WO2016016805A1 - Construction génétique destinée à la transformation de souches de levure - Google Patents

Construction génétique destinée à la transformation de souches de levure Download PDF

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WO2016016805A1
WO2016016805A1 PCT/IB2015/055692 IB2015055692W WO2016016805A1 WO 2016016805 A1 WO2016016805 A1 WO 2016016805A1 IB 2015055692 W IB2015055692 W IB 2015055692W WO 2016016805 A1 WO2016016805 A1 WO 2016016805A1
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gene
seq
sequence
gene construct
transformation
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PCT/IB2015/055692
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English (en)
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Daniele Bianchi
Giuliana Franzosi
Silvia Galafassi
Concetta COMPAGNO
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Versalis S.P.A.
Eni S.P.A.
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Publication of WO2016016805A1 publication Critical patent/WO2016016805A1/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
    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates to a gene construct based on auxotrophy for uracil suitable for the transformation of yeasts of the species Rhodosporidium azoricum.
  • the invention also relates to a vector containing said gene construct, a yeast of the species Rhodosporidium azoricum transformed with said gene construct and a transformation method.
  • yeasts belonging to the group of ascomycetes have been widely studied and characterized.
  • auxotrophic mutants represent a simple system for having available selective markers in DNA transformation with genes of interest.
  • the enzyme orotidine-5' -monophosphate (OMP) decarboxylase normally encoded by the gene URA3 converts OMP to uridine-5'- monophosphate (UMP) in the last passage of the biosynthetic pathway of pyrimidine, essential for the synthesis of DNA and RNA.
  • OMP uridine-5'- monophosphate
  • UMP uridine-5'- monophosphate
  • URA3 uridine-5'- monophosphate
  • Basidiomycetae yeasts such as, for example, Cryptococcus neoformans, Cryptococcus gattii,
  • URA5 mutants are obtained. With respect to the species belonging to the Rhodosporidium genus, URA5 mutants have only been described for the species R. kratochvilovae (in Abbott EP Ianiri G, Castoria R, Idnurm A. 2013. Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Applied Microbiology and Biotechnology 97(1) :283-295) .
  • 5'-FOA is employed, which is a compound used for the selection of uracil auxotroph strains, as it generates mutations in the URA3 and URA5 genes in yeasts commonly studied such as S. cerevisiae and in other yeasts and fungi (see articles cited above) .
  • International patent application WO2009126890 describes recombinant oleaginous fungi for the biological production of carotenoids and/or retinoic acid
  • US patent application US20100305341 describes recombinant oleaginous fungi for the biological production of sterols.
  • Basidiomycetae yeasts those belonging to the Rhodosporidium species are of particular interest as they are known oleaginous capable of accumulating lipids even naturally, so that, when cultivated under particular favourable conditions, they can increase the conversion of carbon sources into fatty acids and triglycerides (as described in literature in M. Khot, S. Kamat, S. Zinjarde, A. Pant, B. Chopade and A. RaviKumar. Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential for biodiesel. Microbial Cell Factories 2012, 11:71. and G. Katre, C. Joshi, M. Khot, S. Zinjarde, A. Ravikumar.
  • yeasts of the Rhodosporidium genus can be improved by acting on specific biosynthetic pathways. This can be obtained by genetic engineering, wherein fragments of exogenous DNA encoding, for example, enzymatic or regulatory activities of interest, are inserted in the genome of the target microorganism by gene recombination.
  • DNA fragments of one species may not be functional in another nearby species, thus indicating the need for specific molecular tools. See for example Applied Microbiology and Biotechnology 97 ( 1 ) : 283-295 (mentioned above) , in which DNA fragments of Basidiomycetous Rhodotorula sloffiae have proved not to be functional in the phylogenetically close yeast Rhodotorula glutinis .
  • a molecular marker containing the sequence of the marker gene URA5 encoding the orotidine monop osp ate phosphorylase enzyme (OMPPase) and including the promoter and terminator sequences, and an uracil auxotroph strain wherein the URA5 gene has a mutation that makes the enzyme inactive, and which therefore makes the microorganism unable to autonomously reproduce itself without uracil.
  • URA5 encoding the orotidine monop osp ate phosphorylase enzyme (OMPPase) and including the promoter and terminator sequences
  • OMPPase monop osp ate phosphorylase enzyme
  • the Applicants have now found a gene construct based on auxotrophy for uracil suitable for the transformation of yeasts of the species Rhodosporidium azoricum auxotrophic for uracil.
  • yeasts belonging to the species Rhodosporidium azoricum are not only oleaginous but also have other advantages.
  • yeasts are capable of utilizing sugars with five carbon atoms and fermentative set-ups have been advantageously developed that allow high concentrations of biomass production.
  • a first aspect of the present invention therefore relates to a gene construct having the characteristics according to claim 1.
  • the invention relates to a method for transforming yeast cells of the species Rhodosporidium azoricum auxotrophic for uracil, wherein said method is effected according to claim 14.
  • the invention relates to a transformation vector as described in claim 15.
  • the invention relates to a yeast cell of Rhodosporidium azoricum transformed as described in claim 16.
  • the invention relates to a yeast cell of Rhodosporidium azoricum as described in claim 17.
  • the expression gene construct refers to a polynucleotide which contains information necessary for the transformation and expression of at least one desired characteristic in the target organism.
  • said polynucleotide is a DNA fragment.
  • This definition also comprises, moreover, the expressions "expression cassette” and "transformation cassette”.
  • An object of the present invention relates to a gene construct comprising a sequence encoding the URA5 gene for the transformation of yeast strains of the species Rhodosporidium azoricum auxotrophic for uracil.
  • the gene construct of the present invention comprises the sequence of the URA5 gene of 696 base pairs with identification number 1 (SEQ ID NO: 1), which was obtained from the genome of Rhodosporidium azoricum by direct sequencing, following amplification of the corresponding fragment by PCR and identified thanks to the construction of degenerated promoters (primers) on the sequences of the URA5 genes of similar yeasts present in public databases.
  • Said sequence of the promoter is preferably the sequence with identification number 2 (SEQ ID NO: 2), of 995 base pairs.
  • Said terminator sequence is preferably the sequence with identification number 3 (SEQ ID NO: 3) of 415 base pairs.
  • the promoter and terminator sequences were also amplified from the regions adjacent to the gene URA5 in the genome of Rhodosporidium azoricum.
  • the gene construct of the present invention comprises the sequence with identification number 4 (SEQ ID NO: 4), wherein the sequence of the URA 5 gene has 770 base pairs, as it also comprises the intron of SEQ ID NO: 5 (see also the explanation of figure 2) .
  • the sequence of the URA5 gene of the gene construct encodes a polypeptide with an amino acid sequence with identification number 6 (SEQ ID NO: 6) .
  • any promoter/terminator pair of constitutive (autologous) genes of Rhodosporidium can be used: according to a preferred aspect, the promoter of the (autologous) phosphoglycerate kinase (PGK) gene with the sequence having identification number 7 (SEQ ID NO: 7) , is used.
  • PGK phosphoglycerate kinase
  • the promoter of the (heterologous ) TEF gene of Ashbya gossypii (Ji, L., Z.- D. Jiang, Y. Liu, C. M. J. Koh and L.-H. Zhang. 2010
  • a simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genetics and Biology, 47:279-287.) with the sequence having identification number 8 (SEQ ID NO: 8) can be used.
  • the terminator of the (autologous) gene of phosphoglycerate kinase (PGK) with the sequence having identification number 9 (SEQ ID NO: 9) or the terminator of the (heterologous) TEF gene of Ashbya gossypii with identification number 10 (SEQ ID NO: 10), can be used as terminators.
  • promoter, URA 5 gene and terminator constructs are those having identification number SEQ ID NO: 11, wherein the promoter and the terminator respectively have SEQ ID NO: 7 and SEQ ID NO: 9 (autologous), or having identification number SEQ ID NO: 12, wherein the promoter and the terminator respectively have SEQ ID NO: 8 and SEQ ID NO: 10
  • promoter- URA5 gene sequence-terminator therefore represents a molecular tool which, if inserted in a transformation vector, allows the transfer and expression of exogenous DNA in a target organism auxotrophic for uracil, relying on a selective system capable of verifying its correct integration, i.e. a selection marker, in this case URA5, whose gene sequence has been obtained from the genome of Rhodosporidium azoricum by means of PCR amplification.
  • said organisms are yeasts of the genus Rhodosporidium, preferably Rhodosporidium azoricum.
  • this gene expression construct can be used for the transformation of a strain of Rhodosporidium azoricum, or more generally a strain of the genus Rhodosporidium, which is auxotrophic for uracil, i.e. having the URA5 gene inactivated.
  • the gene construct of the present invention is included in an expression vector, preferably a plasmid.
  • vectors can be used for effecting the transformation with the gene construct of the invention.
  • said construct can be inserted into different types of bacterial plasmids well-known in the art, such as, for example, the commercially available plasmids TOPO, pUC18, pJET1.2, pSP72 and those of the pGEM family, so as to be able to easily manipulate the construct and obtain a sufficient quantity for the transformation of yeast cells.
  • the cassette can then be excised from the plasmid or the plasmid can be simply linearized to facilitate its recombination in the genome of the target yeast.
  • a further object of the present invention relates to a method for the transformation of yeast cells of the species Rhodosporidium azoricum auxotrophic for uracil and selection of the transformants comprising: - transforming yeast cells with the gene construct as defined in the present invention;
  • the minimal medium mainly used for the culture of yeasts comprises glucose from 5 to 50 g/1, preferably 20 g/1, YNB (Yeast Nitrogen Base) W/O amino acid from 3.35 to 13.4 g/1, preferably 6.7 g/1.
  • the minimal medium preferably envisages the addition of agar 20 g/1 (from 15 to 30 g/1) .
  • a further object relates to a transformation vector comprising the gene construct as defined in the present invention .
  • Another object of the present invention relates to a yeast transformed with the gene construct as defined above, said yeast belonging to the species Rhodosporidium azoricum and being auxotrophic for uracil.
  • the strain belonging to the species Rhodosporidium azoricum uracil auxotroph was deposited under the Budapest Treaty at the Leibniz-Institut DSMZ
  • said gene construct also comprises at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts.
  • the at least one nucleotide sequence and/or gene of interest to be transferred into the genome of target yeasts through the construct of the invention encodes resistance to antibiotics.
  • figure 1 shows:
  • figure 1A gene construct of the invention comprising a gene of interest to be transferred into the genome of target yeasts;
  • figure IB gene construct of figure 1A inserted in a bacterial plasmid
  • figure 1C gene construct of the invention comprising two genes of interest to be transferred into the genome of target yeasts;
  • FIG. 2 shows the nucleotide sequence of the URA5 gene of Rhodosporidium azoricum.
  • the start (ATG) and stop (TAG) codons of the transcript are shown, whereas an intron (non-encoding area situated inside the gene, with sequence SEQ ID NO: 5) , is shown in light grey.
  • the URA5 gene inserted in the gene construct of the invention corresponds to the underlined nucleotide sequence (SEQ ID NO: 1, without intron) .
  • the region upstream of the underlined part is the promoter with SEQ ID NO: 2 and the region downstream of the underlined part is the terminator with SEQ ID NO: 3.
  • FIG. 4 shows examples of mutations in the URA5 gene that can lead to uracil auxotrophy Nucleotide sequences of the URA5 gene of Rhodosporidium azoricum are shown: the wild-type
  • mutant strain (SEQ ID NO: 13) is that from which the mutants are obtained (therefore its sequence encodes a functional enzyme) , whereas the sequences called "mutant U24" (SEQ ID NO: 14) and “mutant U27” (SEQ ID NO: 15) are those relating to the gene not functioning in different mutants indicated as an example (in U24 see residue nr. 420; in U27 see residue nr. 614) .
  • the mutant U27 is that used in the strain deposited with number DSM 28738 on May 6, 2014, at the Leibniz- Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), InhoffenstraBe 7 B 38124 Braunschweig (Germany) .
  • the sequence called “consensus” is automatically generated by the program used for the alignment (MultAlin software, “Multiple sequence alignment with hierarchical clustering” F. CORPET, 1988, Nucl . Acids Res., 16 (22), 10881-10890) : in fact it creates this sequence "consensus” which indicates the "common” base in all the aligned sequences, whereas it inserts a dot where there are modifications.
  • the first step of the strategy used for the sequencing of the gene URA5 of Rhodosporidium azoricum consisted in the search for regions having a high homology among the protein sequences of URA5 deriving from yeasts, close from a phylogenetic point of view ⁇ Malassezia globosa, Cryptococcus gattii, Melampsora larici-populina) , whose sequences were present in public databases.
  • the conserved regions among the different sequences compared were thus identified and degenerated primers were then constructed (following the method proposed by Rose, T.M., E.R. Schultz, J.G. Henikoff, S. Pietrokovski , CM. McCallum and S. Henikoff. 1998. Consensus-degenerate hybrid oligonucleotide primer for amplification of distantly- related sequences. Nucleic Acids Research, 26(7) :1628- 1635) .
  • mutants were obtained with the URA5 gene non-functional.
  • the yeast Rhodosporidium azoricum was grown in a rich medium
  • the uracil auxotroph mutants were transformed with the constructed cassette.
  • the protocol envisaged the collection of a culture of exponentially growing cells at about 1-4 x 10 7 cells ml -1 in YPD medium (100 ml), by centrifuging for 5 minutes at 4, 000 rpm. The pellet was washed with 10 ml of buffer LiAc/TE (0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8) and gently re-suspended in LiAc/TE at a final concentration of 1-4 x 10 9 cells ml -1 .
  • buffer LiAc/TE 0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8
  • test-tube 100 ⁇ of cell suspension were prepared in a sterile eppendorf test-tube for each transformation treatment, to which the DNA was added in a volume of 10 ⁇ .
  • the test-tube was gently shaken and incubated at room temperature for 5 minutes.
  • 280 ⁇ of PEG 4000 50% in LiAc/TE were added to each test-tube
  • test-tubes were inverted 5-6 times to guarantee a complete mixing and then incubated at 30 °C for 45 min.; 1/10 of the volume of dimethylsulfoxide
  • DMSO methyl methacrylate
  • the cells were subjected to thermal "shock" at 42°C for 5 minutes and then immediately re-immersed in ice, in order to stimulate the DNA insertion.
  • the cells were then washed with water 3-5 times to eliminate the residues, subjecting the cells to centrifuges of 15 seconds at 13,000 rpm, to isolate the pellet.
  • the cells were then plated on a minimal medium, with no uracil, so as to only allow the growth of the cells transformed and which had therefore acquired a functional copy of the URA5 gene .
  • Rhodosporidium azoricum which, after transformation, showed a growth capability on plates of minimal medium, were analyzed to verify the real insertion of the wild-type URA5 gene in the genome and that it was not a reversion of the phenotype, an event that can occur spontaneously, even if rarely.
  • a PCR reaction was then carried out, using a pair of primers capable of revealing the presence of URA5 fused to the plasmid TOPO TA used as cloning vector.
  • This PCR revealed the presence of the hexogen gene in all the clones obtained from the transformation of two different ura ⁇ strains, thus demonstrating the functioning of the gene construct object of the invention .
  • the expression of the gene for resistance to the antibiotic geneticin (commonly known as G418, from the name of the commercial product) was chosen, already successfully used in basidiomycete yeasts (Hua, J., J. D. Meyer and J. K. Lodge. 2000. Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans. Clinical and Diagnostic Laboratory Immunology, 7: 125-128) and to which Rhodosporidium azoricum is sensitive.
  • Yeast 10, 1793-1808 which contains the gene encoding an amyloglycoside phosphotransferase isolated from the bacterial transposon Tn903 r flanked by the promoter and terminator sequences of the gene TEE of Ashbya gossypii (SEQ ID NO: 8 and SEQ ID NO: 10, respectively), also already successfully tested in other basidiomycete yeasts (Ji, L., Z.-D. Jiang, Y. Liu, C. M. J. Koh and L.-H. Zhang. 2010 A simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells. Fungal Genetics and Biology, 47:279-287) .
  • the KANMX4 module was amplified by PCR using the primers KANf 5'-ATTGGATCCGATATCAAGCTTGCCTCG-3' and KANr 5'-ATAGGATCCCACTGGATGGCGGCGTTA-3', which, in addition to the sequence homologous to the module, contain restriction sites recognized by the enzyme BamHI .
  • the fragment obtained was purified from enzymes and salts present in the PCR reaction, then digested with the restriction enzyme BamHI, so as to generate, at the ends, single strand sequences.
  • the plasmid TOPO TA was also digested with the same enzyme, in which the gene URA5 of Rhodosporidium azoricum had been previously inserted (see Example 1) and the two fragments were then ligated to obtain a plasmid containing, in sequence, the gene URA5 and the module KANMX5, and the ligation mixture was transformed into E. coli, made competent for the transformation.
  • the transformants obtained were verified by means of a PCR carried out directly on the colonies, with the primers KANf and KANr .
  • a clone containing the module ⁇ 4 correctly inserted was then grown overnight in a liquid medium with ampicillin and the plasmid was extracted the next day by using a column having a high affinity for DNA (Pure Yield plasmid MiniPrep system, Promega) .
  • the plasmid was then cut with the restriction enzyme Spel (which linearizes the same plasmid) to allow its integration in the receiving genome.
  • the uracil auxotroph mutants were then transformed with the gene construct thus constructed.
  • the protocol envisaged the collection of a culture of exponentially growing cells at about 1-4 x 10 7 cells ml -1 in YPD medium (100 ml), by centrifuging for 5 minutes at 4,000 rpm. The pellet was washed with 10 ml of buffer LiAc/TE (0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8) and gently re-suspended in LiAc/TE to a final concentration of 1-4 x 10 9 cells ml -1 .
  • buffer LiAc/TE 0.1 M Lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 8
  • 100 ⁇ of cell suspension were prepared in a sterile eppendorf test-tube for each transformation treatment, to which the DNA was added in a volume of 10 ⁇ .
  • the test-tube was gently shaken and incubated at room temperature for 5 minutes.
  • 280 ⁇ of PEG 4000 50% in LiAc/TE were added to each test-tube (eppendorf) .
  • the test-tubes were inverted 5-6 times to guarantee a complete mixing and then incubated at 30°C for 45 min.; 1/10 of the volume of dimethylsulfoxide (DMSO) , i.e. about 43 ⁇ , were added to each aliquot.
  • DMSO dimethylsulfoxide
  • the cells were subjected to thermal "shock" at 42 °C for 5 minutes and then immediately re-immersed in ice, in order to stimulate the DNA insertion.
  • the cells were then washed with water 3-5 times to eliminate the residues, subjecting the cells to centrifuges of 15 seconds at 13, 000 rpm, to isolate the pellet.
  • the cells were then plated on a minimal medium, containing no uracil, so as to only allow the growth of the cells transformed and which had therefore acquired a functional copy of the URA5 gene.
  • Rhodosporidium azoricum which, after transformation, showed a growth capability on plates of minimal medium, were analyzed to verify the real insertion of the wild-type URA5 gene and KANMX4 in the genome by means of a PCR with the pairs of primers URA5_f and URA5_r and KANf and KA r .
  • the clones obtained were parallely plated on plates containing the antibiotic geneticin (glucose 20 g/1, yeast extract 10 g/1, peptone 20 g/1, agarose 20 g/1, G418 100 mg/1) to verify the effective expression of the gene of interest inserted in the gene construct, demonstrating the correct functioning of the gene construct object of the invention.
  • the antibiotic geneticin glucose 20 g/1, yeast extract 10 g/1, peptone 20 g/1, agarose 20 g/1, G418 100 mg/1

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Abstract

La présente invention concerne une construction génétique basée sur l'auxotrophie à l'uracile qui convient pour la transformation de levures de l'espèce Rhodosporidium azoricum. L'invention concerne également un vecteur contenant ladite construction génétique, une levure de l'espèce Rhodosporidium azoricum transformée avec ladite construction génétique et un procédé de transformation.
PCT/IB2015/055692 2014-08-01 2015-07-28 Construction génétique destinée à la transformation de souches de levure WO2016016805A1 (fr)

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Cited By (1)

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CN116287388A (zh) * 2023-02-17 2023-06-23 首都医科大学附属北京世纪坛医院 一种隐球菌的鉴定方法、引物对及试剂盒

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Cited By (1)

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CN116287388A (zh) * 2023-02-17 2023-06-23 首都医科大学附属北京世纪坛医院 一种隐球菌的鉴定方法、引物对及试剂盒

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