EP0956356A1 - SYSTEME DE TRANSFORMATION DANS $i(CANDIDA UTILIS) - Google Patents

SYSTEME DE TRANSFORMATION DANS $i(CANDIDA UTILIS)

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Publication number
EP0956356A1
EP0956356A1 EP97943729A EP97943729A EP0956356A1 EP 0956356 A1 EP0956356 A1 EP 0956356A1 EP 97943729 A EP97943729 A EP 97943729A EP 97943729 A EP97943729 A EP 97943729A EP 0956356 A1 EP0956356 A1 EP 0956356A1
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EP
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Prior art keywords
yeast
host
candida
defective
gene
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EP97943729A
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German (de)
English (en)
Spanish (es)
Inventor
Luis Apartamento 5B RODRIGUEZ MENOCAL
Francisco Pablo Calle 7ma CHAVEZ ESPINOZA
Maria Elena Calle 26 No. 1002 GONZALEZ MARTINEZ
Tanilo Calle 58B No. 6703 RIVERO BAEZA
Liliana Apartamento 3C BESABE TUERO
Edenia Apartamento 41 PAIFER REYES
Julio Marcos Apartamento 52 DELGADO BOADA
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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Publication of EP0956356A1 publication Critical patent/EP0956356A1/fr
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Classifications

    • 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)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase

Definitions

  • the present invention is related to the field of genetic engineering and biotechnology, and in particular to the development of a host-vector system for the genetic transformation of the yeast Candida or tilis, which allows the expression and secretion of heterologous proteins in this yeast, which can be subsequently used for various purposes.
  • Escherichia coli bacteria have been for some time the most widely used microorganism for these purposes by various biotechnology companies, due to the knowledge they have regarding their genetics, their easy handling and their large-scale culture systems.
  • heterologous proteins in euca ⁇ ot systems has some advantages over prokaryotic systems. Among these we can point out the ability to grow at high cell densities and the possibility of adapting their culture to continuous systems.
  • yeasts are able to secrete proteins to the culture medium in considerable greater amounts compared to E. coli, and the culture media used for yeast growth are cheaper than those used in bacteria (Lemome, Y., 1988. Heterologous expression ⁇ n yeast. 8th International Biotechnology Symposium, Paris, July 17-22).
  • these systems can carry out other post-translational modifications such as glycosylation, which is absent in bacterial systems (Fiers, W., 1988.
  • Saccharomyces cerevisiae protein expression in Saccharomyces has faced problems ranging from low levels of expression obtained using its homologous promoters until hyperglycosylation of the products secreted to the medium, that is why in recent years the search for unconventional yeasts for use in the expression of heterologous proteins has intensified.
  • Candida utilis yeast is particularly interesting because of its peculiar characteristics. First, it uses a wide spectrum of cheap carbon sources such as xylose, sucrose, maltose among others. Another interesting feature is that it is possible to efficiently produce a large number of cells in a continuous culture.
  • Candida utilis like Saccnaromyces cerevisiae and Kl uyveromyces lactis, have been authorized by the FDA (Food an Drug Administration) as safe sources of food additives. At the same time, Candida utilis has been used as a source for the industrial production of L-glutamma, ethyl acetate, mvertase, among other products.
  • a preliminary transformation system in Candida utilis has been described by Ho, I. et. al., 1984, (Biotechnology and Bioengmeenng Symp. 14: 295-301). In the mentioned work there is no direct evidence of the presence of the antibiotic resistance marker, so the direct verification of the transformation is incomplete.
  • Candida utilis could be an attractive organism for commercial use as an expression system for heterologous proteins.
  • the objective of the present invention has been to provide a transformation system that allows to express heterologous proteins in Candida yeast or tilis, based on obtaining auxotrophic mutants of this species as well as in the isolation of genes, from a genomic library , which complement said auxotrophies.
  • the transformation process described here provides means for the introduction of DNA fragments or sequences into a host strain of Candida utilis and allows this yeast to be used for protein expression and production.
  • the isolation of genes whose promoter and thermatoring sequences can serve for the expression of heterologous genes in said yeast has been a purpose of this invention.
  • transformed yeast cells can be identified and selected by the methods described in the present invention.
  • New strains of Candida utilis, vectors and subclones are provided.
  • the new strains are used as hosts for the introduction of recombinant DNA fragments.
  • the invention also relates to the stable transformation and maintenance of said DNA fragments in the host cells, where the marker is integrated by homologous recombination in the yeast genome.
  • the invention consists of a new transformation system for the yeast Can ⁇ ida or tilis, which uses new auxotrophic mutants isolated from strain NRRL Y-1084 of said yeast as hosts.
  • mutants are deficient in the enzyme orot ⁇ dm-5 phosphate decarboxylase of the uracil biosmhesis pathway or in the enzyme imidazole glycerol phosphate dehydratase of the biosmtetic pathway of histidma, which were obtained by using classical mutagenesis caused by physical agents chemists known from the state of the art (Sherman, F. et al., 1986. Laboratory course: Manual for methods m yeast genetics. Cold Sprmg Harbor Laboratory Press, NY). These mutants showed a high stability (reversal frequency approximately 10) and can be efficiently transformed by the method described in the present invention.
  • the URA3 gene coding for the enzyme orotidin 5-monophosphate decarboxylase and the HIS3 gene coding for the enzyme Imidazol-glycerol phosphate dehydratase, were isolated as selection markers for the new Candida or tilis mutants, which were isolated at from a library of Candida utilis constructed in plasmid pUC19 and identified by complement of the pyrF and h ⁇ sb463 mutations of the E strains. coli MC1066 and KC8 respectively.
  • the URA3 gene of C. u til is complement the URA3 mutation of Sa ccnaro yces cerevisiae SEY 2202. The complete sequence of the isolated genes was determined and the predicted amino acid sequence showed a high similarity with that of the same gene from other yeasts and mushrooms .
  • the mtegrative vectors constructed to perform the transformation of this mutant were pURA5 and pUREC3. These plasmids contain the URA3 gene isolated from Candida utilis as a selection marker, in addition to presenting chromosomal DNA regions that are flanking it, thus favoring integration into the Candida utilis locus by homologous recombination.
  • Another novel aspect of the present invention is the isolation of the gene that encodes the enzyme sucrose invertase or P-fructofuranosidase ⁇ INV1) of Candida utilis, as well as the identification of the promoter, thermator regions and the signal sequence of this gene, which can also be advantageously used in the protein expression of different origins in said yeast.
  • the present invention also provides a series of expression vectors based on the system described above, which are used for the transformation of mutants isolated from C. utilis with a view to obtaining heterologous proteins.
  • the transformation system of the present invention uses as new hosts auxotrophic mutants from strain NRRL Y-1084 of Candiaa or tilis, which are mainly defective in the biosynthetic pathway of uracil and histidma, within which they were selected by its characteristics the imitant CUT-35 for uracil, as well as for the histidm the TMN-3 mutant.
  • auxotrophic mutants from strain NRRL Y-1084 of Candiaa or tilis, which are mainly defective in the biosynthetic pathway of uracil and histidma, within which they were selected by its characteristics the imitant CUT-35 for uracil, as well as for the histidm the TMN-3 mutant.
  • a selection marker for the transformation which may be an auxotrophy marker or a dominant marker
  • a classic mutagenesis was carried out in the Candida utilis yeast.
  • cultures of the selected yeast strain (NRRL Y-1084), were inoculated in 100 ml of YPG medium (1% yeast extract, 2% peptone, 2% glucose) and incubated in a shack at 30 ° C of 10-20 hours 50 ml of culture were taken and centrifuged at 3000 rpm for 5 minutes. Later The cells were washed twice with sterile 0.1M citrate buffer (pH 5.5) and then resuspended in 50 ml of the same buffer.
  • the minimum medium (YNB; Yeast Nitrogen Base) used for enrichment with the antibiotic was not supplemented with the metabolite produced by the biosynthetic pathway in which the defect is sought. For example, for the isolation of auxotrophic mutants to uracil, it is not added to the medium. Incubation was continued until the optical density (OD) of the culture reached 20-30 ⁇ of the initial OD. When the culture reached the desired OD, the cell suspension was treated with 25 units / ml of a nystatma solution. The solution treated with the antibiotic was incubated at 30 ° C for 30 minutes without stirring.
  • OD optical density
  • nystatma was removed from the medium by washing the cell suspension twice with distilled water and then the cells were resuspended in a suitable volume to obtain 150 to 200 colonies per plate.
  • Landscape and Selection Plates containing the mutagenized colonies according to the enrichment performed in the previous example were plated in YNB minimal medium with and without uracil. The colonies that did not grow in the absence of uracil were taken for further analysis.
  • ura3 and ura5 mutants were grown in the presence of the toxic acid compound 5-fluorotok (5-FOA). Resistant colonies were selected as ura 3 or ura5 mutants.
  • 5-FOA the toxic acid compound 5-fluorotok
  • Example 2 Isolation of uz a3 mutants.
  • mutants were checked for the frequency of reversion, highlighting a group of about 23 mutants for presenting a reversal frequency of the order of 10 " , which gives them acceptable stability as hosts with a view to being transformed.
  • the ura auxotrophic mutants thus obtained were checked for ODCase activity (activity of the URA3 gene product), by the method described by Yoshimoto et. al., 1978 (Methods Enzymol. 51: 74-79), as well as 8 of them were determined Its growth parameters. The results of the reversal frequency, as well as these last determined characteristics, are shown in Table 1. Table 1. Summary of the characteristics of the most significant ura3 mutants. Name Frequency Activity Growth Reversal OMPDCase
  • Example 3 Isolation of mutants other than the ura phenotype.
  • the cell suspension obtained according to enrichment with nystatin was disseminated in YPG plates, which were incubated at 30 ° C for 50 hours. Subsequently, the colonies contained in the YPG plates were replicated on plates containing YNB minimum medium, and incubated at 30 ° C for 48 hours. Colonies that did not grow on the YNB plates were taken for later analysis.
  • auxotrophic mutants As a result, about 2411 colonies were checked and a 2% appearance of auxotrophic mutants was obtained. These mutants were checked through the Holliday test and the Finchan test, from which it was obtained that 90% of the mutants obtained responded to the his phenotype, " 2% to the lys phenotype " , 1% to the leu phenotype " , 1% at phenotype meth, 1% to ade phenotype ", and 5% did not show a simple auxotrophic phenotype (Naa).
  • the mutants obtained were checked for the frequency of reversal, thus selecting the most stable for presenting a frequency of reversal between 10 'and 10 (Table 2).
  • Example 4 Construction of a genomic bookstore of Candida utilis.
  • the chromosomal DNA extracted from Candida or tilis NRRL Y-1084 was partially digested with the Sau3A enzyme and fragments of sizes between 6 and 9 kb were isolated by low melting agarose gel electrophoresis
  • LGT LGT
  • F ' D Lac x74, hsr, hs, rpsl, galU, galK, trip C 9030F, leuB, pyrF :: tn5
  • Example 5 Isolation of the URA3 gene from Candida utilis.
  • the plasmid was purified and rechecked by transformation in this strain of Escne ⁇ chia coli MC1066 that the restoration of the URA3 phenotype is associated with the presence of the plasmid.
  • the plasmid pURA5 was digested with several restriction enzymes according to the sites present in the vector of the püC19 library. With a view to the boundary of the Candida URA 3 gene, you use the fragments corresponding to the digestions EcoRI (1.9 kb), HincII (1.5 kb), Sacl (l, lkb) were subcloned into pBluesc ⁇ pt SK (+) giving rise to plasmids pUREc-3, püRHmc-1 and pURSac-4, respectively. Of these plasmids only pURSac-4 was not able to complement the pyrF mutation of Escherichia coli ( Figure 2).
  • the 1.9 kb EcoRI fragment (pUREc-3, Figure 3) containing the useful Candida URA 3 gene is double sequenced RAMIFICATION by the method of Sanger et. to the. (1977, Proc. Nati. Acad. Sci USA 74: 5463-5467).
  • the 3'-untranslated region contains a possible TATAAAA polyadenylation signal (AATAAAA consensus) found in the 3 'terminal region of most euca ⁇ otic genes (Guo, Z and Sherman, F., 1995. Mol. Cell. Biol. 15: 5983-5990).
  • AATAAAA consensus TATAAAA polyadenylation signal found in the 3 'terminal region of most euca ⁇ otic genes (Guo, Z and Sherman, F., 1995. Mol. Cell. Biol. 15: 5983-5990).
  • Example 7 Complementation analysis in Saccharomyces cerevisiae.
  • the 2.8 kb Kpnl / Xbal fragment of the purasmid pURA5 was cloned into a pBR322 derivative vector (pBSARTR -3) .
  • the vector pBSARTR 3 contains a Autonomous replication sequence (ARS1) and the TRIP1 selection marker, both from Saccnaromyces cerevisiae.
  • pUT64 vector was obtained for Saccharomyces cerevisiae ( Figure 5) which was used to transform the Saccnaromyces cerevisiae strain SEY2202 (URA3-52-, leu2-111, h ⁇ s3-) using the Lithium Acetate method previously reported by Ito. et. al., 1983 (J. Bacteriol. 153: 163-168). The transformants were obtained 48 hours after the transformation was performed and the presence of the plasmid was checked by hybridization ⁇ e colonies and southern blot experiments.
  • transformation frequency obtained (2-5 x 10 "transf / mg) corresponded to that reported in the literature for other auxotrophic markers obtained in yeasts.
  • the ⁇ tante ara 3 strain of Candi ⁇ a j til is CUT35 (Number of Pending Deposit) was transformed by the lithium acetate method reported by Ito. et. to the. (1983, J. Bacte ⁇ ol. 153: 163-168) and using as a selection marker the Candida URA3 gene or previously isolated tilis.
  • the transformation with plasmids pURA5 and p C URA3 carrying the URA3 gene of Candida utilis was carried out by integrating said gene by homologous recombination in the corresponding region of the Candida utii is genome.
  • the plasmid pOC URA 3 was obtained by cloning the 1.9 kb EcoRI fragment of the URA3 gene from Candida utilis in the vector pUC19 ( Figure 6). To do this, the plasmids were digested with the restriction enzyme Xhol which is located towards the end of the structural gene. The linearization of the plasmid favors the homologous integration in the genomic locus
  • the transformation procedure used which is based on the treatment of intact yeast cells with alkali metal cations, is basically the same used for Saccnaromyces cerevisiae, with 50 mM Lithium Acetate instead of 100 mM.
  • the selection of the transformants was carried out in a minimal medium YNB lacking uracil.
  • the mitotic stability of these transformants is high, due to the homologous integration mechanism.
  • the transformation frequency coincides with that reported for mtegrative plasmids of Saccnaromyces cerevisiae and other unconventional yeasts using integrative vectors (Table 3).
  • the mutant strain ura3 of Candida utilis CUT35 was transformed by the electroporation method reported by Kondo, K. et al., 1995, (J. Bacteriol. 177: 7171-7177) and using the URA3 gene of Candida utilis as the selection marker previously isolated.
  • the transformation with plasmids pURA5 and pxxC URA 3 carriers of the Candida utilis URA3 gene was carried out by means of ⁇ -integration of said gene by homologous recombination in the corresponding region of the Candida utilis genome.
  • the transformation procedure used is based on the treatment of intact yeast cells with an electric field set to the following conditions 0.7 kV (3.5 kV / cm), a resistance of 800 ⁇ and a capacitance of 25 F.
  • both plasmids were digested with the restriction enzyme Xhol which is toward the end of the structural gene which favors the homologous integration event.
  • YNB Yeast Nitrogen Base
  • the HIS3 gene of Candida utilis was isolated and characterized from the library previously described in Example 4.
  • DNA fragments, which contain the HIS3 gene of Candida utilis, were isolated from a genomic library for their ability to complement the mutation h ⁇ sB463 ⁇ e Escherichia col i KC8 (hsd, h ⁇ sB463, leuB6, pyrF :: Tn5 Km, trp (9830 (lact YA), stm, galU, gal), taking into consideration that the HIS3 gene of Saccharomyces cerevisiae complements the h ⁇ sb463 mutation of Escherichia coli, using fortuitous promoter sequences in Escherichia coli.
  • Imidazole glycerol phosphate dehydratase from Candida or til is, approximately 10 " transforming cells were seeded in selective medium (M9 supplemented with uracil, leucma and tryptophan. Plasmid DNA was extracted from colonies capable of growing therein. medium and therefore capable of complementing the h ⁇ sb463 mutation of the Escnericma coli KC8 strain. The plasmids obtained were used to retransform the Escherichia coli KC8 strain. All plasmids capable of supplying the histidma requirement were called pHCU.
  • PCR band of approximately 500 bp that Southerbott demonstrated that hybrid with the genomic DNA of Candida utilis was cloned into T-Vector (pMOSBLUE, Amershan) and the predicted ammoacidic sequence of its nucleotide sequence was highly homologous to H ⁇ s3p from other yeasts and fungi. Plasmid pHCU 37 ( Figure 9), was used to determine the sequence of the HIS3 gene of Candida utilis.
  • Example 11 Sequencing of the HIS3 gene of Candida utilis. The HIS3 gene of Candida or tilis was completely sequenced double RAMIFICATION by the method of Sanger et. to the. (1977). Universal oligonucleotides of the M13mp / pUC series were used.
  • Example 13 Sequencing of the INVl gene from Candida utilis.
  • a total of 2607 bp of clone pCI-6 containing the INVl gene encoding the Candida utilis mvertase was completely sequenced double RAMIFICATION by the method of Sanger et. al., (1977), both universal oligos of the M13mp / pUC series as well as internal oligos derived from the sequence were used for this.
  • the complete sequence of the 2607 bp fragment is shown in Figure 12 (No. Sec. Sec: 5, 6). This fragment contains an open frame ⁇ e reading ⁇ e 1602 bp (534 codons ⁇ .
  • the Candida INVl gene utilis codes for a theoretical molecular mass protein 60 703 Da.
  • Candida utilis mvertase is a periplasmic enzyme, it must have a peptide signal at its N-thermal end. Analyzing the sequence towards the 5 end of the gene reveals two ATG codons (ATGi and ATG 2 in Figure 12) giving rise to ORF coding for proteins that differ only in the size of their N-thermal ends. Applying von Heijne G.'s (1986, Nuc ⁇ . Acids Res.
  • N-glycosylation sites according to the general NXT / S rule are found in the asparagraphs of positions 40, 88, 141, 187, 245, 277, 344, 348, 365, 373, 379 and 399 of the sequence of the mature protein.
  • the 5-non-translated region shows two possible TATA boxes (TATAA consensus), in the -18 to -14 and -212 to -208 regions, in addition to several possible Migl repressor binding sites (SYGGRG consensus).
  • FIG. 1 Plasmid pURA5, resulting from the Candida utilis library, where the Candida utilis DNA fragment that complements a pyrF mutation in Escne ⁇ chia with MC1066 and URA3 of Saccnaromyces cerevisiae SEY 2202 was identified.
  • Figure 2. Location, restriction map and complement analysis of the URA 3 gene of Candida utlilis. Sequencing strategy of said gene.
  • Figure 4 DNA sequence corresponding to the fragment that contains the URA3 gene of Candida or til is.
  • Figure 5. Plasmid pUT64 obtained for the complementation experiments of the ura3 mutation in Sa ccnaromyces cere ⁇ siae.
  • Figure 8 Peptide sequence and DNA corresponding to the oligonucleotides used in the PCR reaction for the isolation of the HIS3 ⁇ e Candida useful gene.
  • Candida u tilis which contains a fragment capable of complementing the h ⁇ sB463 mutation of Esche ⁇ chia coli KC8.
  • Figure 11 Peptide sequence and DNA corresponding to the oligonucleotides used in the PCR reaction for the isolation of the INVl gene from Candida utilis.
  • TYPE Z nucleic acid. RAMIFICATION: simple ⁇ Z, TOPOLOGY: linear di) TYPE OF MOLECULE: DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAGTCATCAC CATCGTACTT TAACGACTTA CTATTCTCAT TGAGTATTGA GAAGAAGGAT 240 AGAGAAATGG CTGAACGAAC GGTGAAACCC CAGAGAAGAG CTCTTGTGAA TCGTACAACA 300
  • CAAGGACTCT ACGACCACTG GTGGCTTTGA TATGATTTCC GCCAGTACT TGTAAGAGGT 1140
  • MOLECULE TYPE DNA (genus ico)
  • HYPOTHETICS NO ' ⁇ v)
  • ANTI-SENSE NO

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Abstract

L'invention concerne un système de transformation utile pour exprimer des protéines hétérologues dans la levure Candida utilis, ce système de transformation se fondant sur l'obtention de mutants auxotrophiques de cette espèce ainsi que sur l'isolement de différents gènes, à partir d'une bibliothèque génomique, qui complètent lesdites auxotrophies. Le système de transformation consiste à utiliser en tant que hôtes de nouveaux mutants auxotrophiques obtenus à partir de la levure NRRL Y-1084 de Candida utilis, lesquels sont déficients essentiellement dans les voies biosynthétiques de uracyl et histidine, lesquels sont transformés avec des plasmides qui contiennent en tant que marqueurs de sélection les gènes $(URA3) et HIS3 de $(Candida utilis). Un autre aspect de l'invention concerne l'isolement du gène codant pour l'enzyme sucrose invertase ou β-fructofuranosidase de Candida utilis, ainsi que l'identification de séquences de promotion, de signalisation de sécrétion et de terminaison dudit gène INV1. Ces séquences peuvent être utilisées de manière avantageuse pour effectuer l'expression de protéines hétérologues dans cette levure.
EP97943729A 1996-10-03 1997-10-03 SYSTEME DE TRANSFORMATION DANS $i(CANDIDA UTILIS) Withdrawn EP0956356A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CU1996082A CU22722A1 (es) 1996-10-03 1996-10-03 Sistema de transformación para la expresión de genes heterólogos en la levadura candida utilis
CU8296 1996-10-03
PCT/CU1997/000005 WO1998014600A1 (fr) 1996-10-03 1997-10-03 SYSTEME DE TRANSFORMATION DANS $i(CANDIDA UTILIS)

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EP0956356A1 true EP0956356A1 (fr) 1999-11-17

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EP (1) EP0956356A1 (fr)
JP (1) JP2001501475A (fr)
CN (1) CN1237208A (fr)
AU (1) AU744698B2 (fr)
BR (1) BR9713313A (fr)
CA (1) CA2268004A1 (fr)
CU (1) CU22722A1 (fr)
RU (1) RU2235127C2 (fr)
WO (1) WO1998014600A1 (fr)

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PE20031013A1 (es) * 2002-03-26 2004-02-05 Ajinomoto Kk CANDIDA UTILIS QUE PRODUCE y-GLUTAMILCISTEINA
US7972809B2 (en) 2002-04-26 2011-07-05 National Institute Of Advanced Industrial Science & Technology Methylotrophic yeast producing mammalian type sugar chain
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JPWO2022201917A1 (fr) 2021-03-22 2022-09-29
CN117203327A (zh) 2021-03-29 2023-12-08 株式会社钟化 产环状脂肽微生物株及环状脂肽的制备方法
KR102556901B1 (ko) * 2021-05-25 2023-07-17 국립낙동강생물자원관 담수에서 분리한 항균력 및 식물생장촉진능을 가지는 에드니아 속 nnibrfg15114 균주 및 이의 용도
WO2023286629A1 (fr) 2021-07-16 2023-01-19 株式会社カネカ Procédé de production d'adn plasmidique à l'aide d'escherichia coli
CN115838645B (zh) * 2022-09-15 2024-03-08 天津大学 一种高产乳清酸的酵母菌株及其应用

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AU4548597A (en) 1998-04-24
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BR9713313A (pt) 2000-10-24
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