Abstract:
The present application relates to genetically modified yeasts for the production of glycoproteins having optimized and homogeneous glycan structures. These yeasts comprise an inactivation of the Och 1 gene, the integration by homologous recombination, into an auxotrophic marker, of an expression cassette comprising a first promoter, and an open reading frame comprising the coding sequence for an α-1,2-mannosidase I, and the integration of a cassette comprising a second promoter different from said first promoter and the coding sequence for an exogenous glycoprotein. These yeasts make it possible to produce EPO with an optimized and 98% homogeneous glycosylation.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a National Phase Entry of International Application No. PCT/EP2008/050888, filed on Jan. 25, 2008, which claims priority to French Patent Application No. 0753050, filed on Feb. 2, 2007, both of which are incorporated by reference herein. 
     BACKGROUND 
     The present invention relates to genetically modified yeasts for producing glycoproteins having optimized and homogeneous glycan structures. These yeasts comprise inactivation of the Och1 gene, integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a first promoter, and an open reading phase comprising the sequence coding for an α-1-2 mannosidase I, and integration of a cassette comprising a second promoter different from said first promoter and the sequence coding for an exogenous glycoprotein. These yeasts allow production of EPO with optimized and homogeneous 98% glycosylation. 
     The production of glycoproteins or glycopeptides having glycans of the complex type, i.e. structures identical with oligosaccharides added during post-translational modifications in humans, has been a sought goal for quite a few years in the pharmaceutical industry. Indeed, many studies have shown the importance of oligosaccharides for optimizing the activity of therapeutic glycoproteins or further for improving their half-life time once they are administered. For example, human erythropoietin (HuEPO) is a glycoprotein of 166 amino acids containing three N-glycosylation sites in positions Asn-24, Asn-38 and Asn-83 and an O-glycosylation site of the mucin type in position Ser-126. EPO is a particularly relevant model for studying N-glycosylation because of its glycosylated structures representing 40% of its molecular weight. The EPO molecule considered as natural is the urinary form (uHuEPO) (Takeuchi et al., 1988, Tusda 1988, Rahbeck-nielsen 1997). Recombinant EPO (rHuEPO) is presently produced in CHO cells (Sasaki H et al., Takeuchi et al., 1988) or in BHK cells (Nimtz et al., 1993). The rHuEPOs expressed in cell lines have N-glycan structures different from the structures found in uHuEPO. These differences may have repercussion in vitro (Higuchi et al., 1992; Takeuchi et al., 1990) but seem to be more sensitive in vivo by a drastic loss of activity for the deglycosylated forms and by an increase of activity correlated with the number of sialic acids present on the structure (Higuchi M et al., 1992). 
     In order to produce glycoproteins having optimum N- or O-glycosylation, many technical solutions have been proposed. Mention may be made of in vitro modifications of glycan structures by adding sugars such as galactose, glucose, fucose or even sialic acid by means of different glycosyl transferases or by suppressing certain sugars such as mannose with mannosidases. This technique is described in WO 03/031464 (Neose). It is however possible to wonder how such a technique may be applied on a large scale since this involves many steps for sequential modification of several given oligosaccharides present on a same glycoprotein. In each step, strict control of the reaction should be carried out and production of homogeneous glycanic structures should be ensured. Now, in the case when many oligosaccharides have to be modified on a glycoprotein, a sequential reaction may result in undesirable and heterogeneous modifications. This technique is therefore not compatible with the preparation of biodrugs. Further, the use of purified enzymes for production on an industrial scale does not seem to represent a viable economical alternative. 
     The same applies with chemical coupling techniques, such as those described in documents WO 2006/106348 and WO 2005/000862. These chemical coupling techniques involve tedious reactions, protection/deprotection steps, multiple checks. In the case when many oligosaccharides have to be modified on a given glycoprotein, a sequential reaction may also result in undesirable and heterogeneous modifications. Other technologies using mammal cell lines such as YB2/0 described in WO 01/77181 (LFB) or further CHO lines genetically modified in WO 03/055993 (Kyowa) have demonstrated that slight fucosylation of the Fc region of the antibodies improves ADCC activity by a factor 100. However, these technologies specifically relate to the production of antibodies. 
     Finally, production of glycoproteins in yeasts or filamentous fungi has been proposed by transformation of these micro-organisms with plasmids allowing expression of mannosidases and of different glycosyl transferases. This approach was described in WO 02/00879 (Glycofi). However, to this day, it has not been demonstrated that these micro-organisms are stable over time in a high capacity fermenter for producing clinical batches. Also, it has not been shown that this transformation enables production of glycoproteins with the desired and homogeneous glycans. 
     With the purpose of producing rHuEPO having N-glycan structures with which optimum activity may be obtained in vivo, we expressed an rHuEPO in genetically modified  S. cerevisiae  and  S. pombe  yeasts. These yeasts showed strong expression of rHuEPO having homogeneous and well-characterized N-glycosylation units. In a second phase, we started with genetically modified yeasts and we incorporated other modifications in order to produce more complex N-glycan units, depending on their sialylation levels. The yeast system is known for its capacity of rapidly producing a large amount of proteins but the modified yeasts described hereafter are also capable of N-glycosylation of the produced proteins in a “humanized” and homogeneous way. Further, these yeasts are found to be stable under conditions of production on a large scale. Finally, in the case of mutations leading to genotype reversion, these yeasts are constructed so as to allow them to be restored identically, which is required within the scope of producing clinical batches. 
     Thus, this is the first example which illustrates targeted integration methods in particular loci which have been used for the whole of the invention, methods allowing control of interrupted and selected genome regions to within one nucleotide, and therefore allowing restoration of the interruption in the case of spontaneous genome reversion. This method should be opposed to the one described in WO 02/00879, consisting of transforming a yeast strain with a bank of sequences and subsequently selecting the best clone without any genomic characterization. Indeed, in WO 02/00879 the integrations are random and the clones are exclusively selected on the basis of the profile of N-glycan structures of the produced proteins, which involves, in the case of mutations, reversion or any other genetic modification, pure and simple loss of the clone of interest. The advantage of the technology according to the invention is to provide increased safety to the user, by providing him/her with a guarantee of controlling, tracking genetic modifications, and especially the possibility of reconstructing a clone which will strictly have the same capacities. 
     Further, for the first time we provide a “Glycan-on-Demand” technology from the Amélie strain as described hereafter. Under these conditions, the homogeneity of the structures is more important than in the CHO systems which glycosylate like mammals. Indeed, the obtained results (see EPO spectrum), report a glycan structure of the Man5GlcNAc2 type representing about 98% of the N-glycans present on the protein. The system is therefore designed so as to force glycosylation in order to obtain a desired unit in very large proportion. The Amélie strain is the clone used as a basis for elaborating any other strain intended to produce humanized, hybrid or complex glycans, which one wishes to obtain. The advantage of this strain is to form a starting point, which was demonstrated as being a stable and homogeneous system producing 98% of Man5GlcNAc2 glycoproteins, which may be reworked for additional modifications such as the introduction of a GlcNAc transferase, of a fucosyl transferase, of a galactosyl and/or sialyl transferase, on demand, rapidly, according to the desired final structures. 
     SUMMARY 
     The construction of an expression cassette is carried out by integrating a promoter sequence in position 5′ and a terminal sequence in position 3′ of the ORF. On the other hand, the integration of these cassettes into the genome of the yeasts is controlled by adding to the ends, sequences homologous to the target locus with the purpose of integration by homologous recombination. For each strain and for each ORF, the promoter sequences as well as the integration sequences, have been determined with the purpose of obtaining stable and optimum expression of the different enzymes allowing homogeneous glycosylation of glycoproteins. The construction of an expression cassette is accomplished in several successive pCR steps, according to this general model shown in  FIG. 2  (assembly PCR for constructing expression cassettes of the ORFs). Certain ORF sequences have been partly modified by integrating sub-cellular localization signals in order to express (address) the protein in a compartment where its activity will be optimum (environment, presence of the donor, and of the substrates, etc. . . . ). 
     Thus, in a first aspect, the present invention relates to genetically modified yeasts, capable of producing glycoproteins having homogeneous glycans having the structure Man5GlcNAc2, said yeasts comprising the following modifications:
     a) inactivation of the Och1 gene coding for α-1,6-mannosyl transferase by insertion by homologous recombination of a heterologous sequence coding for a gene of resistance to an antibiotic (kanamycin) (delta-Och1 strain),   b) integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, an open reading phase comprising the sequence coding for an α-1-2-mannosidase I comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription,   c) integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, said promoter in c) being different from the promoter in b); an open reading phase comprising the sequence coding for an exogenous glycoprotein to be produced and a terminator of the transcription.   

     Preferably, the yeasts described above have integrated α-1-2 mannosidase I of  C. Elegans , notably a sequence comprising SEQ ID NO 1. These yeasts are found to be capable of producing glycoproteins having 98% of Man5GlcNac2 glycans: 
     
       
                 
         
             
             
         
      
     
     Advantageously, α-1-2 mannosidase I is expressed under the control of the promoter pGAP and the exogenous protein glycoprotein is expressed under the control of the promoter pGAL1. In the following description, reference will be made to the abbreviations used in the state of the art Man=mannose, GlcNac=N-acetyl-glucosamine, Gal=galactose, Fuc=fucose and NANA designating sialic acid or further N-acetyl-neuraminic acid. 
     In a second aspect, the yeasts of the invention include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of the GlcNacMan5GlcNAc2 structure: 
     
       
                 
         
             
             
         
      
     
     For this purpose, the above strains further comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, an open reading phase comprising the sequence coding for human N-acetyl-glucosaminyl transferase I, comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription. Preferably, human N-acetyl-glucosaminyl transferase I comprises the sequence SEQ ID NO 2 without the cytoplasmic portion of the enzyme which is replaced with the cytoplasmic portion of Mnn9 for Golgian localization of the protein. This strain is designated as “Arielle”. Arielle should also contain the GlcNAc UDP transporter cassette (described below) in order to synthesize this type of glycan. Advantageously, the promoter pGAP is used. 
     
       
         
               
             
               
             
               
             
           
               
                 Mnn9 
               
             
          
           
               
                 (SEQ ID NO 13) 
               
             
          
           
               
                 
                   
                     Atgtcactttctcttg 
                   
                   tatcgtaccgcctaa 
                   
                     gaaagaacccgtgqgttta 
                   
                 
               
               
                   
               
               
                     ac   : cytoplasmic portion. 
               
             
          
         
       
     
     The Amélie strain above may further comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, an open reading phase comprising the sequence coding for the cassette for the human UDP-GlcNAc transporter and a terminator of the transcription. Preferably, the human UDP-GlcNAc transporter comprises the sequence SEQ ID NO 3. Preferably the promoter is PGK. This strain is designated hereafter as “Agathe”. 
     In a third aspect, the yeasts of the invention include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of the GlcNacMan3GlcNAc2 structure: 
     
       
                 
         
             
             
         
      
     
     As such, the Arielle yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, an open reading phase comprising the sequence coding for a mannosidase II comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription. Preferably, mannosidase II is that of mice, notably a sequence comprising SEQ ID NO 4. Preferably the promoter is TEF. This strain is designated hereafter as “Anaïs”. 
     In a fourth aspect, the yeasts of the invention include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of the GlcNac2Man3GlcNAc2 structure: 
     
       
                 
         
             
             
         
      
     
     In this case, the Anaïs yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID No 16-26 respectively, an open reading phase comprising the sequence coding for an N-acetyl-glucosaminyl transferase II, comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription. Preferably, the N-acetyl-glucosaminyl transferase II is human, notably a sequence comprising SEQ ID NO 5. Preferably the promoter is PMA1, this strain is designated hereafter as “Alice”. 
     In another embodiment, the Alice yeasts of the invention include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of the Gal2GlcNac2Man3GlcNAc2 structure: 
     
       
                 
         
             
             
         
      
     
     In this case, the Alice yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, preferably the promoter CaMV, an open reading phase comprising the sequence coding for a galactosyl transferase I, comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription. Preferably, the galactosyl transferase I is human, notably a sequence comprising SEQ ID NO 6, which is without the human targeting sequence. This strain is designated as “Athena”. 
     Advantageously, the integration of the aforementioned expression cassettes is carried out in an integration marker selected from (auxotrophy marker selected from) URA3, ADE2, LYS2, LEU2, TRP1, CAN1, ADO1, HIS5, HIS3, ARG3, MET17, LEM3, Mnn1, Mnn9, gma12. Even more advantageously, the expression cassette of α-1-2 mannosidase I is integrated into the URA3 gene, the expression cassette of N-acetyl-glucosaminyl transferase I is integrated into the ADE1 or ADE2 gene, the expression cassette of the UDP-GlcNAc transporter is integrated into the LYS2 gene, the expression cassette of α-mannosidase II is integrated in the LEU2 gene, and the expression cassette of N-acetyl-glucosaminyl transferase II is integrated into the LEM3 or TRP1 gene. The expression cassette of β-1,4-galactosyl transferase I is integrated into TRP1 or MET17. Further, a targeting sequence in the endoplasmic reticulum or the Golgi apparatus, derived from the localization sequence of the Mnt1 gene which comprises the sequence SEQ ID NO 14 and the terminator CYC1 comprising the SEQ ID NO 15, is preferably used in the constructs. 
     In another embodiment, the yeasts Alice and Athena, described above, of the invention, include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of a structure selected from
         GlcNac2Man3GlcNAc2,   Gal2GlcNac2Man3GlcNAc2, and   GlcNac2Man3(Fuc)GlcNAc2, Ashley strain   Gal2GlcNac2Man3(Fuc)GlcNAc2, Aurel strain       

     In this case, the yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter selected from pGAP, pGAL1, PGK, TEF, adh1, nmt 1, SV40, PMA1, CaMV, pet56 of  S. cerevisiae  or  S. pombe , and ADH2 having the sequence SEQ ID Nos. 16-26 respectively, or the promoter of the nmt1 gene, an open reading phase comprising the sequence coding for an α-1,6-fucosyl transferase FUT8, comprising a targeting sequence in the endoplasmic reticulum or the Golgi apparatus and a terminator of the transcription, in particular the terminator derived from the CYC1 gene. These strains may advantageously contain the cassette corresponding to the GDP-fucose transporter described below. This cassette may be integrated into CAN1 or HIS5. 
     Preferably, the α-1,6-fucosyl transferase FUT8 is human, notably a sequence comprising SEQ ID NO 7. Further, this strain should comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising the promoter SV40, and open reading phase comprising the sequence coding for a GDP-fucose transporter, notably a sequence comprising SEQ ID NO 8. This cassette may be integrated in TRP1, ARG3 or gma12. 
     In another embodiment, the yeasts GlcNac2Man3GlcNAc2 (Athena) and Gal2GlcNac2Man3(Fuc)GlcNAc2 (Aurel) described above of the invention, include additional modifications in order to produce glycoproteins having more than 75%, or even 80% or further 95% or 98% of a structure selected from
         NANA2Gal2GlcNac2Man3GlcNAc2 Aeron strain   NANA2Gal2GlcNac2Man3(Fuc)GlcNAc2 Avrel strain       

     
       
                 
         
             
             
         
      
     
     In this embodiment, the yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter among those mentioned above or the promoter of the thymidine kinase of the herpes virus comprising the sequence SEQ ID NO 9. An open reading phase comprising the sequence coding for an α-2,3-sialyl transferase (ST3GAL4 gene) and a terminator of the transcription, in particular the terminator derived from the CYC1 gene comprising the sequence SEQ ID NO 15. Preferably the sialyl transferase is human (NM — 006278), notably a sequence comprising SEQ ID NO 10. 
     In another embodiment, the yeasts Gal2GlcNac2Man3GlcNAc2 (Athena) and NANA2Gal2GlcNac2MAN3GLCNAc2 (Aeron) described above of the invention include additional modifications in order to produce glycoproteins having more than 75%, or even 80%, or further 95% or 98% of a structure selected from
         Gal2GlcNac3Man3GlcNAc2 Azalée strain   NANA2Gal2GlcNac3Man3GlcNAc2 A strain       

     
       
                 
         
             
             
         
      
     
     In this embodiment, the yeasts mentioned above comprise integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a promoter from those mentioned above. An open reading phase comprising the sequence coding for a β-1,4-n-acetyl-glucosaminyl transferase III and a terminator of the transcription, in particular the terminator derived from the CYC1 gene comprising the sequence SEQ ID NO 15. Preferably the GNTIII is murine, notably a sequence comprising SEQ ID NO 27. 
     As indicated above, the yeasts according to the invention are integrated into a cassette for expressing an exogenous glycoprotein or glycopeptide. The glycoprotein may be selected from glycoproteins for therapeutic use such as cytokines, interleukins, growth hormones, growth factors, enzymes, and monoclonal antibodies, vaccinal proteins, soluble receptors and all types of recombinant proteins. This may be a sequence coding for EPO, notably a cassette comprising SEQ ID NO 11 coding for an EPO with SEQ ID NO 12 comprising the epitope V5 and an N-terminal poly-HIS unit for purification. 
     The invention also relates to a pharmaceutical composition comprising a glycoprotein having homogeneous glycan structures of more than 75%, 90%, 95% or further 98% of the structure:
     Man5GlcNAc2,   GlcNacMan5GlcNAc2,   GlcNacMan3GlcNAc2,   GlcNac2Man3GlcNAc2,   Gal2GlcNac2Man3GlcNAc2,   NANA2Gal2GlcNac2Man3GlcNAc2,   GlcNac2Man3(Fuc)GlcNAc2,   Gal2GlcNac2Man3(Fuc)GlcNAc2,   NANA2Gal2GlcNac2Man3GlcNAc2,   Gal2GlcNac2Man3GlcNAc2,   NANA2Gal2GlcNac3Man3GlcNAc2,   

     The invention also relates to a pharmaceutical composition comprise EPO as an active ingredient, said EPO having more than 75%, 90%, 95% or further 98% of the structure
     NANA2Gal2GlcNac2Man3GlcNAc2 or   NANA2Gal2GlcNac2Man3(Fuc)GlcNAc2
 
The invention also relates to a culture in a fermenter comprising a basic culture medium of culture media for yeasts and to a yeast described above.
   

     In still another aspect, the invention relates to a method for producing a glycoprotein having homogeneous glycan structures with more than 75%, 90%, 95% or further 98% of the structure
     Man5GlcNAc2,   GlcNacMan5GlcNAc2,   GlcNacMan3GlcNAc2,   GlcNac2Man3GlcNAc2,   Gal2GlcNac2Man3GlcNAc2,   NANA2Gal2GlcNac2Man3GlcNAc2,   GlcNac2Man3(Fuc)GlcNAc2,   Gal2GlcNac2Man3(Fuc)GlcNAc2,   NANA2Gal2GlcNac2Man3(Fuc)GlcNAc2,   Gal2GlcNac3Man3GlcNAc2,   NANA2Gal2GlcNac3Man3GlcNAc2
 
comprising the cultivation of a yeast as described above in a fermenter, and the extraction of said glycoprotein from the culture medium. This method may comprise a purification step.
   

     Finally, the invention also relates to the use of a yeast as described above for producing in a fermenter a glycoprotein having homogeneous glycan structures with more than 75%, 90%, 95% or further 98% of the structure
     Man5GlcNAc2,   GlcNacMan5GlcNAc2,   GlcNacMan3GlcNAc2,   GlcNac2Man3GlcNAc2,   Gal2GlcNac2Man3GlcNAc2,   NANA2Gal2GlcNac2Man3GlcNAc2,   GlcNac2Man3(Fuc)GlcNAc2,   Gal2GlcNac2Man3(Fuc)GlcNAc2,   NANA2Gal2GlcNac2Man3(Fuc)GlcNAc2,   Gal2GlcNac3Man3GlcNAc2,   NANA2Gal2GlcNac3Man3GlcNAc2   

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : PCR-based construction of an OCH1-inactivating cassette. 
         FIG. 2 : Construction of expression cassettes. 
         FIG. 3 : PCR analysis of och1:Kan R  transformants. 
         FIG. 4 : Och1-activity assay in wild-type and PCR-selected transformants; a)  S. cerevisiae , b)  S. pombe.    
         FIG. 5 : MALDI-TOFF mass spectrometry N-glycan analysis of total proteins in Adele and Edgar strains. 
         FIG. 6 : Mannosidase I-activity assay in wild-type and PCR-selected transformants in presence and absence of DMJ, an inhibitor of alpha-1,2-mannosidase I activity; SC= S. cerevisiae , SP= S. pombe.    
         FIG. 7 : GlcNAc Transferase I-activity assay in wild-type and PCR-selected transformants; SC= S. cerevisiae , SP= S. pombe.    
         FIG. 8 : RT-PCR analysis of  S. cerevisiae  (SC) and  S. pombe  (SP) clones transformed with the UDP-GlcNAc transporter expression cassette. 
         FIG. 9 : Mannosidase II-activity assay in wild-type and PCR-selected transformants in presence and absence of swainsonine, an inhibitor of mannosidase II activity; SC= S. cerevisiae , SP= S. pombe.    
         FIG. 10 : RT-PCR analysis of  S. cerevisiae  (SC) and  S. pombe  (SP) clones transformed with the GDP-fucose transporter expression cassette. 
         FIG. 11 : MALDI-TOFF mass spectrometry N-glycan analysis of rhuEPO expressed in the Amèlie strain. 
         FIG. 12 : RT-PCR analysis of rhuEPO expression in pGAL-rhuEPO transformants grown with (lanes 1 and 3) galactose or with glucose (lanes 2 and 4). 
         FIG. 13 : Purification of rhuEPO by ion exchange chromatography (Sephadex C-50). 
         FIG. 14 : SDS-PAGE analysis of the ion exchange chromatography fractions. 
         FIG. 15 : Western blot analysis of the ion exchange chromatography fractions with an anti-EPO antibody. 
     
    
    
     DETAILED DESCRIPTION 
     Example 1 
     Creation of Mutated Strains on the Och1 Gene Coding for α-1,6-Mannosyl Transferase (Delta-Och1 Strain) 
     The gene for resistance to kanamycin was amplified by PCR and homologous flanking regions to the gene Och1 were added in both of these ends ( FIG. 1 ), specific regions of each strain of  S. cerevisiae  or  S. pombe  yeast. The gene Och1 is made non-functional by inserting this gene for resistance to an antibiotic, kanamycin. Integration of the gene into the genome of the yeast is accomplished by electroporation and the gene of interest is then integrated by homologous recombination. The flanking regions have about forty bases and allow integration of the kanamycin gene within the gene Och1 in the genome of the yeast. 
     The strains having integrated the gene for resistance to kanamycin are selected on the medium containing 50 μg/mL of kanamycin. We then checked by PCR the integration of the gene for resistance to kanamycin in the gene Och1. Genomic DNA of the clones having resisted to the presence of kanamycin in the medium, was extracted. Oligonucleotides were selected so as to check the presence of the gene for resistance to kanamycin on the one hand and that this gene was actually integrated into the Och1 gene on the other hand. Genomic DNA of wild strains was also tested; we amplified the Och1 gene of these strains. This gene has 1,100 bp. In the strains having integrated the kanamycin cassette, the observed amplification of the gene Och1 is longer (1,500 bp). 
     Example 2 
     Tests of Activities 
     2.1 Och1 Mannosyl Transferase Activity on Strains of Mutated Yeasts 
     Another validation level: for each gene integrated to the genome of the yeast strains, systematic check of the enzymatic activity was carried out, in order to constantly follow possible fluctuations in the activity levels, due for most of the time to spontaneous mutations and then requiring selection of new clones. 
     The activity of the Och1 enzyme may be detected by an assay in vitro. Prior studies have shown that the best acceptor for transfer of mannose by the Och1 enzyme is Man 8 GlcNAc 2 . From microsomal fractions of yeasts (100 μg of proteins) or from a lysate of total proteins (200 μg), the transfer activity of mannose in the alpha-1,6 position on a Man 8 GlcNAc 2  structure is measured. For this, the Man 8 GlcNAc 2  coupled to an amino-pyridine group (M 8 GN 2 -AP) is used as an acceptor and the GDP-mannose marked with [ 14 C]-mannose as a donor molecule of radioactive mannose. The microsomes or the proteins are incubated with the donor (radioactive GDP-mannose), the acceptor (Man 8 GlcN 2 -AP) and deoxymannojirimycin (inhibitor of mannosidase I) in a buffered medium with controlled pH. After 30 minutes of incubation at 30° C., chloroform and methanol are added to the reaction medium in order to obtain a proportion of CHCl 3 /MeOH/H 2 O of 3:2:1 (v/v/v). The upper phase corresponding to the aqueous phase, contains Man 8 GlcNAc 2 -AP, radioactive Man 9 GlcNAc 2 -AP and GDP-[ 14 C]-mannose. Once dried, the samples are taken up in 100 μL of H 2 O/1% acetic acid and passed over a Sep-Pak C18 (Waters) column, conditioned beforehand in order to separate GDP-mannose from the formed radioactive Man 9 GlcNAc 2 -AP (the AP group allows this compound to be retained on the C18 columns). By eluting with H 2 O/1% acetic acid (20 mL) and then with 20% methanol/1% acetic acid (4 mL), the different fractions may be recovered and counted with the scintillation counter. 
     2.2 Mannosidase Activity 
     Mannosidase activity is measured by incubating for 4 hours at 37° C., with 4 mM of p-nitrophenyl-α-D-mannopyranoside with 100-200 μg of proteins (from total proteins or sub-cellular fractions) in 0.1 M of PBS, pH 6.5+/−120 μM DMJ (alpha-1,2-mannosidase I inhibitor) +/−12 μM SW (specific inhibitor of mannosidase II). Absorbance is measured at 405 nm. 
     2.3 N-Acetylglucosaminyl Transferase Activity 
     GlcNAc transferase activity is measured on microsomal fractions of yeasts. 50 μg of microsomes (BCA assay) are incubated in finally 50 μL after 25 minutes at 30° C. with 0.01 μCi of donor (radioactive UDP-GlcNAc), 0.5 mM of acceptor (3-O-α-D-manno-pyranosyl-D-mannopyranoside) in a medium with 50 mM HEPES, 10 mM MnCl 2 , 0.1% TritonX-100. The reaction is stopped with 400 μL of 10 mM EDTA and the samples are then passed over Dowex AG-1X2 columns. The radioactive acceptor is then eluted from the columns with 3M formic acid and the radioactivity is measured with a scintillation counter. 
     Example 3 
     Expression Cassette for α-1-2 Mannosidase I of  C. Elegans    
     Explanatory diagram for constructing expression cassettes:  FIG. 2 . 
     3.1 Step 1: Obtaining the ORF 
     α-mannosidase I: PCR from bacterial clones having the plasmid pDONR201 
     (Open Biosystem) 
     Program: 
                                                                     8 minutes at 94° C.                35 cycles:   20 s at 94° C.               30 s at 65° C.               2 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 1,644 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 1) 
               
             
          
           
               
                     AAAGCAGGCatgggcctccga   tcacacgaacaacttgtcgtgtgtgtcgg 
               
               
                 agttatgtttcttctgactgtctgcatcacagcgttt 
               
               
                   
               
               
                 ttctttcttccgtcaggcggcgctgatctgtatttccgagaagaaaactc 
               
               
                 cgttcacgttagagatgtgcttatcagagaggaaatt 
               
               
                   
               
               
                 cgtcgtaaagagcaagatgagttacggcggaaagccgaagaagccaatcc 
               
               
                 cattccaattccaaaacctgaaattggagcat 
               
               
                   
               
               
                 cagatgatgcagaaggacgaagaattttcgtgaaacaaatgattaaattc 
               
               
                 gcatgggacggatatcggaaatatgcctggggg 
               
               
                   
               
               
                 gagaatgaattgaggcccaacagtagatcaggacattcttcatcgatatt 
               
               
                 tgggtatggaaagacgggtgcaacaattattgatg 
               
               
                   
               
               
                 ctattgatacattgtatttggttggattaaaagaagaatataaagaggcc 
               
               
                 agagactggattgctgattttgatttcaaaacgtctgc 
               
               
                   
               
               
                 gaaaggagatctatcagtttttgaaacaaatatccgattcactggtggcc 
               
               
                 tactctccgcatttgcacttaccggagacaaaatgtt 
               
               
                   
               
               
                 cttgaagaaagcagaagatgtggcaactattcttcttccggcttttgaaa 
               
               
                 ctccttctggaataccaaattcattaattgatgctcaa 
               
               
                   
               
               
                 acaggaagatccaaaacgtatagttgggcaagcggaaaggcaattctctc 
               
               
                 ggaatacggttcaattcaacttgaattcgattatc 
               
               
                   
               
               
                 tctccaatctgactggaaatccagtttttgctcaaaaagctgataaaata 
               
               
                 agagatgttttaactgcaatggagaaaccagaagg 
               
               
                   
               
               
                 actttatccaatttatattactatggataatccaccaagatggggacaac 
               
               
                 atcttttctcaatgggtgcaatggctgacagttggtat 
               
               
                   
               
               
                 gaatatctgctcaaacaatggattgccactggtaaaaaagatgatcgcac 
               
               
                 gaaaagagaatacgaagaagcgatatttgcaat 
               
               
                   
               
               
                 ggaaaaacgaatgcttttcaaatcggaacagtcgaatctttggtatttcg 
               
               
                 caaaaatgaacggaaatcgcatggaacattcatttg 
               
               
                   
               
               
                 aacatcttgcatgcttttccggtggaatggttgttcttcatgcaatgaat 
               
               
                 gagaaaaataaaacaatatcagatcattatatgacgtt 
               
               
                   
               
               
                 gggaaaagaaattggtcatacatgtcatgaatcgtacgctagatccacaa 
               
               
                 ctggaatcggcccagaatccttccaattcacatc 
               
               
                   
               
               
                 gagtgtagaggcaaaaacagaacgtcgtcaggattcatattatattcttc 
               
               
                 gtcctgaagtcgttgagacatggttctacttgtgga 
               
               
                   
               
               
                 gggctacaaaagacgagaaatatcgacaatgggcttgggatcatgttcaa 
               
               
                 aatttggaggagtattgtaagggcactgccgga 
               
               
                   
               
               
                 tactctggaatccgaaacgtctacgaatcgagcccggaacaagatgatgt 
               
               
                 gcagcagtcattcctcttcgctgagctcttcaaat 
               
               
                   
               
               
                 atctgtatttaattttcagtgaagataacattcttccacttgatcaatgg 
               
               
                 gttttcaataccgaagctcatccattcc   gcattcggcatcacgacgagtt     
               
               
                 
                   
                     gatt 
                   
                 
               
             
          
         
       
     
     The PCR amplification was extracted and purified from agarose gel with SBIOgene kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and insertion of the PCR amplification into the vector by sequencing (plasmid pGLY02.001). 
     3.2 Step 2: Assembling the Expression Cassette 
     Integration of the expression cassette of mannosidase I will be localized in the auxotrophy marker URA3 for both strains. Invalidation of this gene induces resistance to a toxic agent, 5-fluorouracil. Yeasts modified by this cassette will then become resistant to this drug but also auxotrophic for uracil. 
     Expression Cassette for  S. Cerevisiae  
     Amplification of the promoter pGAP from genomic DNA of wild  S. cerevisiae  BS16 (forward) and BS17′ (reverse)   Assembling the promoter pGAP (PCR product) and the ORF (pGLY02.001) BS16 (forward) and BS19′ (reverse)   Amplification of the terminator CYC1 from the plasmid pYES 2.1 BS40b (forward) and BS41 (reverse)   Assembling the ORF (plasmid pGLY02.001) and the terminator CYC1 (PCR product)
 
BS18 (forward) and BS41 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY02.002).
     Assembling promoter-ORF (PCR product) and ORF-terminator (pGLY02.002) with regions homologous to URA3 (from the primers)
 
BS42 (forward)
 
BS43 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY02.004). 
     Expression Cassette for  S. Pombe  
     Amplification of the promoter adh1 from genomic DNA of wild  S. pombe  
 
BS25 and BS26′ (reverse)
   Assembling the promoter adh1 (PCR product) and the ORF (pGLY02.001)
 
BS25 (forward) and BS20 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY02.009).
     Assembling the product promoter-ORF (pGLY02.009) with ORF-CYC1 (PCR product) BS25 (forward) and BS41 (reverse)   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY02.011).
     Amplification of the cassette (pGLY02.011) with flanking regions homologous to URA3 BS76 (forward) and BS77 (reverse)   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing. 
     3.3 Step 3: Transformation of the Yeasts
     Preparation of competent yeasts:   

       S. Cerevisiae  Adèle
     Procedure: Sow 500 ml of yeasts at OD=0.1 and incubate them at 30° C. until 5.5&lt;OD&lt;6.5.
 
Centrifuge the cells at 1500 g for 5 min at 4° C. and re-suspend them in 500 mL of cold sterile water.
 
Centrifuge the cells and re-suspend them in 250 mL of cold sterile water.
 
Centrifuge the cells and re-suspend them in 20 mL of 1M sorbitol. Centrifuge the cells and re-suspend them in 1 mL of 1M sorbitol. Form 80 μL aliquots and store them at −80° C.
   

       S. Pombe  Edgar
     Procedure: Sow 200 mL of yeasts at OD=0.1 and incubate them at 30° C. until OD=1.5.
 
Centrifuge at 3,000 rpm for 5 min at 20° C. Wash the cells in cold sterile water and centrifugate, wash a second time with 1 m sorbitol. Incubate for 15 min by adding DTT in order to reach a final 25 mM (in order to increase electrocompetence). Take up again as a final suspension in cold 1M sorbitol (density 1-5.10 9  cells/mL: about 5 mL). Form 40 μL aliquots and store about 10 vials at −80° C.
   Transformation of the yeasts by electroporation:   
     For each expression cassette, the DNA used for transforming the yeasts either stems from a digestion of the mentioned plasmid with selected restriction enzymes, or directly from the obtained PCR product, purified after complete assembling. 
       S. cerevisiae  cassette: pGLY02.004 is digested by the restriction enzymes BamHI and SmaI. 
     The competent yeasts are transformed with 1 μg of DNA: incubate for 5 min in ice. Give a pulse with V=1,500 V. Immediately add 1 mL of ice-cold sterile 1M sorbitol and transfer the cells with a Pasteur pipette into an Eppendorf tube and then let them relax for at least 1 hour in the Infors device at 30° C. Spread the yeasts on a dish of selection media (YPD containing 10 mM 5-fluorouracil, 5-FU). The transformants appear within 4-6 days. 
       S. pombe  cassette: the competent yeasts are transformed with 100 ng of DNA (PCR product): incubate for 5 min in ice. The cells and the DNA are transferred into an electroporation tank. Give a pulse at V=1,500V and immediately add 0.9 mL of cold 1M sorbitol. The cells are spread as rapidly as possible on the suitable medium (YPD containing 10 mM 5-FU). The transformants appear within 4-6 days. 
     Example 4 
     Amélie and Emma strains+expression cassette for human N-acetyl-glucosaminyl transferase I 
     4.1 Step 1: Obtaining ORF 
     PCR from a commercial plasmid Biovalley (Human ORF clone V1.1) 
     Program: 
                                                                     5 minutes at 94° C.                30 cycles:   60 s at 94° C.               60 s at 56° C.               2 min at 72° C.                5 minutes at 72° C.                        
Amplification of a 1,327 by fragment without the cytoplasmic portion of the enzyme. It will be replaced with the cytoplasmic portion of Mnn9 for Golgian localization of the protein.
 
     Mnn9 cytoplasmic region: PCR from genomic DNA of wild  S. cerevisiae   
                                                                     8 minutes at 95° C.                30 cycles:   20 s at 94° C.               30 s at 58° C.               1 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 51 by fragment (cytoplasmic portion of mnn9).
 
                           (SEQ ID No 13)                Atgtcactttctcttgtatcg   taccgcctaa   gaaagaacccgtggttaac                
From These Two PCR Amplifications:
 
Obtaining a Single Fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 2) 
               
             
          
           
               
                     Atgtcactttctcttgtatcg   taccgcctaagaaagaacccgtgggttaa 
               
               
                 cgcagggcttgtgctgtggggcgctatcctcttt 
               
               
                   
               
               
                 gtggcctggaatgccctgctgctcctcttcttctggacgcgcccagcacc 
               
               
                 tggcaggccaccctcagtcagcgctctcgatgg 
               
               
                   
               
               
                 cgaccccgccagcctcacccgggaagtgattcgcctggcccaagacgccg 
               
               
                 aggtggagctggagcggcagcgtgggctg 
               
               
                   
               
               
                 ctgcagcagatcggggatgccctgtcgagccagcgggggagggtgcccac 
               
               
                 cgcggcccctcccgcccagccgcgtgtgc 
               
               
                   
               
               
                 ctgtgacccccgcgccggcggtgattcccatcctggtcatcgcctgtgac 
               
               
                 cgcagcactgttcggcgctgcctggacaagctg 
               
               
                   
               
               
                 ctgcattatcggccctcggctgagctcttccccatcatcgttagccagga 
               
               
                 ctgcgggcacgaggagacggcccaggccatcg 
               
               
                   
               
               
                 cctcctacggcagcgcggtcacgcacatccggcagcccgacctgagcagc 
               
               
                 attgcggtgccgccggaccaccgcaagttc 
               
               
                   
               
               
                 cagggctactacaagatcgcgcgccactaccgctgggcgctgggccaggt 
               
               
                 cttccggcagtttcgcttccccgcggccgtgg 
               
               
                   
               
               
                 tggtggaggatgacctggaggtggccccggacttcttcgagtactttcgg 
               
               
                 gccacctatccgctgctgaaggccgacccctcc 
               
               
                   
               
               
                 ctgtggtgcgtctcggcctggaatgacaacggcaaggagcagatggtgga 
               
               
                 cgccagcaggcctgagctgctctaccgcacc 
               
               
                   
               
               
                 gactttttccctggcctgggctggctgctgttggccgagctctgggctga 
               
               
                 gctggagcccaagtggccaaaggccttctggga 
               
               
                   
               
               
                 cgactggatgcggcggccggagcagcggcaggggcgggcctgcatacgcc 
               
               
                 ctgagatctcaagaacgatgacctttggcc 
               
               
                   
               
               
                 gcaagggtgtgagccacgggcagttctttgaccagcacctcaagtttatc 
               
               
                 aagctgaaccagcagtttgtgcacttcacccagc 
               
               
                   
               
               
                 tggacctgtcttacctgcagcgggaggcctatgaccgagatttcctcgcc 
               
               
                 cgcgtctacggtgctccccagctgcaggtggag 
               
               
                   
               
               
                 aaagtgaggaccaatgaccggaaggagctgggggaggtgcgggtgcagta 
               
               
                 tacgggcagggacagcttcaaggctttcgc 
               
               
                   
               
               
                 caaggctctgggtgtcatggatgaccttaagtcgggggttccgagagctg 
               
               
                 gctaccggggtattgtcaccttccagttccggg 
               
               
                   
               
               
                 gccgccgtgtccacctggcgcccccactgacgtgggagggctatgatcct 
               
               
                 agctggaa   ttagcacctgcctgtccttc     
               
             
          
         
       
     
     The amplification product of the assembling PCR was purified from agarose gel with the QIAGEN kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.001). 
     4.2 Step2: Assembling the Expression Cassette 
     Expression Cassette for  S. Cerevisiae    
     The integration of the expression cassette of the GlcNAc transferase I for the yeast  S. cerevisiae  will be localized in the auxotrophy marker ADE2. Invalidation of this gene induces a change in the color of the yeasts which become red and also auxotrophy for adenine.
     of the promoter adh 1 from genomic DNA of  S. cerevisiae  
 
BS29 (forward) and BS30 (reverse)
   Assembling the promoter adh 1 (PCR product) and the ORF (pGLY03.001):
 
BS29 (forward) and BS59
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification in the vector by sequencing (pGLY03.002).
     Assembling of the ORF (pGLY03.001) with the terminator CYC1 (PCR product):
 
CA005 (forward)
 
BS41 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.011).
     Assembling the promoter-ORF (pGLY03.002) and the ORF-terminator (PCR product) with the extensions homologous to ADE2:
 
BS67 (forward) and BS68 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.010). 
     Expression Cassette for  S. Pombe    
     The integration of the expression cassette of the GlcNAc transferase I for the yeast  S. pombe  will be localized in the auxotrophy marker ADE1. Invalidation of this gene induces a change in the color of the yeasts which become red and also auxotrophy for adenine.
     Amplification of the promoter hCMV from the plasmid pCDNA 3.1
 
BS62 (forward) and BS58 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.004).
     Assembling hCMV-ORF (pGLY03.004) with the terminator CYC1 (PCR product) BS62 (forward) and BS41 (reverse)   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.005).
     Assembling the expression cassette (pGLY03.005) with the extensions homologous to ADD:
 
BS69 (forward) and BS70 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY03.007). 
     4.3 Step 3: Transformation of the Yeasts 
     Preparation of Competent Yeasts: 
     The Amélie and Emma strains were prepared as indicated above in order to make them competent. 
     Electroporation of the Yeasts  S. Cerevisiae  and  S. Pombe    
     
         
         Procedure:
 
20 μg of plasmids containing the expression cassette for  S. cerevisiae  and  S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced in the yeasts Amélie and Emma by electroporation. The yeasts are selected on an YNB medium containing the required amino acids.
 
       
    
     Example 5 
     Agathe and Egée strains+expression cassette for the UDP-GlcNAc transporter 
     5.1 Step 1: Obtaining the ORF 
     Program: 
                                                                     3 minutes at 94° C.                30 cycles:   20 s at 94° C.               30 s at 58° C.               2 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 916 by Fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 3) 
               
             
          
           
               
                     atgttcgccaacctaaaatacg   tttccctgggaattttggtctttcagac 
               
               
                   
               
               
                 taccagtttggttctaacaatgcgttattccagaact 
               
               
                   
               
               
                 ttaaaagaagaaggacctcgttatctatcttctacagcagtggttgttgc 
               
               
                   
               
               
                 tgaacttttgaagataatggcctgcattttattggtcta 
               
               
                   
               
               
                 caaagacagcaaatgtagtctaagagcactgaatcgagtactacatgatg 
               
               
                   
               
               
                 aaattcttaataaacctatggaaacacttaaactt 
               
               
                   
               
               
                 gctattccatcagggatctatactcttcagaataatttactgtatgtggc 
               
               
                   
               
               
                 actatcaaatctagatgcagctacttatcaggtcacgt 
               
               
                   
               
               
                 atcagttgaaaattcttacaacagcattattttctgtgtctatgcttagt 
               
               
                   
               
               
                 aaaaaattgggtgtataccagtggctgtccctagtaattt 
               
               
                   
               
               
                 tgatgacaggagttgcttttgtacagtggccctcagattctcagcttgat 
               
               
                   
               
               
                 tctaaggaactttcagctggttctcaatttgtaggact 
               
               
                   
               
               
                 catggcagttctcacagcatgtttttcaagtggctttgctggggtttact 
               
               
                   
               
               
                 ttgagaaaatcttaaaagaaacaaaacaatcagtgtg 
               
               
                   
               
               
                 gataagaaatattcagcttggtttctttggaagtatatttggattaatgg 
               
               
                   
               
               
                 gtgtatacatttatgatggagaactggtatcaaagaatg 
               
               
                   
               
               
                 gattttttcagggatataaccgactgacctggatagtagttgttcttcag 
               
               
                   
               
               
                 gcacttggaggccttgtaatagctgctgttattaagtat 
               
               
                   
               
               
                 gcagataatattttaaaaggatttgcaacctctttatcgataatattatc 
               
               
                   
               
               
                 aacattgatctcctatttttggcttcaagattttgtgccaa 
               
               
                   
               
               
                 ccagtgtcttttt   ccttggagccatccttgtaa     
               
             
          
         
       
     
     The PCR amplification was extracted and purified from agarose gel with the QIAGEN kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY04.001). 
     5.2 Step 2: Assembling the Expression Cassette 
     Cassette for  S. Cerevisiae  and for  S. Pombe    
     The integration of the expression cassette of the UDP-GlcNAc transporter will be localized in the auxotrophy marker LYS2 for both strains  S. cerevisiae  and  S. pombe . Invalidation of this gene induces resistance to a toxic agent, alpha-aminoadipic acid. The yeasts modified by this cassette will therefore become resistant to this drug but also auxotrophic for lysine.
     Amplification of the promoter PGK from the plasmid pFL61
 
BS95 (forward) and BS96 (reverse)
   Assembling the ORF (pGLY04.001) with the terminator CYC1 (PCR product)
 
CA017 (forward) and BS41 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY04.002).
     Assembling the promoter PGK (PCR product) with the ORF-terminator CYC1 fragment (pGLY04.002):
 
BS95 (forward) and BS41 (reverse)
   Assembling the expression cassette with the extensions homologous to LYS2
 
 S. cerevisiae:  
 
BS97 (forward) and BS98 (reverse)
 
 S. Pombe  
 
BS99 (forward)
 
BS100 (reverse)
   

     The PCR amplifications were extracted and purified from agarose gel with the Qiagen kit and were introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY04.006) for the  S. cerevisiae  cassette and pGLy04.005 for the  S. pombe  cassette). 
     5.3 Step 3: Modification of the Yeasts 
     Preparation of Competent Yeasts: 
     The Agathe and Egée strains were prepared as indicated above in order to make them competent 
     Electroporation of the Yeasts: 
     20 μg of the plasmids containing the expression cassettes for  S. cerevisiae, S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced into the yeasts Agathe and Egée by electroporation. The yeasts are selected on an YNB medium containing the required amino acids and alpha-aminoadipic acid. 
     Example 6 
     Arielle and Erika Strains+Expression Cassette for α-mannosidase II 
     6.1 Step 1: Obtaining the oRF 
     PCR from cDNA of mouse liver 
     Program: 
                                                                     3 minutes at 94° C.                35 cycles:   20 s at 94° C.               30 s at 58° C.               4 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 3,453 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 4) 
               
             
          
           
               
                     atgaagttaagtcgccagttcacc   gtgtttggcagcgcgatcttctgcgt 
               
               
                   
               
               
                 cgtaatcttctcactctacctgatgctggacagg 
               
               
                   
               
               
                 ggtcacttggactaccctcggggcccgcgccaggagggctcctttccgca 
               
               
                   
               
               
                 gggccagctttcaatattgcaagaaaagattga 
               
               
                   
               
               
                 ccatttggagcgtttgctcgctgagaacaacgagatcatctcaaatatca 
               
               
                   
               
               
                 gagactcagtcatcaacctgagcgagtctgtgga 
               
               
                   
               
               
                 ggacggcccgcgggggtcaccaggcaacgccagccaaggctccatccacc 
               
               
                   
               
               
                 tccactcgccacagttggccctgcaggctg 
               
               
                   
               
               
                 accccagagactgtttgtttgcttcacagagtgggagtcagccccgggat 
               
               
                   
               
               
                 gtgcagatgttggatgtttacgatctgattccttttg 
               
               
                   
               
               
                 ataatccagatggtggagtttggaagcaaggatttgacattaagtatgaa 
               
               
                   
               
               
                 gcggatgagtgggaccatgagcccctgcaagtg 
               
               
                   
               
               
                 tttgtggtgcctcactcccataatgacccaggttggttgaagactttcaa 
               
               
                   
               
               
                 tgactactttagagacaagactcagtatatttttaataa 
               
               
                   
               
               
                 catggtcctaaagctgaaagaagactcaagcaggaagtttatgtggtctg 
               
               
                   
               
               
                 agatctcttaccttgcaaaatggtgggatattatag 
               
               
                   
               
               
                 atattccgaagaaggaagctgttaaaagtttactacagaatggtcagctg 
               
               
                   
               
               
                 gaaattgtgaccggtggctgggttatgcctgatga 
               
               
                   
               
               
                 agccactccacattattttgccttaattgaccaactaattgaagggcacc 
               
               
                   
               
               
                 aatggctggaaaaaaatctaggagtgaaacctcga 
               
               
                   
               
               
                 tcgggctgggccatagatccctttggtcattcacccacaatggcttatct 
               
               
                   
               
               
                 tctaaagcgtgctggattttcacacatgctcatccag 
               
               
                   
               
               
                 agagtccattatgcaatcaaaaaacacttctctttgcataaaacgctgga 
               
               
                   
               
               
                 gtttttctggagacagaattgggatcttggatctgct 
               
               
                   
               
               
                 acagacattttgtgccatatgatgcccttctacagctacgacatccctca 
               
               
                   
               
               
                 cacctgtgggcctgatcctaaaatatgctgccagttt 
               
               
                   
               
               
                 gattttaaacggcttcctggaggcagatatggttgtccctggggagttcc 
               
               
                   
               
               
                 cccagaagcaatatctcctggaaatgtccaaagc 
               
               
                   
               
               
                 agggctcagatgctattggatcagtaccggaaaaagtcaaaacttttccg 
               
               
                   
               
               
                 cactaaagttctgctggctccactgggagacgac 
               
               
                   
               
               
                 tttcggttcagtgaatacacagagtgggatctgcagtgcaggaactacga 
               
               
                   
               
               
                 gcaactgttcagttacatgaactcgcagcctcatc 
               
               
                   
               
               
                 tgaaagtgaagatccagtttggaaccttgtcagattatttcgacgcattg 
               
               
                   
               
               
                 gagaaagcggtggcagccgagaagaagagtagc 
               
               
                   
               
               
                 cagtctgtgttccctgccctgagtggagacttcttcacgtacgctgacag 
               
               
                   
               
               
                 agacgaccattactggagtggctacttcacgtcca 
               
               
                   
               
               
                 gacctttctacaaacgaatggacagaataatggaatctcgtataagggct 
               
               
                   
               
               
                 gctgaaattctttaccagttggccttgaaacaagct 
               
               
                   
               
               
                 cagaaatacaagataaataaatttctttcatcacctcattacacaacact 
               
               
                   
               
               
                 gacagaagccagaaggaacttaggactatttcagc 
               
               
                   
               
               
                 atcatgatgccatcacaggaaccgcgaaagactgggtggttgtggactat 
               
               
                   
               
               
                 ggtaccagactctttcagtcattaaattctttggag 
               
               
                   
               
               
                 aagataattggagattctgcatttcttctcattttaaaggacaaaaagct 
               
               
                   
               
               
                 gtaccagtcagatccttccaaagccttcttagagatg 
               
               
                   
               
               
                 gatacgaagcaaagttcacaagattctctgccccaaaaaattataataca 
               
               
                   
               
               
                 actgagcgcacaggagccaaggtaccttgtggtc 
               
               
                   
               
               
                 tacaatccctttgaacaagaacggcattcagtggtgtccatccgggtaaa 
               
               
                   
               
               
                 ctccgccacagggaaagtgctgtctgattcggga 
               
               
                   
               
               
                 aaaccggtggaggttcaagtcagtgcagtttggaacgacatgaggacaat 
               
               
                   
               
               
                 ttcacaagcagcctatgaggtttcttttctagctc 
               
               
                   
               
               
                 atataccaccactgggactgaaagtgtttaagatcttagagtcacaaagt 
               
               
                   
               
               
                 tcaagctcacacttggctgattatgtcctatataata 
               
               
                   
               
               
                 atgatggactagcagaaaatggaatattccacgtgaagaacatggtggat 
               
               
                   
               
               
                 gctggagatgccataacaatagagaatcccttc 
               
               
                   
               
               
                 ctggcgatttggtttgaccgatctgggctgatggagaaagtgagaaggaa 
               
               
                   
               
               
                 agaagacagtagacagcatgaactgaaggtcc 
               
               
                   
               
               
                 agttcctgtggtacggaaccaccaacaaaagggacaagagcggtgcctac 
               
               
                   
               
               
                 ctcttcctgcctgacgggcagggccagccat 
               
               
                   
               
               
                 atgtttccctaagaccgccctttgtcagagtgacacgtggaaggatctac 
               
               
                   
               
               
                 tcagatgtgacctgtttcctcgaacacgttactcac 
               
               
                   
               
               
                 aaagtccgcctgtacaacattcagggaatagaaggtcagtccatggaagt 
               
               
                   
               
               
                 ttctaatattgtaaacatcaggaatgtgcataacc 
               
               
                   
               
               
                 gtgagattgtaatgagaatttcatctaaaataaacaaccaaaatagatat 
               
               
                   
               
               
                 tatactgacctaaatggatatcagattcagcctagaa 
               
               
                   
               
               
                 ggaccatgagcaaattgcctcttcaagccaacgtttacccgatgtgcaca 
               
               
                   
               
               
                 atggcgtatatccaggatgctgagcaccggctca 
               
               
                   
               
               
                 cgctgctctctgctcagtctctaggtgcttccagcatggcttctggtcag 
               
               
                   
               
               
                 attgaagtcttcatggatcgaaggctcatgcaggat 
               
               
                   
               
               
                 gataaccgtggccttgggcaaggcgtccatgacaataagattacagctaa 
               
               
                   
               
               
                 tttgtttcgaatcctcctcgagaagagaagcgct 
               
               
                   
               
               
                 gtgaacatggaagaagaaaagaagagccctgtcagctacccttccctcct 
               
               
                   
               
               
                 cagccacatgacttcgtccttcctcaaccatccc 
               
               
                   
               
               
                 tttctccccatggtactaagtggccagctcccctcccctgcctttgagct 
               
               
                   
               
               
                 gctgagtgaatttcctctgctgcagtcctctctacctt 
               
               
                   
               
               
                 gtgatatccatctggtcaacctgcggacaatacaatcaaagatgggcaaa 
               
               
                   
               
               
                 ggctattcggatgaggcagccttgatcctccaca 
               
               
                   
               
               
                 ggaaagggtttgattgccagttctccagcagaggcatcgggctaccctgt 
               
               
                   
               
               
                 tccactactcagggaaagatgtcagttctgaaac 
               
               
                   
               
               
                 ttttcaacaagtttgctgtggagagtctcgtcccttcctctctgtccttg 
               
               
                   
               
               
                 atgcactcccctccagatgcccagaacatgagtgaag tcagcctgagcc 
               
               
                   
               
               
                 ccatggagatcagcacgttccgtatc   cgcttgcgttggacctga     
               
             
          
         
       
     
     The amplification of this ORF was obtained by nested PCR (3,453 bp) on cDNA of mouse liver and then purified by the phenol/chloroform method. The PCR product was introduced into a vector TOPO-XL. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY05.001). 
     6.2 Step 2: Assembling the Expression Cassette  S. Cerevisiae  and  S. Pombe  Cassette 
     The integration of the expression cassette of mannosidase II will be localized in the auxotrophy marker LEU2 for both strains. Invalidation of this gene induces a resistance to a toxic agent, trifluoroleucine. The yeasts modified by this cassette will therefore become resistant to this drug but also auxotrophic for leucine.
     Amplification of the promoter TEF from genomic DNA of  S. cerevisiae  
 
BS83 (forward) and BS84 (reverse)
   Assembling the promoter TEF (PCR product) with the ORF (PCR product) and the terminator (PCR product)
 
For  S. cerevisiae  marker LEU2
 
BS111 (forward) and BS112 (reverse)
 
For  S. pombe  marker LEU2
 
BS113 (forward) and BS114 (reverse)
   

     The PCR amplifications were extracted and purified from agarose gel with the Qiagen kit and were introduced in a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLy05.008 for the  S. cerevisiae  cassette, pGly05.009 for the  S. pombe  cassette). 
     6.3 Step 3: Modification of the Yeasts
     Preparation of competent yeasts:   

     The Arielle and Erika strains were prepared as indicated above in order to make them competent 
     Electroporation of the Yeasts 
     
         
         Procedure: 20 μg of the plasmids containing the expression cassette for  S. cerevisiae  and  S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced into the yeasts Arielle and Erika by electroporation. The yeasts are selected on an YNB medium containing the required amino acids as well as tri-fluoroleucine (TFL) of  S. cerevisiae  and  S. pombe    
       
    
     Example 7 
     Anaïs and Enrique Strains+Expression Cassette of N-acety-glucosaminyl transferase II 
     6.1 Step 1: Obtaining the OR 
     PCR from complementary DNA of human fibroblasts—Use of Taq polymerase Isis™ (Q-Biogene) 
     Program: 
                                                           3 minutes at 94° C.                30 cycles:   30 s at 94° C.               30 s at 58° C.               1.30 min at 68° C.                        
Amplification of a 1,344 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 5) 
               
             
          
           
               
                   atgaggttccgcat ctacaaacggaaggtgctaatcctgacgctcgtggt 
               
               
                   
               
               
                 ggccgcctgcggcttcgtcctctggagcagca 
               
               
                   
               
               
                 atgggcgacaaaggaagaacgaggccctcgccccaccgttgctggacgcc 
               
               
                   
               
               
                 gaacccgcgcggggtgccggcggccgcg 
               
               
                   
               
               
                 gtggggaccacccctctgtggctgtgggcatccgcagggtctccaacgtg 
               
               
                   
               
               
                 tcggcggcttccctggtcccggcggtccccca 
               
               
                   
               
               
                 gcccgaggcggacaacctgacgctgcggtaccggtccctggtgtaccagc 
               
               
                   
               
               
                 tgaactttgatcagaccctgaggaatgtagat 
               
               
                   
               
               
                 aaggctggcacctgggccccccgggagctggtgctggtggtccaggtgca 
               
               
                   
               
               
                 taaccggcccgaatacctcagactgctgctg 
               
               
                   
               
               
                 gactcacttcgaaaagcccagggaattgacaacgtcctcgtcatctttag 
               
               
                   
               
               
                 ccatgacttctggtcgaccgagatcaatcagctga 
               
               
                   
               
               
                 tcgccggggtgaatttctgtccggttctgcaggtgttctttcctttcagc 
               
               
                   
               
               
                 attcagttgtaccctaacgagtttccaggtagtgaccc 
               
               
                   
               
               
                 tagagattgtcccagagacctgccgaagaatgccgctttgaaattggggt 
               
               
                   
               
               
                 gcatcaatgctgagtatcccgactccttcggcca 
               
               
                   
               
               
                 ttatagagaggccaaattctcccagaccaaacatcactggtggtggaagc 
               
               
                   
               
               
                 tgcattttgtgtgggaaagagtgaaaattcttcga 
               
               
                   
               
               
                 gattatgctggccttatacttttcctagaagaggatcactacttagcccc 
               
               
                   
               
               
                 agacttttaccatgtcttcaaaaagatgtggaaactg 
               
               
                   
               
               
                 aagcagcaagagtgccctgaatgtgatgttctctccctggggacctatag 
               
               
                   
               
               
                 tgccagtcgcagtttctatggcatggctgacaag 
               
               
                   
               
               
                 gtagatgtgaaaacttggaaatccacagagcacaatatgggtctagcctt 
               
               
                   
               
               
                 gacccggaatgcctatcagaagctgatcgagtg 
               
               
                   
               
               
                 cacagacactttctgtacttatgatgattataactgggactggactcttc 
               
               
                   
               
               
                 aatacttgactgtatcttgtcttccaaaattctggaaag 
               
               
                   
               
               
                 tgctggttcctcaaattcctaggatctttcatgctggagactgtggtatg 
               
               
                   
               
               
                 catcacaagaaaacctgtagaccatccactcagagt 
               
               
                   
               
               
                 gcccaaattgagtcactcttaaataataacaaacaatacatgtttccaga 
               
               
                   
               
               
                 aactctaactatcagtgaaaagtttactgtggtagcc 
               
               
                   
               
               
                 atttccccacctagaaaaaatggagggtggggagatattagggaccatga 
               
               
                   
               
               
                 actctgtaaa agttatagaagactgcagtga   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY08.002). 
     Cytoplasmic region of mmn9: PCR from genomic DNA of wild  S. cerevisiae   
                                                                     8 minutes at 94° C.                30 cycles:   20 s at 94° C.               30 s at 65° C.               1 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 51 by fragment (cytoplasmic portion of mmn9)+homology to
 
     
       
         
               
               
               
             
           
               
                 GNTII 
                     Atgtcactttctcttgtatcg   taccgcctaag   aaagaacccgtgggttaacaggttccgcatctac     
                   
               
             
          
         
       
     
     Assembling Mnn9 (PCR product) and the ORF (pGLY08.002) with Taq Platinium 
     CA005 (forward) and CD005 (reverse) 
     Program: 
     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2 minutes at 95° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 45 s at 95° C. 
               
               
                   
                   
                 45 s at 54° C. 
               
               
                   
                   
                 2 min at 72° C. 
               
             
          
           
               
                   
                 10 minutes at 72° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109), Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY08.007). 
     7.2 Step 2: Assembling Expression Cassette for  S. Cerevisiae  and  S. Pombe  Strains 
     The integration of the expression cassette of the GlcNAc-transferase II will be inserted into the Lem3 marker for  S. cerevisiae  and TRP1 marker for  S. pombe . Invalidation of the gene Lem3 induces a resistance to a toxic agent, miltefosine. The yeasts modified by this cassette will therefore become resistant to this drug. Invalidation of the gene TRP1 induces a resistance to a toxic agent, 5-fluoro-anthranilic acid. The yeasts modified by this cassette will therefore become resistant to this drug but also auxotrophic for tryptophan.
     Amplification of the promoter PMA1   

                             CD001 (forward)           aagcttcctgaaacggag                       CD008 (reverse)           acgatacaagagaaagtgacatattgatattgtttgataattaaat            
PCR from genomic DNA of  S. cerevisiae  
 
Program:
 
                                                                     2 minutes at 95° C.                30 cycles:   45 s at 95° C.               45 s at 54° C.               2 min at 72° C.                5 minutes at 72° C.                        
Assembling the promoter PMA1 (PCR product) with Mnn9-homology ORF (pGLY08.007) with Taq Expand (Roche)
 
     
       
         
               
               
             
           
               
                 CD007 
                 cgtttgtagatgcggaacctgttaacccacgggttcttt 
               
               
                   
               
               
                 CD001 
                 aagcttcctgaaacggag 
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2 minutes at 94° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 45 s at 94° C. 
               
               
                   
                   
                 45 s at 55° C. 
               
               
                   
                   
                 1.15 min at 68° C. 
               
             
          
           
               
                   
                 5 minutes at 68° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector TOPO2.1 (Invitrogen). Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY08.005).
     Assembling Mnn9-ORF (pGLY08.007) with the terminator CYC1 (PCR product) with Taq polymerase Phusion™ (Ozyme)
 
Program
   

                                                           2 minutes at 98° C.                3 cycles:   10 s at 98° C.               30 s at 52° C.               40 s at 72° C.                        
addition of the primers and then
 
     
       
         
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 30 cycles 
                 10 s at 98° C. 
               
               
                   
                   
                 30 s at 61° C. 
               
               
                   
                   
                 40 s at 72° C. 
               
             
          
           
               
                   
                 5 min at 72° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY08.04).
     Assembling the promoter PMA1-Mnn9 (pGLY08.009) with Mnn9-ORF-terminator CYC1 (PCR product) with the ends homologous to the marker Lem3 for  S. cerevisiae  with Taq polymerase Phusion™ (Ozyme)   

                         CB053: Atggtaaatttcgatttgggccaagttggtgaagtattccaagcttcctgaaacggag (forward)                   CB070: Ttctaccgccgaagagccaaaacgttaataatatcaatggcagcttgcaaattaaagc (reverse)            
Program
 
                                                           2 minutes at 98° C.                3 cycles:   10 s at 98° C.               30 s at 52° C.               4 min at 72° C.                        
addition of the primers and then
 
     
       
         
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 30 cycles 
                 10 s at 98° C. 
               
               
                   
                   
                 30 s at 61° C. 
               
               
                   
                   
                 1 min at 72° C. 
               
             
          
           
               
                   
                 5 min at 72° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY08).
     Assembling the ends homologous to the marker TRP1 for  S. pombe  from pGLY08.012   

     
       
         
               
               
               
             
           
               
                 CD009 (forward) 
                 taaagttgattccgctggtgaaatcatacatggaaaagtttaagcttcctgaaacggag 
                   
               
               
                   
               
               
                 CD010 (reverse) 
                 atgtgaaatttccttggccacggacaagtccacttttcgtttggcagcttgcaaattaaagc 
               
             
          
         
       
     
     7.3 Step 3: Modification of the Yeasts
     Preparation of competent yeasts:   

     The Anaïs and Enrique strains were prepared as indicated above in order to make them competent.
     Electroporation of the yeasts   Procedure: 20 μg of the plasmids containing the expression cassette for  S. cerevisiae  and  S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced into the yeasts Anaïs and Enrique by electroporation. The yeasts are then spread over a gelosed YPD medium containing the required selection drug.   

     Example 8 
     Alice and Elga Strains+Expression Cassette of Galactosyl Transferase I 
     8.1 Step 1: Obtaining the ORF Without the Human Localization Sequence 
     PCR from cDNA of human lymphoblasts 
     CD025 (forward) CD026 (reverse) 
                                                                     2 minutes at 94° C.                30 cycles:   45 s at 94° C.               45 s at 58° C.               1.15 min at 72° C.                5 minutes at 72° C.                        
Amplification of a 1,047 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 6) 
               
             
          
           
               
                     ccccaactggtcggagt   ctccacaccgctgcagggcggctcgaacagtgc 
               
               
                   
               
               
                 cgccgccatcgggcagtcctccggggagc 
               
               
                   
               
               
                 tccggaccggaggggcccggccgccgcctcctctaggcgcctcctcccag 
               
               
                   
               
               
                 ccgcgcccgggtggcgactccagcccagt 
               
               
                   
               
               
                 cgtggattctggccctggccccgctagcaacttgacctcggtcccagtgc 
               
               
                   
               
               
                 cccacaccaccgcactgtcgctgcccgcctgc 
               
               
                   
               
               
                 cctgaggagtccccgctgcttgtgggccccatgctgattgagtttaacat 
               
               
                   
               
               
                 gcctgtggacctggagctcgtggcaaagcagaa 
               
               
                   
               
               
                 cccaaatgtgaagatgggcggccgctatgcccccagggactgcgtctctc 
               
               
                   
               
               
                 ctcacaaggtggccatcatcattccattccgca 
               
               
                   
               
               
                 accggcaggagcacctcaagtactggctatattatttgcacccagtcctg 
               
               
                   
               
               
                 cagcgccagcagctggactatggcatctatgttat 
               
               
                   
               
               
                 caaccaggcgggagacactatattcaatcgtgctaagctcctcaatgttg 
               
               
                   
               
               
                 gctttcaagaagccttgaaggactatgactacacc 
               
               
                   
               
               
                 tgctttgtgtttagtgacgtggacctcattccaatgaatgaccataatgc 
               
               
                   
               
               
                 gtacaggtgtttttcacagccacggcacatttccgttg 
               
               
                   
               
               
                 caatggataagtttggattcagcctaccttatgttcagtattttggaggt 
               
               
                   
               
               
                 gtctctgctctaagtaaacaacagtttctaaccatcaat 
               
               
                   
               
               
                 ggatttcctaataattattggggctggggaggagaagatgatgacatttt 
               
               
                   
               
               
                 taacagattagtttttagaggcatgtctatatctcgcc 
               
               
                   
               
               
                 caaatgctgtggtcgggaggtgtcgcatgatccgccactcaagagacaag 
               
               
                   
               
               
                 aaaaatgaacccaatcctcagaggtttgaccg 
               
               
                   
               
               
                 aattgcacacacaaaggagacaatgctctctgatggtttgaactcactca 
               
               
                   
               
               
                 cctaccaggtgctggatgtacagagatacccattg tatacccaaatcac 
               
               
                   
               
               
                 agtgga   catcgggacaccgacctag   . 
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY11.003). 
     Mnt1 localization: PCR from gDNA of  S. cerevisiae   
                                                                     3 minutes at 94° C.                30 cycles:   20 s at 94° C.               30 s at 58° C.               45 s at 72° C.                10 minutes at 72° C.                        
Amplification of a 246 by fragment—SEQ ID NO 14
 
     
       
         
               
             
           
               
                     atggccctctttctcagtaa   gagactgttgagatttaccgtcattgcagg 
               
               
                   
               
               
                 tgcggttattgttctcctcctaacattgaattccaac 
               
               
                   
               
               
                 agtagaactcagcaatatattccgagttccatctccgctgcatttgattt 
               
               
                   
               
               
                 tacctcaggatctatatcccctgaacaacaagtcatct 
               
               
                   
               
               
                 ctgaggaaaatgatgctaaaaaattagagcaaagtgctctgaattcagag 
               
               
                   
               
               
                 gcaag   cgaagactccgaagcc     
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing. 
     8.2: Step 2: Assembling Expression Cassettes for the  S. Cerevisiae  and  S. Pombe  Strains 
     The integration of the expression cassettes of Galactosyl transferase I will be localized in the marker TRP1 for  S. cerevisiae  Alice. Invalidation of this gene induces resistance to a toxic agent, fluoroanthranilic acid. The yeasts modified by this cassette will therefore become resistant to this drug. The integration of the expression cassette of Galactosyl transferase I will be localized in the marker Met17 for  S. cerevisiae  Ashley.
     Amplification of the promoter CaMV from the plasmid pMDC:
 
CD035 (forward)
 
CD037 (reverse)
 
Program
   

     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2 minutes at 94° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 45 s at 94° C. 
               
               
                   
                   
                 45 s at 65° C. 
               
               
                   
                   
                 2 min 30 s at 72° C. 
               
             
          
           
               
                   
                 5 minutes at 72° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY11.001).
     Assembling the promoter CaMV (pGLY11.001) with the Mnt1 localization sequence (PCR product):
 
CD035 (forward) and CD028 (reverse)
 
Program:
   

                                                                     2 minutes at 94° C.                30 cycles:   45 s at 94° C.               45 s at 59° C.               1 min 15 s at 72° C.                3 minutes at 72° C.                        
The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY11.002).
     Assembling the promoter CaMV-Mnt1 localization (pGLY011.002) with the ORF (PCR product).
 
CD035 (forward)
 
CD029 (reverse)
 
Program:
   

     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 2 minutes at 94° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 45 s at 94° C. 
               
               
                   
                   
                 45 s at 56° C. 
               
               
                   
                   
                 2 min 30 s at 72° C. 
               
             
          
           
               
                   
                 3 minutes at 72° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY011.004).
     Assembling the promoter CaMV-localization Mnt1-ORF (pGLY011.004) with the terminator CYC1 (PCR product) with Taq Expand (Roche)
 
CD035 (forward) BS41 (reverse)
 
Program:
   

     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 3 minutes at 95° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 30 s at 95° C. 
               
               
                   
                   
                 30 s at 57° C. 
               
               
                   
                   
                 2 min 30 s at 68° C. 
               
             
          
           
               
                   
                 10 minutes at 68° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector pTarget (Promega). Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY011.005).
     Assembling the integration cassette for  S. cerevisiae  with Taq Expand (Roche)   

                             CD063 (forward)   agatgccagaaacaaagcttgttgcaggtggtgctgctcatgcctgcaggtcaacatggt                   CD064 (reverse)   gtgtcgacgatcttagaagagtccaaaggtttgactggatgcagcttgcaaattaaagcc            
Program
 
     
       
         
               
               
             
               
               
               
             
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 3 minutes at 95° C. 
               
             
          
           
               
                   
                 30 cycles: 
                 30 s at 95° C. 
               
               
                   
                   
                 30 s at 57° C. 
               
               
                   
                   
                 2 min 30 s at 68° C. 
               
             
          
           
               
                   
                 10 minutes at 68° C. 
               
               
                   
                   
               
             
          
         
       
     
     The PCR amplification was purified by the phenol/chloroform method and introduced into the vector TOPO 2.1 (Invitrogen). Competent bacteria (TOP10 Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY011.008). 
     For  S. pombe , integration into the sequence PET6 
     Use of Taq Expand (Roche) 
     CD038 (forward) and CD039 (reverse) 
     The PCR products were introduced into a vector TOPO-XL. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of PCR amplification into the vector by sequencing (pGLY11.006 for  S. pombe  and pGLY11.007 for  S. cerevisiae ). 
     8.3 Step 3: Modification of the Yeasts
     Preparation of competent yeasts:   

     The Alice and Elga strains were prepared as indicated above in order to make them competent.
     Electroporation of the yeasts   Procedure: 20 μg of the plasmids containing the expression cassettes for  S. cerevisiae  and  S. pombe  were digested by restriction enzymes KpnI and XhoI. The linearized cassette was introduced into the yeasts Alice and Elga by electroporation. The yeasts were then spread on a gelosed YPD medium containing the required selection.   

     Example 9 
     Strains Anaïs and Enrique+Expression Cassettes for Fucosylation (α1,6-Fucosyl Transferase FUT8 and GDP-Fucose Transporter) 
     9.1 Expression Cassette of FUT8 
     9.1.1 Step 1: Obtaining the ORF 
     PCR from cDNA of human pancreas and lungs 
     Program: 
                                                                     3 minutes at 94° C.                35 cycles:   20 s at 94° C.               30 s at 58° C.               2 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 1,801 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 7) 
               
             
          
           
               
                     caggactccagggaagtgag   ttgaaaatctgaaaatgcggccatggactg 
               
               
                   
               
               
                 gttcctggcgttggattatgctcattctttttgcc 
               
               
                   
               
               
                 tgggggaccttgctgttttatataggtggtcacttggtacgagataatga 
               
               
                   
               
               
                 ccatcctgatcactctagccgagaactgtccaagat 
               
               
                   
               
               
                 tctggcaaagcttgaacgcttaaaacagcagaatgaagacttgaggcgaa 
               
               
                   
               
               
                 tggccgaatctctccggataccagaaggccct 
               
               
                   
               
               
                 attgatcaggggccagctataggaagagtacgcgttttagaagagcagct 
               
               
                   
               
               
                 tgttaaggccaaagaacagattgaaaattacaa 
               
               
                   
               
               
                 gaaacagaccagaaatggtctggggaaggatcatgaaatcctgaggagga 
               
               
                   
               
               
                 ggattgaaaatggagctaaagagctctggttt 
               
               
                   
               
               
                 ttcctacagagtgaattgaagaaattaaagaacttagaaggaaatgaact 
               
               
                   
               
               
                 ccaaagacatgcagatgaatttcttttggatttagg 
               
               
                   
               
               
                 acatcatgaaaggtctataatgacggatctatactacctcagtcagacag 
               
               
                   
               
               
                 atggagcaggtgattggcgggaaaaagaggcc 
               
               
                   
               
               
                 aaagatctgacagaactggttcagcggagaataacatatcttcagaatcc 
               
               
                   
               
               
                 caaggactgcagcaaagccaaaaagctggtgt 
               
               
                   
               
               
                 gtaatatcaacaaaggctgtggctatggctgtcagctccatcatgtggtc 
               
               
                   
               
               
                 tactgcttcatgattgcatatggcacccagcgaac 
               
               
                   
               
               
                 actcatcttggaatctcagaattggcgctatgctactggtggatgggaga 
               
               
                   
               
               
                 ctgtatttaggcctgtaagtgagacatgcacagac 
               
               
                   
               
               
                 agatctggcatctccactggacactggtcaggtgaagtgaaggacaaaaa 
               
               
                   
               
               
                 tgttcaagtggtcgagcttcccattgtagacagt 
               
               
                   
               
               
                 cttcatccccgtcctccatatttacccttggctgtaccagaagacctcgc 
               
               
                   
               
               
                 agatcgacttgtacgagtgcatggtgaccctgcagt 
               
               
                   
               
               
                 gtggtgggtgtctcagtttgtcaaatacttgatccgcccacagccttggc 
               
               
                   
               
               
                 tagaaaaagaaatagaagaagccaccaagaagc 
               
               
                   
               
               
                 ttggcttcaaacatccagttattggagtccatgtcagacgcacagacaaa 
               
               
                   
               
               
                 gtgggaacagaagctgccttccatcccattgaag 
               
               
                   
               
               
                 agtacatggtgcatgttgaagaacattttcagcttcttgcacgcagaatg 
               
               
                   
               
               
                 caagtggacaaaaaaagagtgtatttggccacaga 
               
               
                   
               
               
                 tgacccttctttattaaaggaggcaaaaacaaagtaccccaattatgaat 
               
               
                   
               
               
                 ttattagtgataactctatttcctggtcagctggactg 
               
               
                   
               
               
                 cacaatcgatacacagaaaattcacttcgtggagtgatcctggatataca 
               
               
                   
               
               
                 ttttctctctcaggcagacttcctagtgtgtactttttc 
               
               
                   
               
               
                 atcccaggtctgtcgagttgcttatgaaattatgcaaacactacatcctg 
               
               
                   
               
               
                 atgcctctgcaaacttccattctttagatgacatctact 
               
               
                   
               
               
                 attttgggggccagaatgcccacaatcaaattgccatttatgctcaccaa 
               
               
                   
               
               
                 ccccgaactgcagatgaaattcccatggaacctg 
               
               
                   
               
               
                 gagatatcattggtgtggctggaaatcattgggatggctattctaaaggt 
               
               
                   
               
               
                 gtcaacaggaaattgggaaggacgggcctatatc 
               
               
                   
               
               
                 cctcctacaaagttcgagagaagatagaaacggtcaagtaccccacatat 
               
               
                   
               
               
                 cctgaggctgagaaataaagctcagatggaag agat aaacgaccaaact   
               
               
                   
               
               
                 
                   cagttcga 
                 
               
             
          
         
       
     
     The PCR amplification (1,801 by from cDNA of human lungs and pancreas) was extracted and purified from agarose gel with the QBIOgene kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing. 
     9.1.2 Step2: Assembling the Expression Cassette for  S. Cerevisiae  
     Amplification of the promoter mnt1 from genomic DNA of  S. pombe  
 
BS86 (forward) and BS84 (reverse)
   Assembling the promoter mnt1 (PCR product) with the ORF (pGLY06.001)
 
BS86 (forward) and BS88 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY06.003). 
     Assembling the ORF (pGLY06.001) with the terminator CYC1 (PCR product) 
     CA011 (forward) and BS41 (reverse) 
     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY06.002).
     Assembling nmt1-ORF (pGLY06.003) with ORF-terminator CYC1 (PCR product)
 
BS86 (forward) and BS41 (reverse)
   Assembling the ends for integration into auxotrophy markers of yeasts:   

     9.1.2.1  S. Cerevisiae    
     The integration of the expression cassette of FUT8 was localized in the marker CAN1 for  S. cerevisiae  strain. Invalidation of the gene CAN1 induces auxotrophy for canavanine. 
     BS147 (forward) and BS148 (reverse) 
     The PCR amplifications were extracted and purified from agarose gel with the Qiagen kit and were introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY06.005). 
     9.1.2.2  S. Pombe    
     The expression cassette for fucosyl transferase 8 was produced in tandem with a cassette for resistance to an antibiotic, phleomycin. This double cassette is inserted in a simple auxotrophy marker HIS5. Insertion of this cassette into this locus will induce resistance to phleomycin as well as auxotrophy for histidine. 
     The expression cassette of FUT8 obtained previously is assembled with an expression cassette of phleomycin comprising the promoter of SV40, the ORF of the resistance to phleomycin as well as the terminator TEF. These tandem cassettes are inserted into the marker HIS5. 
     9.1.3 Step 3: Modification of the Yeasts
     Preparation of competent yeasts:   

     The Anaïs and Enrique strains were prepared as indicated above in order to make them competent.
     Electroporation of the yeasts   Procedure: 20 μg of plasmids containing the expression cassette for  S. cerevisiae  and  S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced into the yeasts Anaïs and Enrique by electroporation. The yeasts are spread with a limiting dilution on a gelosed YPD medium, the deleted markers do not impart resistance to a drug. Once the clones have been released, replicates on a minimum medium are established in order to select the clones which can no longer grow without the required amino acid.   

     9.2 Expression Cassette of the GDP-Fucose Transporter 
     9.2.1 Obtaining the ORF 
     PCR from cDNA of human lungs 
     Program: 
                                                                     3 minutes at 94° C.                35 cycles:   20 s at 94° C.               30 s at 58° C.               2 min at 72° C.                10 minutes at 72° C.                        
Amplification of a 1,136 by fragment
 
     
       
         
               
             
               
             
           
               
                 (SEQ ID No 8) 
               
             
          
           
               
                     tgacccagctcctctgctac   catgaatagggcccctctgaagcggtccag 
               
               
                   
               
               
                 gatcctgcacatggcgctgaccggggcctca 
               
               
                   
               
               
                 gacccctctgcagaggcagaggccaacggggagaagccctttctgctgcg 
               
               
                   
               
               
                 ggcattgcagatcgcgctggtggtctccctct 
               
               
                   
               
               
                 actgggtcacctccatctccatggtgttccttaataagtacctgctggac 
               
               
                   
               
               
                 agcccctccctgcggctggacacccccatcttcgt 
               
               
                   
               
               
                 caccttctaccagtgcctggtgaccacgctgctgtgcaaaggcctcagcg 
               
               
                   
               
               
                 ctctggccgcctgctgccctggtgccgtggact 
               
               
                   
               
               
                 tccccagcttgcgcctggacctcagggtggcccgcagcgtcctgcccctg 
               
               
                   
               
               
                 tcggtggtcttcatcggcatgatcaccttcaata 
               
               
                   
               
               
                 acctctgcctcaagtacgtcggtgtggccttctacaatgtgggccgctca 
               
               
                   
               
               
                 ctcaccaccgtcttcaacgtgctgctctcctacctg 
               
               
                   
               
               
                 ctgctcaagcagaccacctccttctatgccctgctcacctgcggtatcat 
               
               
                   
               
               
                 catcgggggcttctggcttggtgtggaccaggag 
               
               
                   
               
               
                 ggggcagaaggcaccctgtcgtggctgggcaccgtcttcggcgtgctggc 
               
               
                   
               
               
                 tagcctctgtgtctcgctcaacgccatctacac 
               
               
                   
               
               
                 cacgaaggtgctcccggcggtggacggcagcatctggcgcctgactttct 
               
               
                   
               
               
                 acaacaacgtcaacgcctgcatcctcttcctgc 
               
               
                   
               
               
                 ccctgctcctgctgctcggggagcttcaggccctgcgtgactttgcccag 
               
               
                   
               
               
                 ctgggcagtgcccacttctgggggatgatgacg 
               
               
                   
               
               
                 ctgggcggcctgtttggctttgccatcggctacgtgacaggactgcagat 
               
               
                   
               
               
                 caagttcaccagtccgctgacccacaatgtgtcg 
               
               
                   
               
               
                 ggcacggccaaggcctgtgcccagacagtgctggccgtgctctactacga 
               
               
                   
               
               
                 ggagaccaagagcttcctctggtggacgagc 
               
               
                   
               
               
                 aacatgatggtgctgggcggctcctccgcctacacctgggtcaggggctg 
               
               
                   
               
               
                 ggagatgaagaagactccggaggagcccag ccccaaagacagcgagaag 
               
               
                   
               
               
                 ag cgccatgggggtgtgagc   accacaggcaccctggat     
               
             
          
         
       
     
     The PCR amplification was extracted and purified from agarose gel with the QIAGEN kit and was introduced into a vector TOPO2.1. Competent bacteria (TOP10, Invitrogen) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY07.001). 
     9.2.2 Step 2: Assembling the Expression Cassette for  S. Cerevisiae  and  S. Pombe  
     Amplification of the promoter SV40 from pTarget:
 
BS109 (forward) and BS110 (reverse)
   Assembling the ORF (pGLY07.001) with the terminator CYC1 (PCR product) CA013 (forward) and BS41 (reverse)   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY07.002). 
     9.2.2.1  S. Cerevisiae    
     The integration of the expression cassette of the GDP-fucose transporter will be localized in the auxotrophy marker TRP1 for the  S. cerevisiae  strain. Invalidation of the gene TRP1 induces resistance to a toxic agent, 5-fluoro-anthranilic acid. The yeasts modified by this cassette will therefore become resistant to this drug but also auxotrophic for tryptophan.
     Assembling the promoter SV40 cassette (PCR product), the ORF (PCR product) and the terminator CYC1 (PCR product)
 
BS136 (forward) and BS137 (reverse)
   

     The PCR amplification was extracted and purified from agarose gel with the Qiagen kit and was introduced into a vector pTarget. Competent bacteria (JM109, Promega) were transformed with this vector. The transformation was checked by PCR and the insertion of the PCR amplification into the vector by sequencing (pGLY07.003). 
     9.2.2.2  S. Pombe    
     The expression cassette for the GDP-fucose transporter was produced in tandem with a cassette for resistance to an antibiotic, hygromycin. This double cassette is inserted in a gene coding for a protein involved in the maturation of N-glycans of  S. pombe , GMA12. Insertion of this cassette in this locus will induce resistance to hygromycin but also the deletion of the gene gma12. 
     The expression cassette of the GDP-fucose transporter obtained previously is assembled with an expression cassette of resistance to hygromycin comprising the promoter of SV40, the ORF of the resistance to hygromycin as well as the terminator TEF. These tandem cassettes are inserted into the marker gma12. 
     9.2.3 Step 3: Modification of the Yeasts
     Preparation of competent yeasts:   

     The Apolline and Epiphanie strains were prepared as indicated above in order to make them competent.
     Electroporation of the yeasts   Procedure: 20 μg of plasmids containing the expression cassettes for  S. cerevisiae  and  S. pombe  were digested by the restriction enzyme EcoRI. The linearized cassette was introduced into the yeasts Apolline and Epiphanie by electroporation. The  S. cerevisiae  yeasts are spread on an YPD medium containing 5-fluoro-anthranilic acid. The  S. pombe  yeasts are spread with limiting dilution on a gelosed YPD medium, the deleted marker not imparting resistance to a drug. Once the clones are released, replicates on a minimum medium are established in order to select the clones which cannot grow without the required amino acid.   

     Example 10 
     Athena and Etienne Strains+Expression Cassette of N-Acetylglucosaminyl-Transferase III 
     10.1 Obtaining the ORF 
     PCR from complementary DNA of murine brain 
                                 CB007 (forward)   Atgaagatgagacgctacaa                       CB036 (reverse)   ctagccctccactgtatc            
Program:
 
                                                                     2 minutes at 94° C.                30 cycles:   45 s at 94° C.               45 s at 56° C.               2 min at 72° C.                5 minutes at 72° C.                        
Amplification of a by fragment without the cytoplasmic portion of the enzyme: it will be replaced with the cytoplasmic portion of Mnt1 for Golgian localization of the protein.
 
     10.2 Expression Cassette Assembling for the  S. Cerevisiae  and  S. Pombe  Strains 
     10.2.1 Assembling for  S. Cerevisiae    
     Amplification of the promoter nmt1 
                     CB013: tatagtcgctttgttaaatcatatggccctctttctcagtaa               CB014: agcgaagactccgaagcccacttctttaagaccttatcc            
Amplification of the terminator CYC1
 
Amplification of the expression cassette with the ends CAN1
 
     
       
         
               
               
               
             
           
               
                   
                 CB030 (forward): 
                 cagaaaatccgttccaagag 
               
               
                   
                   
               
               
                   
                 CB031 (reverse): 
                 tgccacggtatttcaaagct 
               
             
          
         
       
     
     10.2.2 Assembling for  S. Pombe    
     Expression cassette of GNTIII in tandem with a cassette for resistance to hygromycin. Insertion in GMA12. 
     10.3 Transformation of the Yeasts 
     Example 11 
     Deletion of Genes Involved in Hypermannosylation in  S. Cerevisiae  and  S. Pombe    
     11.1 Step 1: Deletion of the Mnn1 Gene in the Yeasts Amélie, Arielle, Anaïs, Alice, Abel, Ashley, Athena, Azalée and Aurel 
     11.1.1 Construction and Insertion of a Cassette Containing a Gene for Resistance to Hygromycin into the Mnn1 Gene 
     11.1.1.1 Construction of the Expression Cassette 
     Amplification of the promoter CaMV with the Mnn1 5′ end 
                     CB39:       TTTATATTAAACCAAAGGTCTTTGAGATCGTGTACCATACTGCCTGCAGG               TCAACATG               CB40:       TTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTACCCATCCGGGGATC               CTCTAGAGTC            
Hygromycin-terminator TEF amplification with homology to the promoter CaMV and the Mnn1 3′ end
 
                     CB41:       TTCATTTGGAGAGGACCTCGACTCTAGAGGATCCCCGGATGGGTAAAAAG               CCTGAACTC               CB42:       GGTGTTATCTTTATTAGCATGTGACCAAACAGTGTTGACATCGACACTGG               ATGGCGGCGTATGGGTAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGA               AGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCG               GAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATA               TGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATG               TTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGAC               ATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACA               GGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGC               AGCCGGTCGCGGAGGCCATGGATGCGATGGCTGCGGCCGATCTTAGCCAG               ACGAGCGGGTTCGGCCCATTCGGACCGCAAGGGGCGTGATTTCATATGCG               CGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACG               GTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGA               GGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACA               ATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAG               GCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAG               GCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGC               ATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATT               GGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGC               AGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGA               CTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGAT               GGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCG               TCCGAGGGCAAAGGAATAATCAGTACTGACAATAAAAAGATTCTTGTTTT               CAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTT               AATCAAATGTTAGCGTGATTTATATTTTTTTTAGATGCGAAGTTAAGTGC               GCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATAC               TGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGa            
Assembling the insertion cassette for deletion of the Mnn 1 gene in  S. cerevisiae :
 
     
       
         
               
             
           
               
                 CB39: 
               
               
                 TTTATATTAAACCAAAGGTCTTTGAGATCGTGTACCATACTGCCTGCAGG 
               
               
                   
               
               
                 TCAACATG 
               
               
                   
               
               
                 CB42: 
               
               
                 GGTGTTATCTTTATTAGCATGTGACCAAACAGTGTTGACATCGACACTGG 
               
               
                   
               
               
                 ATGGCGGCGT 
               
             
          
         
       
     
     11.1.1.2 Transformation of the Yeasts
     Preparation of competent yeasts:
 
The strains were prepared as indicated above in order to make them competent.
   Electroporation of  S. cerevisiae  yeasts   Procedure:   20 μg of the plasmids containing the expression cassettes for  S. cerevisiae  were digested by a restriction enzyme. The lenearized cassette was introduced into the yeasts by electroporation. The yeasts are selected on a YPD medium containing hygromycin.   

     11.1.2 Deletion of the Mnn9 Gene in the Yeasts Amélie, Arielle, Anaïs, Alice, Abel, Ashley, Athena, Azalée and Aurel 
     11.1.2.1 Construction of a Cassette for Integration Into the Mnn9 Gene Containing a Gene for Resistance to Phleomycin 
     Amplification of the promoter SV40 with the Mnn9 5′ enc 
                     CB46:       AAAGATCTTAACGTCGTCGACCATGTGGTTAAGCACGACGCAGGCAGAAG               TATGCAAA               CB47:       AAATGTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGCCATAGCTTTTTG               CAAAAGCCTAG            
Phleomycin-terminator TEF amplification with homology to the promoter SV40
 
     
       
         
               
             
           
               
                 CB48: 
               
               
                 GCTGTGGAATGTGIGTCAGTTAGGGTGTGGAAAGTCCCCAATGGCCGACC 
               
               
                   
               
               
                 AAGCGACGCCC 
               
               
                   
               
               
                 CB49: 
               
               
                 GGTGTTATCTTTATTAGCATGTGAGCAAACAGTGTTGACATCGACACTGG 
               
               
                   
               
               
                 ATGGCGGCGT 
               
               
                   
               
               
                 atggccgaccaagcgacgcccaacctgccatcacgagatttcgattccac 
               
               
                   
               
               
                 ggccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccg 
               
               
                   
               
               
                 gctggatgatcctccagcgcggggatctcaagctggagttcttcgcccac 
               
               
                   
               
               
                 cccgggctcgatcccctcgcgagttggttcagctgctgcctgaggctgga 
               
               
                   
               
               
                 cgacctcgcggagttctaccggcagtgcaaatccgtcggcatccaggaaa 
               
               
                   
               
               
                 ccagcagcggctatccgcgcatccatgcccccgaactgcaggagtgggga 
               
               
                   
               
               
                 ggcacgatggccgctttggtcgacccggacgggacgctcctgcgcctgat 
               
               
                   
               
               
                 acagaacgaattgcttgcaggcatctcatgatcagtactgacaataaaaa 
               
               
                   
               
               
                 gattcttgttttcaagaacttgtcatttgtatagtttttttatattgtag 
               
               
                   
               
               
                 ttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctc 
               
               
                   
               
               
                 gacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatg 
               
               
                   
               
               
                 cgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatact 
               
               
                   
               
               
                 aacgccgccatccagtgtcgaaaacgagctctcgagaacccttaat 
               
             
          
         
       
     
     11.1.2.2 Transformation of the Yeasts
     Preparation of competent yeasts:
 
The yeasts are prepared as indicated above in order to make them competent
   Electroporation of  S. cerevisiae  yeasts   Procedure:   20 μg of the plasmids containing the expression cassette for  S. cerevisiae  were digested by a restriction enzyme. The linearized cassette was introduced into the yeasts by electroporation. The yeasts are selected on a YPD medium containing phleomycin.   

     11.2 Step 2: Dejection of the GMA12 Gene in the Yeasts  S. Pombe , Emma, Erika, Enrique, Elga, Etienne 
     11.2.1 Construction of the Integration Cassette Containing the Gene for Resistance to Hygromycin 
     ext-gma12/prom-CaMV/hph/Tef-term/ext-gma12 
     
       
         
               
             
           
               
                 CB51: 
               
               
                 CAAAGATCTTAACGTCGTCGACCATGTGCTTAAGCACGACTGGCTGCAGG 
               
               
                   
               
               
                 TGAACATG 
               
               
                   
               
               
                 CB52: 
               
               
                 ATATGATCCTTTTCTTGAGCAGACATCCAATCGGATCCTTTCGACACTGG 
               
               
                   
               
               
                 ATGGCGGCGT 
               
             
          
         
       
     
     11.2.2 Transformation of the Yeasts
     Preparation of competent yeasts:
 
The strains are prepared as indicated above in order to make them competent.
   Electroporation of  S. cerevisiae  yeasts.   Procedure:   20 μg of the plasmids containing the expression cassette for  S. pombe  were digested by a restriction enzyme. The linearized cassette was introduced by electroporation. The yeasts are selected on an YPD medium containing hygromycin.   

     Example 12 
     Strains+Expression Cassettes for Sialylation of the N-Glycans of  S. Cerevisiae  and  S. Pombe    
     With the purpose of obtaining effective sialylation of N-glycans of proteins produced in  S. cerevisiae  and  S. pombe , first of all the biosynthesis route for sialic acid has to be introduced into the same yeasts. To do this, we introduced into the genome of the yeasts, the route for the biosynthesis of CMP-sialic acid of  N. meningitidis , enzymes localized in the cytosol. Preparation of cassettes in tandem for sialylation (from the strains Athena, Aurel and Azalée): 
     12.1 Cassette S1 
     Construction of a tandem cassette consisting of the promoter PET56, of the ORF of sialic acid synthase, of the terminator CYC1 and then of the promoter PET565, of the ORF of CMP-sialic acid synthase and of the terminator CYC1.
     Obtaining the ORFs:
 
Sialic acid synthase (1,050 bp)
   

                     Atgcaaaacaacaacgaatttaaaattggtaatcgttcagtaggttacaa               ccacgaaccattgattatctgtgaaatcggcatcaatcatgaaggctctt               taaaaacagcttttgaaatggttgatgctgcctataatgcaggcgctgaa               gttgttaaacatcaaacacacatcgttgaagacgaaatgtctgatgaggc               caaacaagtcattccaggcaatgcagatgtctctatttatgaaattatgg               aacgttgcgccctgaatgaagaagatgagattaaattaaaagaatacgta               gagagtaagggtatgatttttatcagtactcctttctctcgtgcagctgc               tttacgattacaacgtatggatattccagcatataaaatcggctctggcg               aatgtaataactacccattaattaaactggtggcctcttttggtaagcct               attattctctctaccggcatgaattctattgaaagcatcaaaaagtcggt               agaaattattcgagaagcaggggtaccttatgctttgcttcactgtacca               acatctacccaaccccttacgaagatgttcgattgggtggtatgaacgat               ttatctgaagcctttccagacgcaatcattggcctgtctgaccatacctt               agataactatgcttgcttaggagcagtagctttaggcggttcgattttag               agcgtcactttactgaccgcatggatcgcccaggtccggatattgtatgc               tctatgaatccggatacttttaaagagctcaagcaaggcgctcatgcttt               aaaattggcacgcggcggcaaaaaagacacgattatcgcgggagaaaagc               caactaaagatttcgcctttgcatctgtcgtagcagataaagacattaaa               aaaggagaactgttgtccggagataacctatgggttaaacgcccaggcaa               tggagacttcagcgtcaacgaatatgaaacattatttggtaaggtcgctg               cttgcaatattcgcaaaggtgctcaaatcaaaaaaactgatattgaataa            
CMP-sialic acid synthase (687 bp):
 
     
       
         
               
             
           
               
                 atggaaaaacaaaatattgcggttatacttgcgcgccaaaactccaaagg 
               
               
                   
               
               
                 attgccattaaaaaatctccggaaaatgaatggcatatcattacttggtc 
               
               
                   
               
               
                 atacaattaatgctgctatatcatcaaagtgttttgaccgcataattgtt 
               
               
                   
               
               
                 tcgactgatggcgggttaattgcagaagaagctaaaaatttcggtgtcga 
               
               
                   
               
               
                 agtcgtcctacgccctgcagagctggcctccgatacagccagctctattt 
               
               
                   
               
               
                 caggtgtaatacatgctttagaaacaattggcagtaattccggcacagta 
               
               
                   
               
               
                 accctattacaaccaaccagtccattacgcacaggggctcatattcgtga 
               
               
                   
               
               
                 agctttttctctatttgatgagaaaataaaaggatccgttgtctctgcat 
               
               
                   
               
               
                 gcccaatggagcatcatccactaaaaaccctgcttcaaatcaataatggc 
               
               
                   
               
               
                 gaatatgcccccatgcgccatctaagcgatttggagcagcctcgccaaca 
               
               
                   
               
               
                 attacctcaggcatttaggcctaatggtgcaatttacattaatgatactg 
               
               
                   
               
               
                 cttcactaattgcaaataattgtttttttatcgctccaaccaaactttat 
               
               
                   
               
               
                 attatgtctcatcaagactctatcgatattgatactgagcttgatttaca 
               
               
                   
               
               
                 acaggcagaaaacattcttaatcacaaggaaagctaa 
               
             
          
         
       
         
         Expression cassette:
 
Promoter PET56
 
       
    
     
       
         
               
             
           
               
                 CTTTGCCTTCGTTTATCTTGCCTGCTCATTTTTTAGTATATTCTTCGAAG 
               
               
                   
               
               
                 AAATCACATTACTTTATATAATGTATAATTCATTATGTGATAATGCCAAT 
               
               
                   
               
               
                 CGCTAAGAAAAAAAAAGAGTCATCCGCTAGGTGGAAAAAAAAAAATGAAA 
               
               
                   
               
               
                 ATCATTACCGAGGCATAAAAAAATATAGAGTGTACTAGAGGAGGCCAAGA 
               
               
                   
               
               
                 GTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTT 
               
             
          
         
       
     
     12.2 Cassette S2 
     The expression cassette of the CMP-sialic acid transporter was produced in tandem with a cassette for resistance to an antibiotic, hygromycin. This double cassette is inserted into the mnn1 gene. Insertion of this cassette into this locus will induce resistance to hygromycin but also the deletion of the mnn1 gene.
     Obtaining the ORF
 
CMP-sialic acid transporter from mus musculus (1,011 bp)
   

     
       
         
               
             
           
               
                 atggctccggcgagagaaaatgtcagtttattcttcaagctgtactgctt 
               
               
                   
               
               
                 ggcggtgatgactctggtggctgccgcttacaccgtagctttaagataca 
               
               
                   
               
               
                 caaggacaacagctgaagaactctacttctcaaccactgccgtgtgtatc 
               
               
                   
               
               
                 acagaagtgataaagttactgataagtgttggcctgttagctaaggaaac 
               
               
                   
               
               
                 tggcagtttgggtagatttaaagcctcattaagtgaaaatgtcttgggga 
               
               
                   
               
               
                 gccccaaggaactggcgaagttgagtgtgccatcactagtgtatgctgtg 
               
               
                   
               
               
                 cagaacaacatggccttcctggctctcagtaatctggatgcagcagtgta 
               
               
                   
               
               
                 ccaggtgacctatcaactgaagatcccctgcactgctttatgtactgttt 
               
               
                   
               
               
                 taatgttaaatcgaacactcagcaaattacagtggatttccgtcttcatg 
               
               
                   
               
               
                 ctgtgtggtggggtcacactcgtacagtggaaaccagcccaagcttcaaa 
               
               
                   
               
               
                 agtcgtggtagcgcagaatccattgttaggctttggtgctatagctattg 
               
               
                   
               
               
                 ctgtattgtgctctggatttgcaggagtttattttgaaaaagtcttaaag 
               
               
                   
               
               
                 agttccgacacttccctttgggtgagaaacattcagatgtatctgtcagg 
               
               
                   
               
               
                 gatcgttgtgacgttagctggtacctacttgtcagatggagctgaaattc 
               
               
                   
               
               
                 aagaaaaaggattcttctatggctacacgtattatgtctggtttgttatc 
               
               
                   
               
               
                 ttccttgctagtgtgggaggcctctacacgtcagtggtggtgaagtatac 
               
               
                   
               
               
                 agacaacatcatgaaaggcttctctgctgccgcagccattgttctttcta 
               
               
                   
               
               
                 ccattgcttcagtcctactgtttggattacagataacactttcatttgca 
               
               
                   
               
               
                 ctgggagctcttcttgtgtgtgtttccatatatctctatgggttacccag 
               
               
                   
               
               
                 acaagatactacatccattcaacaagaagcaacttcaaaagagagaatca 
               
               
                   
               
               
                 ttggtgtgtga 
               
             
          
         
       
         
         Construction of the expression cassette: 
       
    
     The expression cassette of the CMP-sialic acid transporter (promoter CaMV, ORF, terminator CYC1) is assembled with an expression cassette of resistance to hygromycin comprising the promoter of CaMV, the ORF of the resistance to hygromycin, as well as the terminator TEF. These tandem cassettes are inserted into the marker mnn1. 
     12.3 Cassette S3 
     The expression cassette for sialyl transferase ST3GAL4 was produced in tandem with a cassette for resistance to an antibiotic, phleomycin. This double cassette is inserted into the gene mnn9. Insertion of this cassette into the locus will induce resistance to hygromycin but also deletion of the mnn9 gene.
     Obtaining the ORF
 
Human sialyl transferase ST3GAL4 (990 bp)
   

                     atggtcagcaagtcccgctggaagctcctggccatgttggctctggtcct               ggtcgtcatggtgtggtattccatctcccgggaagacagtttttattttc               ccatcccagagaagaaggagccgtgcctccagggtgaggcagagagcaag               gcctctaagctctttggcaactactcccgggatcagcccatcttcctgcg               gcttgaggattatttctgggtcaagacgccatctgcttacgagctgccct               atgggaccaaggggagtgaggatctgctcctccgggtgctagccatcacc               agctcctccatccccaagaacatccagagcctcaggtgccgccgctgtgt               ggtcgtggggaacgggcaccggctgcggaacagctcactgggagatgcca               tcaacaagtacgatgtggtcatcagattgaacaatgccccagtggctggc               tatgagggtgacgtgggctccaagaccaccatgcgtctcttctaccctga               atctgcccacttcgaccccaaagtagaaaacaacccagacacactcctcg               tcctggtagctttcaaggcaatggacttccactggattgagaccatcctg               agtgataagaagcgggtgcgaaagggtttctggaaacagcctcccctcat               ctgggatgtcaatcctaaacagattcggattctcaaccccttcttcatgg               agattgcagctgacaaactgctgagcctgccaatgcaacagccacggaag               attaagcagaagcccaccacgggcctgttggccatcacgctggccctcca               cctctgtgacttggtgcacattgccggctttggctacccagacgcctaca               acaagaagcagaccattcactactatgagcagatcacgctcaagtccatg               gcggggtcaggccataatgtctcccaagaggccctggccattaagcggat               gctggagatgggagctatcaagaacctcacgtccttctga            
Murine sialyl transferase ST3GAL4 (1,002 bp)
 
     
       
         
               
             
           
               
                 atgaccagcaaatctcactggaagctcctggccctggctctggtccttgt 
               
               
                   
               
               
                 tgttgtcatggtgtggtattccatctcccgagaagataggtacattgagt 
               
               
                   
               
               
                 tcttttattttcccatctcagagaagaaagagccatgcttccagggtgag 
               
               
                   
               
               
                 gcagagagacaggcctctaagatttttggcaaccgttctagggaacagcc 
               
               
                   
               
               
                 catctttctgcagcttaaggattatttttgggtaaagacgccatccacct 
               
               
                   
               
               
                 atgagctgccctttgggactaaaggaagtgaagaccttcttctccgggtg 
               
               
                   
               
               
                 ctggccatcactagctattctatacctgagagcataaagagcctcgagtg 
               
               
                   
               
               
                 tcgtcgctgtgttgtggtgggaaatgggcaccggttgcggaacagctcgc 
               
               
                   
               
               
                 tgggcggtgtcatcaacaagtacgacgtggtcatcagattgaacaatgct 
               
               
                   
               
               
                 cctgtggctggctacgagggagatgtgggctccaagaccaccatacgtct 
               
               
                   
               
               
                 cttctatcctgagtcggcccactttgaccctaaaatagaaaacaacccag 
               
               
                   
               
               
                 acacgctcttggtcctggtagctttcaaggcgatggacttccactggatt 
               
               
                   
               
               
                 gagaccatcttgagtgataagaagcgggtgcgaaaaggcttctggaaaca 
               
               
                   
               
               
                 gcctcccctcatctgggatgtcaaccccaaacaggtccggattctaaacc 
               
               
                   
               
               
                 ccttctttatggagattgcagcagacaagctcctgagcctgcccatacaa 
               
               
                   
               
               
                 cagcctcgaaagatcaagcagaagccaaccacgggtctgctagccatcac 
               
               
                   
               
               
                 cttggctctacacctctgcgacttagtgcacattgctggctttggctatc 
               
               
                   
               
               
                 cagatgcctccaacaagaagcagaccatccactactatgaacagatcaca 
               
               
                   
               
               
                 cttaagtctatggcgggatcaggccataatgtctcccaagaggctatcgc 
               
               
                   
               
               
                 catcaagcggatgctagagatgggagctgtcaagaacctcacatacttct 
               
               
                   
               
               
                 ga 
               
             
          
         
       
     
     The expression cassette of the CMP-sialic acid transporter (promoter CaMV, ORF, terminator CYC1) is assembled with an expression cassette of resistance to hygromycin comprising the promoter of CaMV, the ORF of the resistance to hygromycin as well as the terminator TEF. These tandem cassettes are inserted into the marker mnn1. 
     Example 13 
     Production of Homogeneously Glycosylated EPO 
     13.1 Amplification of the Nucleotide Sequence of Human Erythropoietin (EPO) 
     Amplification of the nucleotide sequence of human EPO was obtained from complementary DNA of human kidney with suitable primers. 
     13.2 Cloning of the Sequence of the EPO in an Expression Vector of  S. Cerevisiae    
     The nucleotide sequence of human huEPO truncated of its STOP codon (585 base pairs) is integrated into an expression vector of the yeast  S. cerevisiae . The continuity of the reading frame between the introduced sequence and the sequence of the plasmid pSC (epitope V5 and poly-histidine tag) was confirmed by sequencing the obtained plasmid (pSC-EPO). The expression of the protein EPO is found under the control of the promoter pGAL1, a promoter inducible by galactose for  S. cerevisiae  strains. The selection of the yeasts having the plasmid is performed by return of prototrophy for uracil (presence of the URA3 sequence in the plasmid). 
     Sequence obtained in the expression plasmid: 
                                       (SEQ ID No 11)                  1   ATGGGGGTGC ACGAATGTCC TGCCTGGCTG TGGCTTCTCC TGTCCCTGCT                    51   GTCGCTCCCT CTGGGCCTCC CAGTCCTGGG CGCCCCACCA CGCCTCATCT               101   GTGACAGCCG AGTCCTGGAG AGGTACCTCT TGGAGGCCAA GGAGGCCGAG               151   AATATCACGA CGGGCTGTGC TGAACACTGC AGCTTGAATG AGAATATCAC               201   TGTCCCAGAC ACCAAAGTTA ATTTCTATGC CTGGAAGAGG ATGGAGGTCG               251   GGCAGCAGGC CGTAGAAGTC TGGCAGGGCC TGGCCCTGCT GTCGGAAGCT               301   GTCCTGCGGG GCCAGGCCCT GTTGGTCAAC TCTTCCCAGC CGTGGGAGCC               351   CCTGCAGCTG CATGTGGATA AAGCCGTCAG TGGCCTTCGC AGCCTCACCA               401   CTCTGCTTCG GGCTCTGGGA GCCCAGAAGG AAGCCATCTC CCCTCCAGAT               451   GCAGCCTCAG CTGCTCCGCT CCGAACAATC ACTGCTGACA CTTTCCGCAA               501   ACTCTTCCGA GTCTACTCCA ATTTCCTCCG GGGAAAGCTG AAGCTGTACA               551   CAGGGGAGGC CTGCAGGACA GGCGACAGA A AGGGCGAGCT TCGAGGTCAC                 601     CCATTCGAAG GTAAGCCTAT CCCTAACCCT CTCCTCGGTC TCGATTCTAC                 651     GCGTACCGGT    CATCATCACC ATCACCAT TG A            
Protein sequence of the sequenced EPO in the expression plasmid (SEQ ID No 12)
 
     
       
         
               
             
           
               
                 MGVHEGPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE 
               
               
                   
               
               
                 NITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEA 
               
               
                   
               
               
                 VLRGQALLVNSSQPWEPQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDA 
               
               
                   
               
               
                 ASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRKGELRGHP 
               
               
                   
               
               
                 FEGKPIPNPLLGLDSTRTGHHHHHH* 
               
               
                                    Epitope V5     poly-HIS 
               
             
          
         
       
     
     13.3 Extraction of the RNAs 
     Centrifuge the yeasts 16000 g for 5 min. Remove the supernatant and re-suspend the pellets in 500 μL of TES buffer (10 mM TrisHCl pH7.5 10 mM EDTA, 0.5% SDS). Add 200 μL of phenol and 200 μL of chloroform and then incubate for 20 min at 65° C. by vortexing for 30 s every 5 min and incubate for 1 hr at −80° C. Centrifuge for 20 min at 13,200 rpm and then recover the aqueous phase and add 335 μL of phenol and 67 μL of chloroform. Vortex and centrifuge for 5 min at 11,000 rpm. Recover the aqueous phase and add 300 μL of chloroform. Vortex and centrifuge for 2 min at 13,200 rpm. Recover the aqueous phase and add 30 μL of 3M sodium acetate at pH 5.2 and 600 μL of absolute ethanol. Incubate for 1 hr at −20° C. Centrifuge for 15 min at 13,200 rpm. Remove the supernatant while being careful with the pellet. Leave them to dry, take them up in 100 μL of EDPC water and then place them in a tube containing the violet Nucleospin (Nucleospin RVAII) filtration unit. Centrifuge for 1 min at 11,000 g. Remove the filter and add 350 μL of 70% ethanol. Load the Nucleospin RNAII column. Centrifuge for 30 s at 8000 g. Place the column in a new tube, add 350 μL of Membrane Desalting Buffer. Centrifuge for 1 min at 11,000 g. Deposit 95 μL of DNase solution at the centre of the column, and then incubate for 15 min at room temperature. Add 200 μL of RA2 solution (inactivate the DNase) and centrifuge for 30 s at 8,000 g. Add 600 μL of RA3 solution to the centre of the column. Centrifuge for 30 s at 8,000 g. Place the column in a new tube, add 250 μL of RA3 solution. Centrifuge for 2 min at 11,000 g in order to drive the column. Place the column in a 1.5 nL tube and add 50 μL of DEPC water. Centrifuge for 1 min at 11,000 g. Store the samples at −80° C. 
     13.4 Reverse transcription: Super Script III First-Strand Synthesis System for RT-PCR 
                                                     5 μg of RNA       At most 8 μL           Random hexamer   50 ng/μL   1 μL           dNTP mix   10 mM   1 μL           DEPC water       qsp 10 μL           Incubate for 5 min at 65° C.           Add 10 μL of transcription mix:           RT Buffer   10X   2 μL           MgCl 2     25 mM   4 μL           DTT   0.1M   2 μL           RNase Out   40 U/μL   1 μL           SuperScript   200 U/μL   1 μL           Incubate           For 10 min at 25° C.           For 50 min at 50° C.           For 5 min at 85° C.                        
Recover in ice and add 1 μL of RNaseH. Leave to incubate for 20 min at 37° C. and then store the cDNAs at −20° C.
 
     13.5 Extraction of the Proteins 
     After centrifugation at 1,500 g for 5 min at 4° C., the cell pellet is taken up in 500 μL of sterile H 2 O and then centrifuged at maximum speed for 30 s at 4° C. The pellet is taken up into 500 μL of sodium phosphate 50 mM lysis buffer, pH 7.4, 5% glycerol, 1 mM PMSF, centrifuge for 10 min at 1,500 g at 4° C. The pellet is then taken up in a volume of lysis buffer required for obtaining an OD comprised between 50 and 100. The samples are then vortexed for 4×30 s with glass beads and centrifugation for 10 min at maximum speed is carried out in order to separate the beads and the cell debris from the protein supernatant. A BCA assay is carried out on the supernatant. 
     13.6 Purification of EPO 
     The total proteins are first of all dialyzed against the 10 mM Tris HCl buffer, pH 6.0. After equilibration of a cation exchanger SP Sephadex C50 column with 10 mM Tris-HCl pH 6.0, the total dialyzed proteins are loaded on the column. After rinsing the column with 10 mM Tris HCl buffer pH 6.0, the proteins are eluted with 10 mM Tris HCl buffer pH 6.0, 250 mM NaCl. The absorbance of each fraction is determined at 280 nm as well as the amount of proteins eluted by a Bradford assay. The proteins are then analyzed by SDS-PAGE electrophoresis on 12% acrylamide gel. 
     13.7 Detection of the EPO Protein—Western Blot 
     The total proteins are transferred onto a nitrocellulose membrane in order to proceed with detection by the anti-EPO antibody (R&amp;D Systems). After the transfer, the membrane is saturated with a blocking solution (TBS, 1% blocking solution (Roche)) for 1 hour. The membrane is then put into contact with the anti-EPO antibody solution (dilution 1:500) for 1 hour. After three rinses with 0.1% Tween 20-TBS the membrane is put into contact with the secondary anti-mouse-HRP antibody in order to proceed with detection by chemiluminescence (Roche detection solution). 
     141. Results 
     14.1: Validation of the Clones Having Integrated the Kanamycin Cassette in the Och1 Gene 
     For suppressing the Och1 activity, the introduced cassette was entirely sequenced after its integration in order to map the affected genomic region in the genome of the yeast. Absence of enzymatic activity is then achieved, enhancing the previous results, and then the structure of the glycans is determined by mass spectrometry. 
     The analysis on 1% agarose-TBE gel of the PCR reaction carried out from genomic DNA of  S. cerevisiae  clones having resisted to the presence of kanamycin in the culture medium shows an amplified 2 kb fragment with a specific pair of oligonucleotides of the kanamycin cassette. The size of this fragment corresponds to the theoretical size of the kanamycin cassette. The second fragment was amplified by means of an oligonucleotide internal to the kanamycin cassette and an oligonucleotide external to the cassette hybridizing with the Och1 gene. The theoretical size of the expected fragment is 1.5 kb which corresponds to the size of the obtained fragment. We may therefore conclude that the clones 1, 2, 3 and 4 have actually integrated the kanamycin cassette and that the latter was integrated into the Och1 gene (see  FIG. 3 ). 
     The same type of PCR reaction was carried out on the  S. pombe  strains having resisted to the presence of kanamycin in the culture medium. Thus, two mutated clones of each strain were isolated and tested for loss of α1,6-mannosyl transferase enzymatic activity. 
     Test of Mannosyl Transferase Och1 Activity on Strains of Mutated Yeasts 
     Validation of the loss of Och1 activity in the mutant Δoch1 obtained by homologous recombination in the  S. cerevisiae  and  S. pombe  yeasts: 
     After validation by PCR of the insertion of the expression cassette of kanamycin in wild  S. cerevisiae  and wild  S. pombe  yeasts, the positive clones to this insertion are tested for their loss of mannosyl transferase Och1 activity. The Och1 activity was tested on microsomes of the  S. cerevisiae  and  S. pombe  yeasts.  FIG. 4  shows the Och1 activity test on a—the microsomal fraction of the wild strain and of the selected clones of  S. cerevisiae , b—the microsomal fraction of the wild strain and of selected clones of  S. pombe . According to  FIG. 4 , we may observe a loss of activity of the Och1 enzyme in the selected clones of  S. cerevisiae  (a) and  S. pombe  (b). 
     Validation of the Strains by Analyses of N-Glycans 
     The total proteins from both modified strains were reduced and alkylated and then digested by trypsin. The free polysaccharides are removed by passing over SepPak C18. The recovered peptides and glycopeptides are subject to PNGase. The glycans are purified on SepPak C18 and then methylated before being analyzed by mass spectrometry in the Maldi-Tof mode.  FIG. 5  shows the mass spectrum carried on N-glycans from the strains Adele and Edgar. 
     Nomenclature of the Yeasts:
 
 S. cerevisiae  Δoch1=Adèle
 
 S. pombe  Δoch1=Edgar
 
     Both strains have N-glycans with oligomannoside forms from Man 7  to Man 10 , shorter forms than in the wild strain of  Saccharomyces cerevisiae  indicating the loss of wild polymannosylated forms. 
     The predominant structures for the Edgar strain (Δoch1) are Man 9  and Man 8 , structures which are conventionally encountered in mammals after transit of the neosynthesized protein into the endoplasmic reticulum. This suggests blocking of glycosylation due to the impossibility of action of the Golgian mannosyl transferases which only graft mannose on glycans on which the enzyme Och1 has grafted a mannose attached in the α1,6 position. 
     14.2: Validation of the Clones Having Integrated the Mannosidase I Cassette into the URA3 Gene 
     The Adèle and Edgar yeasts, positive for the insertion of the expression cassette of mannosidase I in the gene URA3, are tested for their mannosidase I biochemical activity.  FIG. 6  shows the assay of mannosidase activity in microsomes of  S. cerevisiae  and  S. pombe  yeasts. The experiment was conducted in triplicate. We may observe in the wild  S. cerevisiae  and  S. pombe  strains, a mannosidase activity non-inhibited by DMJ. Conversely, the selected strains have significant inhibition of mannosidase activity measured during a DMJ treatment. Further, as the measured mannosidase I activity is present in the microsomes of yeasts, we may infer that this enzyme is expressed in the secretion route at the cis-Golgi/endoplasmic reticulum, indicating that the HDEL retention signal integrated in the C-terminus of the protein is well recognized by the cell system. 
     Nomenclature of the Yeasts:
 
 S. cerevisiae  Adèle+mannosidase  I =Amélie
 
 S. pombe  Edgar+mannosidase  I =Emma
 
     14.3 Validation of the Clones Having Integrated the N-Acetylglucosaminyl Transferase (GlcNAc Transferase I) Cassette in the Modified Yeasts 
       FIG. 7  shows the GlcNAcTransferase I activity in microsomes of wild and modified yeasts. 
     In the microsomes or fractions of the Amélie-GlcNacTI and Emma-GlcNacTI yeasts, we observe an increase in the labeling of the acceptor by transfer of a radioactive GlcNAc group compared with the labeling observed in control yeasts (wild and/or Δoch1-MdseI yeasts). This transfer involves the presence of N-acetylglucosaminyl transferase activity in the yeasts modified by expression of GlcNAcTI. 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Amélie+GlcNAcTI=Agathe
 
 S. pombe  Emma+GlcNAcTI=Egée
 
     14.4 Validation of the Clones Having Integrated the Cassette of the UDP-GlcNAc Transporter in the Modified Yeasts 
     The expression of the UDP-GlcNAc transporter was analyzed by RT-PCR on parent or modified yeast cultures. After a reverse transcription step on the total extracted RNAs, the cDNAs were analyzed by PCR by using specific primers of the UDP-GlcNAc transporter (nested PCR). Therefore, an expression of the mRNA of this transporter is observed in the yeasts modified by the expression cassette of the UDP-GlcNAc transporter ( FIG. 8 ). 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Agathe+UDP-GlcNAc transporter=Arielle
 
 S. pombe  Egée+UDP-GlcNAc transporter=Erika
 
     14.5 Validation of Clones Having Integrated the Cassette of Mannosidase II in the Modified Yeasts 
     a—Validation by PCR Amplification 
     The selected clones for  S. cerevisiae  and  S. pombe  were tested by PCR in order to check the presence of expression cassettes of mannosidase II in the genome of the yeasts. 
     b—Expression of Mannosidase II 
     The expression of mannosidase II was analyzed by RT-PCR on parent or modified yeast cultures. After a step of reverse transcription on the extracted RNAs, the cDNAs were analyzed by PCR by using specific mannosidase II primers (nested PCR). In the yeasts modified by the expression cassette of mannosidase II an expression of the mRNA of this protein is therefore observed. 
     c—Measurement of the Activity of Mannosidase II 
     The Adèle and Edgar yeasts, positive for insertion of the expression cassette of mannosidase II, are tested for their mannosidase II biochemical activity. 
     According to  FIG. 9 , we may observe in the parent  S. cerevisiae  and  S. pombe  strains a mannosidase activity insensitive to the inhibitory action of swainsonine. Conversely, the selected strains have significant inhibition of mannosidase activity measured upon treatment with swainsonine. Further as, the measured mannosidase II activity is detected in Golgian yeast fractions, we may infer that this enzyme is properly expressed in the secretion route at the Golgian system. 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Arielle+Mannosidase  II =Anaïs
 
 S. pombe  Erika+Mannosidase  II =Enrique
 
     14.6 Validation of the Clones Having Integrated the N-Acetylglucosaminyl Transferase II Cassette (GlcNAc Transferase II) in Modified Yeasts 
     a—Validation by PCR Amplification 
     The clones selected for  S. cerevisiae  and  S. pombe  were tested by PCR in order to check for the presence of expression cassettes of the GlcNAc transferase II in the genome of the yeasts (results not shown). 
     b—Expression of GlcNAc Transferase II 
     The expression of GlcNAc transferase II was analyzed by RT-PCR on parent or modified yeast cultures. After a step of reverse transcription on the extracted RNAs, the cDNAs were analyzed by PCR by using specific primers of GlcNAc transferase II (nested PCR). An expression of the transcribed mRNA is therefore observed in the yeasts modified by the expression cassette of GlcNAc transferase II (results not shown). 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Anaïs+GlcNAc transferase  II =Alice
 
 S. pombe  Enrique+GlcNAc transferase  II =Elga
 
     14.7 Validation of the Clones Having Integrated the Galactosyl Transferase I Cassette 
     a—Validation by PCR Amplification 
     The clones selected for  S. cerevisiae  and  S. pombe  were tested by PCR in order to check for the presence of expression cassettes of the GalTI in the genome of the yeasts (results not shown). 
     b—Expression of Galactosyl Transferase I 
     The expression of GalTI was analyzed by RT-PCR on parent or modified yeast cultures. After a step of reverse transcription on the extracted RNAs, the cDNAs were analyzed by PCR by using specific primers of GalTI (nested PCR). An expression of the transcribed mRNA is therefore observed in the yeasts modified by the expression cassette of GalTI (results not shown). 
     c—Activity of the GalTI 
     After extraction of the total proteins of the modified yeasts, 2 μg of proteins are deposited on a nitrocellulose membrane. The membrane is then incubated with  erythrina cristagalli  lectin coupled with biotin, a lectin specifically recognizing the galactose of the Gal-β-1,4-GlcNAc unit present on glycans of glycoproteins. The membrane is put into contact with streptavidin coupled to horse radish peroxidase (HRP) in order to proceed with detection by chemiluminescence (Roche detection solution). 
     14.8 Validation of the Clones Having Integrated the Cassette of the GDP-Fucose Transporter 
     a—Validation by PCR Amplification 
     The clones selected for  S. cerevisiae  and  S. pombe  were tested by PCR in order to check for the presence of expression cassettes of the GDP-fucose transporter in the genome of the yeasts. 
     b—Expression of GDP-Fucose Transporter 
     The expression of GDP-fucose transporter was analyzed by RT-PCR on parent modified yeast cultures. After a step of reverse transcription on the extracted RNAs, the cDNAs were analyzed by PCR by using specific primers of the GDP-fucose transporter (nested PCR). An expression of the transcribed mRNA is therefore observed in the yeasts modified by the expression cassette of GDP-fucose transporter ( FIG. 10 ). 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Anaïs+GDP-fucose transporter=Apolline
 
 S. pombe  Enrique+GDP-fucose transporter=Epiphanie
 
     14.9 Validation of the Clones Having Integrated the Cassette of Fucosyl Transferase 8 (FUT8) 
     a—Validation by PCR Amplification 
     The clones selected for  S. cerevisiae  and  S. pombe  were tested by PCR in order to check for the presence of expression cassettes of FUT8 in the genome of the yeasts. 
     b—Expression of the FUT8 
     The expression of the FUT8 was analyzed by RT-PCR on parent or modified yeast cultures. After a step of reverse transcription on the extracted RNAs, the cDNAs were analyzed by PCR by using specific primers of FUT8 (nested PCR). An expression of the transcribed mRNA is therefore observed in the yeasts modified by the expression cassette of FUT8 (results not shown). 
     Nomenclature of the Modified Yeasts:
 
 S. cerevisiae  Apolline+FUT8=Ashley
 
 S. pombe  Epiphanie+FUT8=Esther
 
     14.10 Particular Case of EPO Expression in the Amélie Strain 
     The Amélie strain has the capability of exclusively producing the N-glycan Man 5 GlcNAc 2  ( FIG. 11 ), a structure encountered in mammals, described as a glycan of a simple type; and being used as a basis for elaborating more complex glycans bearing galactose, fucose or sialic acid. The presence of each genomic modification in this strain is described above. Each of these steps enters a “package” of verifications consisting of selecting the best producing clone and of maximizing the percentage of chances in order to obtain an exploitable clone. The method used allows a complete control of the genetic modification procedure: the sequence to be integrated is perfectly known, just like the target genomic region of the future integration. The latter site is moreover subject to extensive research as to the effects of possible breakage, this is why the whole of the targets is finally selected for the absence of phenotype effects obtained after their breakage. 
     An entire procedure for tracking the genomic stability of the producing clones is performed: after each production: regular planting out of the clones on the drastic media initially used for their selection, and starting out again the validation procedure. All the expression cassettes are cloned so that in the case of genomic rearrangement of a given strain, it may be proceeded with genetic upgrade of the organism. The procedures for integrating cassettes are not standardized and it is possible to imagine production of the strains “on demand” in order to achieve specific glycosylation, as ordered by the user. 
     The Amélie strain is the clone which should be used as a basis for elaborating any other strain intended for producing humanized hybrid or complex glycans. 
     The plasmid used for the expression of EPO in the modified yeasts contains the promoter Gal1. This promoter is one of the strongest promoters known in  S. cerevisiae  and is currently used for producing recombinant proteins. This promoter is induced by galactose and repressed by glucose. Indeed, in a culture of  S. cerevisiae  yeasts in glycerol, addition of galactose allows induction of the GAL genes by about 1,000 times. If glucose is added to this culture in the presence of galactose, the GAL genes will no longer be induced, only to 1% of the level obtained with galactose alone (Johnston, M. (1987) Microbiol. Rev.). The integrated sequence of human EPO in our plasmid was modified in 5′ by adding an epitope V5 as well as a polyhistidine tag in order to facilitate detection and purification of the produced protein. 
     The yeasts used for producing human EPO are first of all cultivated in a uracil drop out YNB medium, 2% glucose until an OD&gt;12 is reached. After 24-48 hours of culture, 2% galactose is added to the culture in order to induce the production of our protein of interest. Samples are taken after 0, 6, 24 and 48 hours of induction. 
     Expression of the mRNA of EPO in Modified Yeasts 
     RT-PCR analysis of the total extracted RNAs shows expression of the messenger RNA or EPO in the clones of yeasts transformed after induction by galactose ( FIG. 12  bands 1 and 3) unlike what is observed in yeasts modified without induction by galactose ( FIG. 12  bands 2 and 4). The presence of galactose therefore causes induction of the transcription of the EPO gene. The sequencing of this amplified fragment confirms the production of a proper mRNA. 
     Purification of the EPO Protein Expressed in the Modified Yeasts 
     The total proteins obtained after induction of the expression of the rhuEPO protein by galactose are then deposited on a Sephadex C50 resin equilibrated to pH 6. Absorbance at 280 nm is determined at the column outlet ( FIG. 13 ). The proteins eluted from the column are analyzed by SDS-PAGE electrophoresis on 12% acrylamide gel. 
     After migration of the SDS-PAGE gel, analysis of the proteins is accomplished either by staining with Coomassie blue ( FIG. 14 ) or by western blot. In this case, the proteins are transferred on a nitrocellulose membrane in order to proceed with detection by the anti-EPO antibody (R&amp;D Systems). 
       FIG. 15  shows the presence of a protein at about 35 kDa. This protein is the majority protein in Coomassie staining and is revealed by an anti-EPO antibody in a western blot analysis (tube 29 at the column outlet). 
     All these results therefore show production of EPO protein by genetically modified yeasts.