the expressed protein or peptide from said host cell or said medium by any technique known in the art for purifying said polypeptide or peptide. According to yet another embodiment, the present invention relates to a composition comprising as an active ingredient a polypeptide or peptide having pectinase activity as defined above together with at least one of a carrier, and/or diluent, or excipient.
Said polypeptide is preferably a recombinant polypeptide. Suitable diluents or carriers include any diluent or carrier known in the art of pectinase activity.
According to another aspect, the present invention relates to the use of a pectinase polypeptide of the invention or a composition comprising the same as defined above for use in any type of food processing, such as the preparation of fruit juices. It is to be understood that the pectinases of the present invention may also be used for any other known use for pectinases.
A further embodiment of the present invention relates to an antibody, more particularly a monoclonal antibody, characterized in that it is specifically directed against an antigenic determinant of a pectinase polypeptide of the present invention as defined above.
According to an alternative embodiment, the present invention also relates to an antigen-binding fragment of the antibody, said fragment being of the F(ab')2, Fab or single chain Fv type, or any type of recombinant antibody derived from said specific antibodies or monoclonal antibodies. The terms "antigenic determinant" or "epitope" refer to that portion of a molecule that is specifically bound by an antibody combining site. Antigenic determinants may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art. The expression "specifically directed against" means that binding between the antigen as a ligand and a molecule containing an antibody combining site, such as a Fab portion of a whole antibody, is specific, signifying that no cross-reaction occurs.
Antibodies according to this preferred embodiment of the invention include specific polyclonal antisera prepared against the pectinase polypeptides of the invention, more particularly the yeast pectinases of the invention, and having no cross-reactivity to others proteins, or monoclonal antibodies prepared against the pectinase (particularly the yeast pectinase) polypeptides of the invention.
The monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the polypeptides according to the invention, defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which have been initially used for the immunization of the animals.
The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.
Also fragments derived from these monoclonal antibodies such as Fab, F(ab)'2 and ssFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses.
The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
FIGURE LEGENDS Figure 1. Physical map of cosmid 109 showing the position and name of the subclones. restriction sites Apa I, BamHI, Kpnl, Pstl and Smal are respectively noted by A, B, K, P and S.
Figure 2. Complete sequence of the insert of cosmid 109 (SEQ ID NO 3).
Figure 3. Position and direction of the ORFs found ( > 100 amino acids long). Homology with known genes or genes with homology with known deposited sequences are noted from 1 to 5.
Figure 4. Relevant characteristics of the ORFs as shown in Figure 3.
Figure 5. Complete nucleotide sequence and amino acid translation of the 4,3 kb Pstl fragment (SEQ ID NO 1/2).
Figure 6. Homology of the different polygalacturonases (EC 3.2.1.15 data) with PEC (SEQ ID NO 1 /2) on amino acid level.
Figure 7. Amino acid sequence homology between deposited pectinase sequences and the sequence of PEC encoded by SEQ ID NO 1 .
Figure 8. Map of pJDB207PECs
Figure 9. Map of pJDB207PECas
Figure 10. Map of pYES2PEC EXAMPLES
Example 1.
Sequence analysis of Saccharomyces cerevisiae cosmid 109
Genomic DNA obtained from Cosmid 109 from Saccharomyces cerevisiae FY1679, a diploid strain issued from the cross between strains FY23 (MATa, ura 3-52, trp 1 D63, leu201 , GAL2) and FY73 (MATα, ura 3-52, his 30200, GAL2). CHIEF-purified chromosome 10 was digested by Sau3AI. Fragments were ligated to the BamHI linearized pWE15 cosmid vector (Huang et al., 1994, DNA-Seq., 4:293-300). One of the obtained cosmid clones is cosmid 109. A physical map of cosmid 109 (p109) constructed by single and multiple restriction digestion analysis is presented in Figure 1 . Smaller fragments of cosmid 109 were subcloned in pSK + . These subclones are indicated on Figure 1 . To clone the small areas of the cosmid for which no subclones were available, PCR was carried out using primers flanking these gaps. The PCR products were cloned in pGEMT (Promega, Madison, Wl, USA) and the sequence was verified. The complete sequence of the insert of cosmid 109 could be generated by combining the sequence data obtained from all the subclones of cosmid 109. Sequencing was performed according to routine sequencing techniques including PCR, primer walking, use of the Exo-lll-SI unidirectional deletion technique, shotgun library technology, etc in combination with the dideoxy chain termination method (Sanger, 1977, PNAS, 74:5463- 5467). A range of 166 primers was used for different purposes: (a) cosmid walking for fragment assembling, (b) primer walking for gap filling, indetermination solving, or double- strand sequencing, (c) PCR in combination with another primer for gap cloning, gap size evaluation or generation of templates for sequencing. Table 2 summarizes the position and orientation of each designed primer referring to each consensus sequence. The full DNA sequence of the insert of cosmid 109 is 40,876 bases long and is shown in Figure 2 (SEQ ID NO 3).
Example 2.
Putative ORF analysis of the sequence of cosmid 109. A search was done for homology with ORFs having a polypeptide length of greater than 100 amino acids at the DNA level (database EMBL40) and at the protein level (databases PIR41 and Swiss Prot29).
The position of the ORFs found in cosmid 109 is shown in Figure 3. Figure 4 summarizes this ORF information.
Example 3.
Analysis of the ORF with partial homology to pectinases.
The ORF spanning positions 3461-4546 of cosmid 109 was chosen for further analysis. The protein encoded by the gene carrying this ORF is predicted to have a pectinase activity and more precisely a polygalacturonase activity: random hydrolysis of 1 ,4-α-D-galactosiduronic linkages in pectate and other galacturonans. Pectinases belong to a family of 28 glycosyl hydrolases and are classified in 3 groups regarding their specific enzymatic activity : polygalacturonase (EC 3.2.1 .15), pectin esterase (EC 3.1 .1 .1 1 ), and pectin lyase (EC 4.2.2.10). Figure 6 summarizes some other polygalacturonase (EC 3.2.1.15) data as well as the percentage of amino acid sequence homology with our pectinase. The homology strongly decreases with more distantly related organisms. Even with only 50% homology, the homology is significantly important in predicting that the protein coded by this gene has pectinase activity. The homology comparison analysis reveals that 4 cysteines are completely conserved. Within the first part of the sequence which corresponds to the signal sequence and the propeptide, less homology is observed.
Figure 7 shows the homology between our pectinase sequence (PEC) and 4 deposited pectinase amino acid sequences : 2 sequences from Aspergillus niger (S 1 7980 and S12895), Aspergillus tubigensis (S1 1391 ), and Achliobolus carbonum (S28771 ). Example 4.
Expression of the PEC gene in S. cerevisiae under the control of its own promoter
Cosmid 109 has been cut with Pstl and the 4.3 kb Pstl fragment containing the PEC gene, flanked by its 5' and 3' regulatory sequence, has been purified from an agarose gel. This fragment was ligated into plasmid pJDB207 (Beggs, Multiple -copy yeast plasmid vectors. Molecular Genetics Yeast. Alfred Benzon Symposium 16, Ed. D. Von Wettstein et al., Copenhagen, 1981 , p. 383-389), which has been treated with Pstl and Calf Intestinal Phosphatase. The resulting plasmids pJDB207PECs and pJDB207PECas are shown in Figures 8 and 9. These two plasmids contain the same 4.3 kb fragment but in opposite direction.
We used these two plasmids to transform S. cerevisiae strain GRF18. Leucine prototrophs were grown in minimal medium lacking leucine and containing 3% glucose for 2 days at 28 °C. They were then used to inoculate cultures of complex medium at a ratio of 1 : 150 and these cultures were grown at 28 °C at 180 rpm. After 7 days of culture, aliqouts of 10 ml were removed, centrifuged at 2500 g for 10 minutes and pectinase activity determined in the cell and in the culture supernatant.
To measure the activity of PEC excreted into the medium, the proteins were extracted from the medium by TCA precipitation at 4°C and centrifuged for 10 min. at 10000g. The protein pellet was washed with 80% ethanol solution, and redissolved in a 1 ml of 50 mM phosphate buffer, pH 5.5. 100 //I of this protein solution was added to 200 μl of 1 % polygalacturonic acid, 50 mM phosphate buffer, pH 5.5, incubated at 30°C and the galacturonase activity was determined according to the method of Nelson (J. Biol. Chem., 1944, 153: 375), modified by Milner and Avigad (Carbohyd. Res., 1967, 4-359- 361 ). In order to determine the amount of PEC which was cell associated, the cell pellets were vortexed in a Braun homogenizer for 5 min. with glass beads in 1 ml 50 mM phosphate buffer, pH 5.5. After separation of the cellular debris by centrifugation at 12500 g, the galacturonase activity was determined as described above.
Total pectinase activity was 1 U/ml of culture (one unit releases 1 .0 /vmole of reducing sugar from polygalacturonic acid per hour at pH 5.5 at 30°C) for both constructs. Between 50 and 70% of total activity is secreted. No significant pectinase activity was found in GRF18 transformed with pJDB207. These results demonstrate that the 4.3 kb fragment contains all information for the expression and secretion of a yeast enzyme having a pectinase activity.
Example 5.
Expression of the PEC ORF under the control of the GAL 1 promoter
The exact coding region of the PEC gene with its signal sequence was isolated by
PCR. The PCR was done with the Taq polymerase enzyme using cosmid 109 as a template and the oligonucleotides 4053 and 4054 as primers. The sequences of the oligonucleotides used were the following: 4053 5'- AGGGAAGCTTATGATTTCTGCTAATTCATTAC -3' (SEQ ID NO 4) 4054 5'- TCCTTCTAGATTAACAGCTTGCACCAG -3*, (SEQ ID NO 5) where primer 4053 contain the ATG codon and primer 4054 the STOP codon. In addition, a tail was designed at the 5' end of each oligo, which allows excision with resp. Hindlll and Xbal at the 5' end the 3' end.
PCR was performed using the following conditions: one ng of cosmid 109 was used as template in 50 μ\ PCRs, with 1 unit of Taq polymerase (Stratagene, wesburg, NL), supplied with standard Taq DNA polymerase buffer (10 mM Tris-HCl, pH8.3, 1 .5 mM MgCI2, 50 mM KCl), 200 nM of each primer, and 0.5 mM dNTP (Pharmacia, Brussels, Belgium). A Perkin-Elmer 480 thermocycler (Perkin-Elmer, Brussels, belgium) was used under the following conditions: 95°C, 1 min; 55°C, 1 min; and 72°C, 1 min. twenty amplification cycles were performed.
The 1.2 kb PCR product was cloned into the pGEM-T vector (Promega), the sequence verified, and subcloned between the Hindlll and Xbal site of pYES2 vector (Invitrogen, CA, USA).
The resulting PEC expression vector pYES2PEC contains the expression cassette of the regulated GAL 1 promoter, the PEC signal sequence, the mature PEC coding region and the CYC1 terminator. This vector is shown in Figure 10.
Yeast strain INVSCI was transformed, and transformants were obtained on uracil selective plates.
Cells from late-log cultures grown on synthetic medium containing glucose were shifted to synthetic medium containing 2 % galactose. After 24 hours of induction, the culture was harvested, and the pectinase (galacturonase) activity determined as described in example 4.
Total pectinase activity was 0.5 U/ml of culture. In this example, more than 60% of the activity is found in the medium.
Example 6. Expression of a heterologous protein under the control of the PEC promoter
The promoter region of the PEC gene has been isolated by PCR. The PCR was done with Taq polymerase using cosmid 109 as a template and the oligonucleotides IGA1 and IGA2 as primers.
The sequences of the oligonucleotides used were the following: IGA1 5'-AGGGAAGCTTAACTTGGCAGGACCACA-3' (SEQ ID NO 6)
IGA2 5'-TCCTTGGATCCTGCGTTTGTCCATCAATGTG-3' (SEQ ID NO 7)
In addition, a tail was designed at the 5' end of each oligo, allowing excision with respectively Hindlll and BamHI at the 5' and 3' end.
The 1 ,8 kD PCR product was cloned into the pGEM-T vector (Promega), the sequence verified, and subcloned in the Hindlll and BamHI site of the yeast vector YepZ36 (Oberto and Davison, 1985, Gene, 40: 57-65). This vector contains the LEU2 marker gene and the 2μ yeast plasmid replication origin with the LacZ gene coding for the E. coli β- galactosidase. The resulting vector pPECGAL contains the LacZ gene under the control of the PEC promoter. Yeast strain GRF18, transformed with pPECGAL, was grown in minimal medium at
28 °C and the amount of intracellular β-galactosidase determined in the cell lysate as described by Miller (1972). The clone produced β-galactosidase to a level of at least 0.5 % of total protein.
TABLE 1
Amino acids Synonymous groups
Ser (S) Ser, Thr, Gly, Asn
Arg (R) Arg, His, Lys, Glu, Gin
Leu (L) Leu; Ile, Met, Phe, Val, Tyr
Pro (P) Pro, Ala, Thr, Gly
Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gin Ala (A) Ala, Pro, Gly, Thr
Val (V) Val, Met, lie, Tyr, Phe, Leu
Gly (G) Gly, Ala, Thr, Pro, Ser lie (I) lie. Met, Leu, Phe, Val, Ile, Tyr
Phe (F) Phe, Met, Tyr, lie, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, lie, Val, Leu
Cys (C) Cys, Ser, Thr, Met
His (H) His, Gin, Arg, Lys, Glu, Thr
Gin (Q) Gin, Glu, His, Lys, Asn, Thr, Arg
Asn (N) Asn, Asp, Ser, Gin Lys (K) Lys, Arg, Glu, Gin, His
Asp (D) Asp, Asn, Glu, Gin
Glu (E) Glu, Gin, Asp, Lys, Asn, His, Arg
Met (M) Met, lie, Leu, Phe, Val
Table 2-1 : Position and orientation of each designed primer on the first consensus sequence
3184(246-264) 3232(6872-6889) 2948(19247-19264) 3111'(25574-25593)
3357(507-524) 3049(7072-7088) 2960(19266-19290) 3110(25587-25607)
3733'(568-591) 3883(7175-7195) 2840(19751-19768) 3959(26190-26208)
3724'(951-976) 3112'(7250-7268) 2842'(19919-19935) 3784'(26190-26208)
3723(951-976) 3882'(7615-7634) 2946(20100-20116) 3053(26359-26376)
3998'(1111-1130) 3047'(7722-7738) 3365(20156-20173) 2970(26464-26481)
3637'(1378-1400) 3233(8104-8121) 2759(20157-20174) 3052(26694-26711)
3183X1393-1411) 3046(8461-8477) 3741'(20544-20565) 3116(26765-26783)
3997' (1705-1724) 3735*(8583-8602) 3740(20544-20565) 3783'(26942-26963)
3355(2041-2060) 3367(8812-8829) 3739(20544-20565) 3791 '(27026-27045)
3726(2572-2589) 3234"(8875-8892) 2843(20900-20916) 2967' (27233-27250)
3725'(2572-2589) 3113(9008-9027) 2947'(20902-20918) 3054'(27303-27319)
3727(2936-2957) 3813(9213-9234) 2881'(21189-21206) 2969(27531-27549)
3889'(3476-3495) 3368(9338-9355) 3537(21300-21321) 3055'(27811-27827)
3728'(3521-3547) 3236(9368-9385) 3891'(21803-21821) 3107(27841-27860)
3356'(3898-3917) 3369'(9664-9681) 3729(21803-21820) 3106'(28220-28239)
3228'(4330-4346) 3237(11106-11123) 3963(21949-21970) 3782'(28384-28406)
2954'(4612-4630) 3238(11565-11582) 3962'(22296-22317) 2957(28451-28469)
3229(4864-4881) 3239(11878-11894) 3892'(22411-22430) 2956(28474-28491)
3734(4903-4924) 3240'(12355-12373) 3961(22649-22670) 3117(28529-28545)
3048'(5280-5297) 3811'(15196-15217) 3893(22926-22946)
3888(5312-5333) 3050(16064-16080) 3536'(23175-23194)
3887(5559-5581) 3114(16171-16188) 3360'(23179-23196)
3231(5757-5773) 3364'(17117-17134) 3535(23297-23315)
3886'(5813-5834) 3736(17198-17218) 3370(23337-23354)
3230'(5880-5896) 3738(17664-17685) 2762'(23862-23878)
3885(5996-6017) 3737'(17664-17685) 3534'(23932-23951)
3884*(6419-6439) 2763'(18076-18092) 3108(24066-24085)
3358'(6477-6494) 2838(18878-18895) 3785'(24624-24645) Table 2-2: Position and orientation of each designed primer on the second consensus sequence
3533(111-130) 3794'(6451-6472)
3781 '(331-353) 3795'(6940-6961)
3780(695-719) 3894'(7506-7525)
3779*(695-719) 3958(8158-8177)
3362'(1355-1372) 3895'(8651-8670)
3778'(1365-1387) 3957(9055-9076)
2761'(1722-1739) 3796'(9141-9161)
3118(3005-3024) 3896'(9556-9575)
2953'(3228-3246) 2764(9818-9834)
3115(3364-3383) 2877'(9965-9981)
2952'(3533-3550) 2878(10502-10520)
3786(3562-3581) 2949'(10566-10583)
3363(3797-3814) 2879(10768-10784)
3129'(4150-4170) 3798'(11127-11144)
3787'(4232-4251) 3797(11127-11144)
3788'(4549-4572) 2880'(11436-11452)
3790'(4844-4863) 2760(11501-11518)
3532(4952-4970) 2845(11678-11694)
3789(5113-5132) 2837'(11678-11694)
3792'(5503-5523) 2951 '(11756-11772)
3531'(5631-5652) 2950(11933-11949)
3793(5742-5763) 3799(12031-12050)
3361 '(5744-5762) 2844'(12033-12049)
2765' (6077-6093)

Claims

1 . A polynucleic acid sequence in substantially isolated form comprising at least part of a sequence selected from:
(a) a polynucleic acid sequence as shown in figure 5 (SEQ ID NO 1 ), (b) a polynucleic acid sequence which hybridizes to the polynucleic acid as shown in
Figure 5 (SEQ ID NO 1 ),
(c) a polynucleic acid sequence which is degenerate as a result of the genetic code to the polynucleic acid sequences as defined in (a) or (b) and which either codes for a polypeptide as defined in claim 8, or which hybridizes to a polynucleic acid as defined in (a) or (b).
2. A recombinant vector, particularly for cloning and/or expression, with said recombinant vector comprising at least part of a polynucleic acid sequence of claim 1 .
3. A recombinant vector according to claim 2, more particularly a plasmid, further comprising a nucleotide sequence encoding a heterologous protein or peptide which is desired to be expressed inserted in said vector sequence in such a way that said heterologous protein is expressed under the control of regulatory sequences of polynucleic acids as defined in claim 1 .
4. A recombinant vector according to claim 2, more particularly a plasmid, comprising a polynucleic acid sequence of claim 1 for homologous expression of at least part of a polypeptide of claim 8.
5. A recombinant expression vector, more particularly a plasmid, comprising an insert consisting of at least part of a coding region comprised in a polynucleic acid sequence according to claim 1 operably linked to a heterologous regulatory region for heterolgous expression of at least part of a polypeptide of claim 8.
6. A host cell transformed with a recombinant vector of any of claims 2 to 5.
7. A method for producing a desired heterologous or homologous protein or peptide in a host cell, comprising at the least the following steps:
- transforming said host cell with a recombinant vector of claim 2 to 5,
- culturing the transformed host cell in a suitable medium under conditions allowing expression of said protein or peptide, and, - recovering the expressed protein or peptide from said host cell or said medium.
8. A polypeptide having a pectinase activity comprising at least part of a polypeptide substantially corresponding to an amino acid sequence encoded by a polynucleic acid sequence of claim 1 , or fragments or derivatives thereof having pectinase activity.
9. A composition comprising as an active ingredient a polypeptide according to claim 8 together with at least one of a carrier, and/or diluent, or excipient.
10. Use of a polypeptide or composition of claim 8 or 9 for food processing.
PCT/EP1996/003091 1995-07-14 1996-07-15 New saccharomyces cerevisiae pectinase sequences and heterologous expression systems derived therefrom WO1997004106A2 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2774999A1 (en) * 1998-02-18 1999-08-20 Pascal Biotech New polygalacturonase prepared by expressing genes from Saccharomyces cerevisiae in micro-organisms, used to degrade pectin, e.g. for clarifying fruit juice
FR2880896A1 (en) * 2005-01-18 2006-07-21 Univ Reims Champagne Ardenne Procedure form converting a strain of yeast that does not produce endopolygalacturonase into one that does uses targeted modification of its genome
WO2007079428A3 (en) * 2006-01-03 2008-08-14 Massachusetts Inst Technology Promoter engineering and genetic control
CN107446903A (en) * 2017-09-13 2017-12-08 云南师范大学 A kind of salt tolerant pectase of resistance to ethanol and its gene with 3 optimal pHs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0552728A1 (en) * 1992-01-20 1993-07-28 Japan Tobacco Inc. Novel pectinase, low-molecular pectin, and food and drink which contain low-molecular pectin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0552728A1 (en) * 1992-01-20 1993-07-28 Japan Tobacco Inc. Novel pectinase, low-molecular pectin, and food and drink which contain low-molecular pectin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 122, no. 3, 16 January 1995 Columbus, Ohio, US; abstract no. 26478, BLANCO, P. ET AL: "Production and partial characterization of an endopolygalacturonase from Saccharomyces cerevisiae" XP002014943 & CAN. J. MICROBIOL. (1994), 40(11), 974-7 CODEN: CJMIAZ;ISSN: 0008-4166, *
DATABASE EMBL Emfun:Scyjr153w SCARCEZ, T XP002014942 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2774999A1 (en) * 1998-02-18 1999-08-20 Pascal Biotech New polygalacturonase prepared by expressing genes from Saccharomyces cerevisiae in micro-organisms, used to degrade pectin, e.g. for clarifying fruit juice
FR2880896A1 (en) * 2005-01-18 2006-07-21 Univ Reims Champagne Ardenne Procedure form converting a strain of yeast that does not produce endopolygalacturonase into one that does uses targeted modification of its genome
WO2007079428A3 (en) * 2006-01-03 2008-08-14 Massachusetts Inst Technology Promoter engineering and genetic control
CN107446903A (en) * 2017-09-13 2017-12-08 云南师范大学 A kind of salt tolerant pectase of resistance to ethanol and its gene with 3 optimal pHs

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