EP0208706A1 - Sequence d'adn utile pour la production et la secretion de peptides et de proteines a partir de levures - Google Patents

Sequence d'adn utile pour la production et la secretion de peptides et de proteines a partir de levures

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Publication number
EP0208706A1
EP0208706A1 EP19860900218 EP86900218A EP0208706A1 EP 0208706 A1 EP0208706 A1 EP 0208706A1 EP 19860900218 EP19860900218 EP 19860900218 EP 86900218 A EP86900218 A EP 86900218A EP 0208706 A1 EP0208706 A1 EP 0208706A1
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EP
European Patent Office
Prior art keywords
melibiase
gene
region
sequence
yeast cell
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EP19860900218
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German (de)
English (en)
Inventor
Martin Sumner-Smith
Richard Paul Bozzato
Roger Wayne Davies
Nigel Armstrong Skipper
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Allelix Biopharmaceuticals Inc
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Allelix Inc
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Publication of EP0208706A1 publication Critical patent/EP0208706A1/fr
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    • 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)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • 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)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to recombinant DNA technology.
  • it relates to sequences of DNA which in one aspect function to promote expression of a gene and, in another aspect, function to signal secretion of the protein coded by the expressed gene.
  • sequences are valuable in manipulating yeast cells.
  • Secretion a process by which particular products of gene expression are exported from the cell to the culture medium, is a natural property of living cells.
  • the feature of secretion of these products is desirable since the products are produced in a partially purified and readily accessible state by virtue of their extracellular location.
  • Protein and polypeptides produced in cells are destined either to remain within the cell or to be secreted from it.
  • the mechanism by which the destinies of these two categorically different types of proteins is determined has been experimentally shown to reside in a functional "signal" peptide coded for by a nucleotide sequence i.e. a signal sequence which in almost all known cases precedes the actual sequence of the gene coding for the protein to be secreted.
  • immature proteins translated from the complementary nucleotide sequence and bound for secretion initially bear a precursorial sequence of amino acids or "signal" which is recognized by receptors on the membraneous secretory pathway within the cell thereby permitting the immature protein to pass into the secretory pathway.
  • the signal sequence of the precursor protein is proteolytically cleaved to provide the active protein which may be released from the cell, after subsequent modification of its structure, e.g. glycosylation.
  • the cell may be sustained in a viable condition by supplying sufficient growth medium while continuing to secrete the desired protein.
  • the protein may be extracted directly from the nutrient broth within which the engineered cells are incubated, on a continuous basis according to known chemical procedures.
  • secretion of products expressed in yeast from genetically-modified expression vehicles is, in principle, assured by providing the foreign gene inserted in the vehicle with a nucleotide sequence encoding a signal peptide appropriate to yeast.
  • This signal peptide should be contiguous with, and at the N-terminal end of the peptide comprising the gene product of interest.
  • region upstream of the coding sequence of the gene typically includes sequences necessary for the initiation of transcription, as well as sequences which enhance and/or repress i.e. regulate, the level of transcription, often in combination with particular proteins present in the cell, together with other sequences which affect the transcription and translation of the gene.
  • This region is commonly called the 'promoter', although as stated it includes more than the sequence which is strictly defined as that region which promotes transcription.
  • This signal sequence is a short polypeptide stretch which is typically encoded in a sequence at the beginning of the translated portion of the gene.
  • the promoter and/or signal sequence as described above may be added, or used to replace analogous foreign sequences with sequences which are functional in yeast.
  • Emr et al (2) made gene fusions between nucleotide sequences coding for the leader region of the secreted yeast peptide, alpha factor, and nucleotide sequences coding for invertase, a yeast protein normally secreted into the periplasmic space, i.e.
  • Brake et al (3) constructed gene fusions between the alpha factor leader region and a synthetic DNA sequence encoding human epidermal growth factor (EGF) and found that this fusion resulted in efficient secretion from yeast of mature EGF, provided a limited region of the sequence of the alpha factor leader-coding region was altered, by in vitro mutagenesis.
  • GEF human epidermal growth factor
  • Bitter et al (4) constructed fusions between the alpha factor leader region and each of the proteins ß-endorphin and a "consensus" human interferon; they found secretion of these foreign proteins but also some degradation due to proteolysis during the secretion events.
  • yeast secretion siqnals contain information that facilitates secretion from yeast of foreign peptides and proteins, albeit peptides and proteins that are secretory gene products in their natural hosts.
  • the present invention provides in a first aspect, the signal sequence of the melibiase gene native to yeast, preferably derived from Saccharomyces carlsbergensis.
  • the present invention provides the regulatable promoter region of the premelibiase gene native to yeast, preferably derived from Saccharomyces carlsbergensis or a synthetic or semi-synthetic equivalent thereof.
  • a coding region which codes for premelibiase and melibiase native to yeast, preferably derived from Saccharomyces carlsbergensis.
  • vector constructs comprising at least one of the promoter region and the signal sequence for use in transforming a host yeast cell.
  • both the promoter region and the signal sequence are integrated on the vector construct and operatively associated with a coding region either native or foreign to the host yeast cell to be transformed.
  • the vector construct so formed is used to transform a foreign host.
  • FIGURE 1 represents the nucleotide sequence of a portion of plasmid pMP550 from Hind III to the first Bam HI site, including the 5' flank of the premelibiase gene including the promoter, the premelibiase gene sequence, showing in the lower line the amino-acid sequence of pre-melibiase and in the upper line the corresponding nucleotide sequence, and extending into the 3' untranslated sequence to the first Bam HI restriction site;
  • FIGURE 2 represents schematically a section of DNA segment shown in Figure 1;
  • FIGURE 2a provides the nucleotide sequence of the natural melibiase secretion signal represented in Figure 2;
  • FIGURE 2b provides the nucleotide sequence of a modified melibiase secretion signal in which the natural sequence depicted in Figure 2a is coupled with a synthetic linker sequence;
  • FIGURE 3 illustrates, via plasmid maps, a scheme for creating plasmid p4
  • FIGURE 4 illustrates, via plasmid maps, a scheme for creating plasmid p5;
  • FIGURE 4a provides the nucleotide sequence of a segment of plasmid p5 shown in Figure 4.
  • FIGURE 5 illustrates, via plasmid maps, a scheme for creating each of plasmids p6 and p7.
  • the signal sequence preferred herein acts as the signal sequence for melibiase secretion and is obtained from S . carlsbergensis by methods described hereinafter.
  • the particular amino acid sequence coded by the signal sequence is reproduced below:
  • nucleotide sequence corresponding to the above amino acid sequence is reproduced below:
  • the first six nucleotides 3' of reference numeral 30 shown in Figure 1 represent a Hind III site on pMP550.
  • Reference numeral 25 identifies the first Bam HI site downstream of Hind III site 30.
  • Within this 5' Hind III - Bam HI 3' region are located the promoter region and signal sequence of the melibiase gene as well as the melibiase coding region.
  • the entire, separated and isolated amino acid sequence of the premelibiase coding redion is shown in the lower lines startinq at reference numeral 10 and ending at reference numeral 1 2 .
  • the corresponding nucleotide sequence is shown in the lines above this particular amino acid sequence. In Fig.
  • a represents adenine nucleotide
  • t represents thymine nucleotide
  • g represents guanine nucleotide
  • c represents cytosine nucleotide.
  • the amino-acid corresponding to each codon has been entered using the normal triplet abbreviations therefor.
  • the structural gene for premelibiase is the sequence starting at reference numeral 10 with the atg codon equivalent to the initial methionine of premelibiase and ends with the tct codon at reference numeral 12 equivalent to serine, and immediately preceding the tga translation-stop sequence. This sequence consists of 1,413 nucleotides as shown in Figure 1.
  • nucleotide sequence In the naturally occurring material, atg starting codon 16 is preceded by a promoter DNA sequence (included in the sequence between reference numerals 20 and 10) to which reference is made hereinafter.
  • the entire protein illustrated in Fig. 1 has a molecular weight of 52,101 (between reference numerals 10 - 12) .
  • the nucleotide sequence from reference numerals 10-14 indicated on Fig. 1 encodes the pre-region, or signal peptide, of premelibiase.
  • This sequence starts with the atg codon equivalent to the initial methionine 16 of premelibiase at reference numeral 10 , and ends with the ggg codon at reference numeral 14, equivalent to the qlycine residue 18 adjacent to the start of the secreted melibiase.
  • This sequence consists of 54 nucleotides.
  • This signal sequence for melibiase has been found to have an unexpectedly large ability to direct the secretion from yeast cells of gene products coded by regions associated with the MEL sequence. Whilst it is not intended that the invention should in any way be limited or bound by any particular theory or mode of operation, it is postulated that the MEL signal has some ability to direct gene products produced under its influence into superior secretion pathways or via other modes of efficient operation.
  • promoters which are useful either in connection with the signal sequence described above or independently thereof are those which are capable of promoting transcription, in a regulatable fashion, of the premelibiase gene native to S. carlsbergensis.
  • the promoter region is identified in and derived from this yeast according to procedures described herein although it may be synthesized to provide a nucleotide sequence analogous to the naturally occurring one.
  • the premelibiase promoter region regulates transcription in response to certain proteins, including the products of the GAL4 and GAL80 genes. Specifically an increase in the level of expression of the GAL4 gene within the host containing the promoter region has been demonstrated to enhance the level of expression of a structural gene over which the promoter exerts its transcription-promoting function. Also, it has been demonstrated that where normal GAL80 product production within the host containing the promoter region is disrupted, expression of genes associated with the promoter region is enhanced and further that expression of such genes is no longer dependent on the presence of galactose in the medium as is the case in the presence of the GAL80 protein.
  • the promoter sequence is shown in Figure 1, between reference numerals 2 0 and 10.
  • these sequences can either be identified and isolated from yeast cells, in whole or in part, and/or produced by synthetic methods.
  • the promoter region for example, may be integrated into a vector and operatively linked either with a polypeptide coding region per se, where only expression is desired or with a signal sequence for secretion which in turn is operatively linked to and in the same reading frame as a coding region which encodes a desired protein.
  • the signal sequence is preferably as particularly described herein.
  • Examples of host yeasts in which this process may be used include S . carlsbe rgensis , S. ce revisiae , Saccharomycopsis lipolytica (also known as Candida lipolytica or Yarrowia lipolytica) etc. It will be understood that S. carlsbergensis is also known as S. uvarum.
  • genes useful in the constructs according to the invention are genes foreign to the aforementioned yeasts, include human, animal and bacterial genes, such as those for human proteins and peptides such as interferons, insulins, hormones, growth factors, enzymes etc., bacterial and fungal proteins such as cellulases etc., plant proteins, etc., peptide flavors and enhancers, antigens, etc.
  • Suitable expression vehicles or plasmids for introducing the constructs of the invention are those which contain genes or portions thereof appropriate to their maintenance and selection in the chosen host yeast and in E. coli, and yeast sequences appropriate to expression in the inserted foreign sequence, namely a strong yeast promoter, to ensure efficient transcription of the inserted nucleotide sequence, and a yeast sequence downstream of the insert to ensure adequate transcription termination.
  • yeast sequences appropriate to the expression of the product, other than the promoter and signal sequence described here may include a transcription terminator etc. as well as such modifications as are required to create the desired construct in a functional form.
  • Example 1 Nucleotide sequencing of promoter region, signal sequence, coding region and flanking regions of melibiase and premelibiase
  • plasmid PMP550 which is plasmid Yep24 (7 and 26) containing an additional fragment of yeast DNA, confers the melibiase-positive phenotype on otherwise melibiase-negative yeast when introduced into them by transformation, implying that the DNA insert contained in pMP550, an approximately 2.9 kilobase EcoRI to Bam HI fragment from a melibiase - positive yeast, contains the structural gene for premelibiase.
  • Plasmid pMP550 obtained from J.E.
  • DNA molecules with a decreasing size of the pMP550 insert sequence adjoined to the Ml3mpl8 sequence were prepared from selected phage and sequenced by the dideoxy technique (12), as detailed in the
  • Figure 1 also shows the predicted amino acid sequence of premelibiase, by translation of the long open reading frame.
  • the calculated molecular weiqht of premelibiase, from the nucleotide sequence, is 52,101.
  • the amino acids from methionine represented by reference numeral 16 to glycine represented by the reference numeral 18 comprise the melibiase signal peptide. Therefore, the calculated molecular wt. of secreted melibiase is 50,104. This agrees well with the molecular weight of melibiase that was purified from culture supernatants of yeast transformed with plasmid pMP550
  • the sequence of the remainder of the S. carlsbergensis flank was determined as follows: two ⁇ g pMP550 and 2 ⁇ g of M13mpl8 were each digested with Xmal and Sphl (New England Biolabs) and electrophoresed in 0.8 percent w/v low melting-point agarose. Gel slices containing the approximately 1.8 kilobase pMP550 fragment and the approximately 7.2 kilobase M13mp18 fraqment were melted and a few ⁇ l of each mixed, ligated and used to transform JM109 essentially as described above. One plaque was selected for further work after verification of the presence of the required insert.
  • a series of deletion clones were made extending through the insert essentially according to the method of Dale et al (1985). Briefly, 5 ⁇ g of single-stranded DNA were hybridized with a primer (RD29), and then digested with Hind III overnight at 50°C. The 3' end of the single-stranded DNA was deleted to varying degrees with T4 DNA polymerase (New England Biolabs), tailed with terminal transferase (IBI) and dATP (Sigma), annealed to RD29 primer and ligated. Following transfection of
  • DNA was prepared and sequenced as described above. A further nine-hundred and twenty-nine nucleotides of sequence from pMP550 were determined by this method. The two methods therefore yielded the complete sequence of the S. carlsbergensis-derived
  • Plasmid pMP550 was introduced into the melibiase-negative yeast strain S . cerevisiae 284 ( ⁇ ,leu 2-3, leu 2-112, ura 3-52, ade 1, mel°), supplied by J. E. Hopper, Pennsylvania State University, by transformation, according to the method described by Ito et al (16). The transformed cells were spread onto agar plates that select for the growth of cells independent of exogenous uracil and incubated for 2-3 days at 30°C.
  • Composition per litre of this medium was: glucose, 20g; agar, 17g; yeast nitrogen base without amino acids, 6.7g; succinic acid, 5.8g; dipotassium hydrogen phosphate, 8.7g, adenine, arginine, histidine, isoleucine, leucine, methionine, tryptophan and tyrosine, 0.02g each, lysine, 0.03g; phenylalanine, 0.05g; threonine, 0.10g and valine, 0.15g; pH was 4.6-4.8.
  • Colonies that appeared on selective medium I were transferred to fresh selective medium I, allowed to grow at 30°C, then transferred to agar plates containing selective medium II, that is selective medium I with 20g/L lactic acid, 30g/L glycerol, and 20g/L galactose, in the place of 20g/L glucose. This was to allow induction by galactose of melibiase synthesis (5) . The colonies were then used to inoculate liquid medium consisting of selective medium II without agar. Following cell growth at
  • the cultures were assayed for secreted melibiase by the following procedure: 500 ⁇ l culture was centrifuged to pellet the cells, then 10 ⁇ l of the supernatant fluid added to 190 ⁇ l pH 4 buffer (30mM citric acid, 38mM dibasic sodium phosphate) containing 12.5mM p-nitrophenyl ⁇ -D-galactoside (p-NPG; Sigma
  • this material contained 2 bands, representing 98% of the protein.
  • One band was diffuse, encompassing the size range 75 to 100 kdaltons, midpoint 82 kdaltons, the second sharp and with a calculated molecular weight of 70 kdaltons.
  • the molecular weight of the melibiase polypeptide chain and to determine if it was pure, covalently-linked carbohydrate, known to be part of the melibiase secreted from S.
  • carlsbergensis (Lazo et al, 19; Lazo et al, 20) was enzymatically removed from the sample with endo-ß-N-acetylglucosaminidase H (endo H) from Streptomyces griseus, as follows; 40 ⁇ q of protein was incubated with 5 milliunits of endo H (Boehringer Mannheim Canada) at 37°C for 25h in a 40 ⁇ l reaction containing 0.05 M citric acid and 0.1 M disodium hydrogen phosphate. The sample was lyophilized then analysed by SDS-polyacrylamide gel electrophoresis. A single protein band, with a calculated molecular weight of 50 kdaltons, was observed.
  • the melibiase signal sequence is defined as follows:
  • Example 3 Use of an expression cassette containing the melibiase promoter and signal sequence to effect production from Saccharomyces cerevisiae of a novel extracellular protein
  • the expression cassette is illustrated schematically in Figure 2. Its essential element are (i) flanking restriction sites, in this example Bam HI, which allow for its ready insertion into appropriate E. coli/S. cerevisiae shuttle vectors; (ii) the DNA sequences for the melibiase promoter 32
  • the segment designated 34 in Figure 2 includes additional sequences such as those specifying transcription, termination, cleavage and polyadenylation.
  • the expression cassette was constructed as described below and as shown in Figure 3 to which reference is now made, using conventional recombinant DNA techniques.
  • Plasmid pMP550 ( Figure 3) is plasmid pBR322 (Bolivar et al; 22) containing a 4.3 kilobase DNA fragment 40 which includes the melibiase gene and its flanking DNA sequences (Post.
  • the nucleotide sequence of fragment 40 is specified in Figure 1, as bounded by the Hind III site 30 and Bam HI site 25. It should be noted that the first 346 nucleotides (reference numerals 30 to 20 in Figure 1) are derived from plasmid pBR322. This is because of the cloning strategy used, and is incidental to the function of the expression cassette derived from the 4.3 kilobase DNA fragment. Plasmid pMP550 was restricted with Hind
  • Plasmid p2 was restricted with Sphl which cuts uniquely within the DNA sequence that encodes the melibiase signal peptide. The linear ized plasmid was then ligated to a synthetic oligonucleotide 42 of the sequence
  • Plasmid p3 was then restricted with Bam HI and the 4.3 kb fragment cloned into the Bam HI site of the shuttle plasmid Yep 24(26), generating plasmid p4.
  • Plasmid p4 contains the 4.3 kilobase fragment originally from pMP550 but the DNA sequence encoding the melibiase signal peptide was now modified, as specified in Figure 2.
  • the 4.3 kilobase Bam HI fragment 40 in p4 ( Figure 3) constitutes the expression cassette which is an example of a preferred embodiment of the present invention.
  • a DNA sequence known to encode a celluase (an endoqlucanase or endo-1, 4- ⁇ -D-glucanase, EC 3.2.1.4) in the soil bacterium Cellulomonas fimi ATCC 484 was used to demonstrate the utility of the expression cassette in Saccharomyces cerevisiae.
  • the endoqlucanase codinq sequence is contained in a 2.4 kilobase Bam HI fraqment 46 from the plasmid pEC2.2 ( Figure 4; Skipper et al; 27) provided by Dr. Robert C. Miller, University of British Columbia.
  • the coding sequence is known to be missing the codons for the first 76 amino-terminal amino acids of the bacterial protein, including the 31 amino acid bacterial signal peptide (28).
  • Saccharomyces cer-gy-isiae cells transformed with a plasmid containing the 2.4 kilobase Bam HI fraqment from pEC2.2 in an operative association with the yeast alcohol dehydroqenase 1 promoter produced a very low level of extracellular endoglucanase activity (Skipper et al; 27) .
  • the addition of an operative signal peptide provided by codons specifying the amino terminus of the yeast preprotoxin protein resulted in a greatly enhanced level of extracellular endoglucanase activity (Skipper et al; 27).
  • the 2.4 kilobase Bam HI fragment 46 from pEC2.2 was inserted into plasmid p4 ( Figure 4) by cohesive-end ligation at the Bgl II site of this plasmid.
  • Recombinant plasmids conta ining the endoglucanase insert were restricted with Bgl II, to identify those containing the insert in the appropriate orientation, and several of those sequenced across the melibiase/endoqlucanase junction to identify those containing the predicted qene fusion.
  • One of these plasmids, p5 was selected for further analysis. It contained the endoqlucanase coding sequence fused to the sequence encoding the melibiase siqnal peptide, in the correct translational reading frame (see
  • Plasmid p5 is Yep24 containinq the melibiase: endouiucanase gene fusion. Since Yep24 carries the URA3 gene, it can be used to transform ura3 yeast strains to uracil-independence. To extend the range of recipient
  • Saccharomyces cerevisiae strains for assessment of endoglucanase expression from the melibiase elements the 6.7 kilobase Bam HI fragment 48 from p5 was cloned into each of 2 further shuttle vect ors , Yep21 and Yep 24.TRP1 ARS , generat ing plasmids p6 and p7, respectively ( Figure 5).
  • Plasmid Yep21 (26) carries the yeast LEU2 gene for transformation of leu2 strains to leucine-independence.
  • Plasmid Yep24.TRP1 ARS was constructed from plasmids Yep24 (26) and Yrp7 (26), as illustrated in Figure 5. It is useful for transferring trp1 yeast to tryptophan-independence, and ura3 yeast to uracil-independence.
  • Saccharomyces cerevisiae strain 20B12 ( ⁇ , trpl, pep4.3; Jones,
  • Example 4 Use of different configurations of yeast chromosomal genes to effect altered regulation of the expression of a novel protein from the expression cassette
  • GALl, GAL2 , GAL7 , and GAL10 expression of the MEL-1 (melibiase) gene is induced by galactose and repressed by glucose. This control by the carbon source appears to be mediated by the products of the GAL4 and GAL80 genes.
  • GAL4 produces a product which is required for transcription of all the genes. The action of GAL4 is inhibited by the GAL80 product, which is thought to bind to it or to its site of action on the DNA in the absence of galactose. In the presence of galactose the binding of GAL80 product is prevented and all the qenes are turned on.
  • the site o f b i nding o f the GAL4 produc t is in the 5 ' -flanking reg ion of each targ et gene , a reg ion call UAS or upstream activation sequence (see Sumner-Smith et a l; 23 , for br ief review) .
  • Manipulations of the GAL4 and GAL80 genes are therefore expected to modify the regulation of expression of proteins dependent on the melibiase promoter, of which extracellular endoglucanase is used as an example here.
  • Strain Sc295 (ura 3-52, ade 1, gal80-deletion) lacks the GAL80 gene, so that galactose is unnecessary for full expression of the melibiase promoter.
  • Strain Sc300 (leu 2-3, leu 2-112, ade 1, gal80-deletion, gall-ga17-gal10-deletion, ADH1:GAL4) lacks GAL80 and in addition has 2 modifications intended to increase the availability of the GAL4 protein to the melibiase promoter. First, the GAL1, GAL7 and GAL10 gene cluster is deleted.
  • an additional copy of the GAL4 gene is present, expressed from the strong promoter of the yeast alcohol dehydrogenase 1 gene; this is estimated to increase the synthesis of GAL4 protein by a factor of 150 over cells containing the normal single chromosomal GAL4 gene.
  • the phenotype of strain Sc300 with respect to melibiase promoter function is not known in any detail, but it could be reasonably predicted to be both constitutive promoter function, due to the GAL80 deletion, and an elevated maximal function, due to the increased availability of GAL4 product.
  • the 3 yeast strains were each transformed with an operative expression cassette/endoglucanase gene fusion, contained in an E . coli/S. cerevisiae shuttle vecto r appropriate to the select ion of t ransfo rmants (Table 1) .
  • Transformants were then grown in liquid media containing carbon sources either non-inducing, inducing or repressing for the melibase promoter in a normal GAL4 , GAL80 strain, then assayed for extracellular endoglucanase activity. The results are illustrated in Table 1 and discussed below:
  • Transformant 113.1 (strain Sc284) .
  • the absolute amount of extracellular endoglucanase produced by this transformant was low, making it difficult to assess the regulatory effect of the various combinations of carbon sources with any accuraccy.
  • the response of endoglucanase expression to the carbonsources was consistent with that expected for a strain carrying wild-type GAL4 and GAL80 genes: induction by galactose, repression by glucose.
  • Transformant 149.1 (strain Sc295). Endoglucanase expression was maximal in the absence of galactose, consistent with the expected effect of the GAL80 deletion, and greatly reduced by 2 percent glucose, consistent with normal glucose repression. The presence of galactose in fact partially repressed endoglucanase synthesis, an effect that has also been seen in GAL80 deletion strains expressing extracellular melibiase (unpublished obserations).
  • Transformant 142.11 (strain Sc300) . Endoglucanase expression in this strain was the highest seen in any yeast recipient. Unexpectedly, galactose was required for maximal endoglucanase expression, and glucose was only partially effective as a represser of its synthesis. Both these unpredicted effects are presumably due to the increased availability of the GAL4 product.
  • NI non-inducing in 2 percent lactic acid, 3 percent glycerol, 0.05 percent glucose; I (inducinq) is NI plus 2 percent qalactose; R (repressing) is I plus 2 percent glucose.
  • C tryptophan medium contained, in grams per litre, yeast nitrogen base, 6.7; adenine, arginine, isoleucine, tyrosine, uracil and leucine 0.02 each; lysine, 0.03; phenylalanine, 0.05; threonine, 0.10; valine, 0.15; Na 2 HPO 4 , 3.0; KH 2 PO 4 , 3.0;
  • uracil medium contained, in grams per litre, yeast nitrogen base, 6.7; adenine, arginine, isoleucine, tyrosine, tryptophan, and leucine, 0.02 each; lysine, 0.03; phenylalanine, 0.05; threonine, 0.10; valine, 0.15; Na 2 HPO 4 , 3.0; KH 2 PO 4 , 1.5.
  • leucine medium contained, in grams per litre, yeast nitrogen base, 6.7; adenine, arginine, isoleucine, tyrosine, tryptophan, and uracil, 0.02 each; lysine,

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Abstract

Composition, notamment la séquence de nucléotides du gène d'ADN codant la prémélibiase de levure et la séquence d'acides aminés contenue dans la protéine de prémélibiase. L'invention décrit la séquence d'ADN du gène de prémélibiase, en combinaison avec la séquence d'acides aminés de la mélibiase sécrétée, et définit ainsi la séquence d'acides aminés de la prérégion et la séquence de nucléotides codant cette séquence. La prérégion de prémélibiase peut être utilisée pour obtenir, par des techniques de génie biologique, des peptides et des protéines autres que la mélibiase à partir de levures modifiées génétiquement, étant donné que les fusions effectuées entre sa séquence de nucléotides et la séquence de codage de peptides et protéines étrangers assurent la sécrétion de ces produits génétiques dans les levures, lorsqu'elles sont contenues dans des véhicules d'expression génétique appropriés. Est également décrite la séquence de nucléotides de la région de promotion du gène de prémélibiase, cette séquence pouvant être régulée par des produits protéiques des gènes GAL4 et GAL80. La région de promotion peut être intégrée dans une structure de vecteur avec un gène structural et comprenant éventuellement la séquence de signaux définie ci-dessus, afin de pouvoir réguler l'expression d'une protéine dans un hôte de levure transformée.
EP19860900218 1984-12-24 1985-12-23 Sequence d'adn utile pour la production et la secretion de peptides et de proteines a partir de levures Withdrawn EP0208706A1 (fr)

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US68570484A 1984-12-24 1984-12-24
US685704 1984-12-24

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EP (1) EP0208706A1 (fr)
JP (1) JPS62502025A (fr)
AU (1) AU5304286A (fr)
CA (2) CA1252256A (fr)
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WO (1) WO1986003777A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2907393A1 (fr) 2014-02-17 2015-08-19 DMK Deutsches Milchkontor GmbH Produits du lait dénué du lactose

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5068185A (en) * 1986-07-10 1991-11-26 Merck & Co., Inc. Trains of yeast for the expression of heterologous genes
US5055401A (en) * 1987-04-10 1991-10-08 Alko Ltd. Construction of new α-galactosidase producing yeast strains and the industrial application of these strains
FR2615527B1 (fr) * 1987-05-22 1989-08-18 Lesaffre Soc Ind Procede d'integration d'une sequence connue d'adn dans les levures ascosporogenes, vecteurs mis en oeuvre et nouvelles souches de levures
DK38893D0 (da) 1993-03-31 1993-03-31 Novo Nordisk As Dna
JPH08322575A (ja) * 1995-05-30 1996-12-10 Takara Shuzo Co Ltd プロモーター
ES2351296B8 (es) * 2009-04-08 2012-07-03 Universidade Da Coruña Cepas de s. cerevisiae capaces de crecer en medios con melibiosa, estaquiosa y rafinosa.
US11692016B2 (en) * 2012-03-09 2023-07-04 Vestaron Corporation High gene expression yeast strain
CN110982721B (zh) * 2019-12-09 2022-04-26 宜昌东阳光生化制药有限公司 提高酿酒酵母代谢产物产量的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8603777A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2907393A1 (fr) 2014-02-17 2015-08-19 DMK Deutsches Milchkontor GmbH Produits du lait dénué du lactose

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JPS62502025A (ja) 1987-08-13
DK403186A (da) 1986-08-22
DK403186D0 (da) 1986-08-22
CA1284959C (fr) 1991-06-18
AU5304286A (en) 1986-07-22
WO1986003777A1 (fr) 1986-07-03
CA1252256A (fr) 1989-04-11

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