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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to the field of cellular engineering, in particular eukaryotic cells. More particularly, the invention relates to eukaryotic cells having improved growth and / or metabolic properties, as well as their uses for the production of compounds of interest. In particular, the invention relates to eukaryotic cells expressing a specific combination of chaperones. The invention finds applications in particular in the field of the production of recombinant proteins.
• the present invention provides eukaryotic cells with improved performance, allowing the development of optimized expression systems.
• the inventors surprisingly observed that coexpression in yeast of a set of three bacterial chaperones (RbcX, GroES and GroEL) conferred eukaryotic cells with remarkable metabolic properties, especially in terms of expression and functional folding of heterologous or endogenous proteins. Pursuing their investigations, the inventors have also demonstrated that this combination of chaperones also allowed to accelerate the growth of yeasts and remove the toxic effects of other engineering, for example when PRK kinase is co-expressed in the cell. The inventors have moreover verified and obtained the same effects on the performances on various eukaryotic cells, confirming the interest and the great potential of these unexpected results.
• An object of the present invention therefore relates to a eukaryotic cell, characterized in that it expresses RbcX, GroES and GroEL chaperones.
• the present invention relates to a transformed eukaryotic cell, characterized in that it contains:
• the invention also relates to a eukaryotic cell containing:
• the eukaryotic cells of the invention do not contain a coding sequence for the RbcL and / or RbcS subunit of a RuBisCO enzyme of form I of bacterial origin.
• the invention notably proposes a yeast transformed as indicated above.
• the subject of the invention is also the use of a combination of expression cassettes allowing the expression of an RbcX chaperone and GroES and GroEL chaperones, to improve the physiology of a eukaryotic cell, and in particular to increase the growth rate of said eukaryotic cell and / or increase the resistance of said eukaryotic cell to environmental stress and / or increase the resistance of said cell to the toxicity of a compound synthesized by the eukaryotic cell and / or for the production of a recombinant protein.
• the subject of the invention is also a biotechnological process for producing at least one compound selected from chemical molecules and proteins, characterized in that it comprises a step of culturing a eukaryotic cell according to the invention under conditions allowing the synthesis, by said eukaryotic cell, of this compound, and a step of recovering said compound.
• the invention more particularly proposes a method for producing a recombinant protein comprising (i) inserting a sequence encoding said protein into a eukaryotic cell expressing RbcX, GroES and GroEL, (ii) culturing said cell in conditions permitting the expression of said sequence and optionally (iii) the recovery or purification of said protein.
• Figure 1 Kinetics of ethanol production strains lb, 18b, 102, 15, 14b.
• the error bar represents a standard deviation of 3 independent cultures.
• bacterial chaperones can be expressed in the cytosol of a eukaryotic cell and retain their chaperone function in said eukaryotic cell.
• the bacterial chaperone function is added to the cytosolic chaperone function already present in the eukaryotic cell.
• the inventors have furthermore discovered that the expression of a particular triplet of chaperones, namely the bacterial chaperones GroEL and GroES and the RbcX chaperone, confers on the transformed eukaryotic cell which expresses them properties of particular interest in terms of growth, d expression and functional folding of proteins.
• the observed effects require obligatorily the simultaneous presence of the three chaperones mentioned, preferably coming from at least two different microorganisms. If it was known that coexpression of chaperones is likely to improve in a bacterial system the folding of heterologous proteins and therefore potentially the performance of a bioprocess dependent, it was not foreseeable that a specific interorganism combination of three bacterial chaperone proteins, can be particularly effective in being expressed in eukaryotic context. The molecular mechanisms leading to the effects of the invention are currently unknown even if they are probably, at least in part, related to protein folding or control of their intracellular fate.
• a surprising generalist role is played by the protein of the RbcX family (described in the prior art as specialized for the functional association of the RuBisCO complex of photosynthetic organisms), in the presence of GroES and GroEL chaperones which play complementary roles.
• This feature of the invention suggests that the observed effect can not be fully attributed to a facilitated folding role and may involve other mechanisms such as modulation of the lifetime of specific proteins, assembly of complexes or modification of their properties, or even specific effects of a nature to be defined.
• the invention therefore proposes to transform eukaryotic cells so that they express a particular triplet of chaperones, namely the bacterial chaperones GroEL and GroES and chaperone RbcX.
• Such transformed cells may find many applications, especially in the context of the production of recombinant proteins.
• the subject of the invention is therefore a eukaryotic cell, characterized in that it expresses RbcX, GroES and GroEL chaperones.
• the GroEL and GroES chaperones belong to the family of heat shock proteins (HSP). These chaperones are present in many bacteria.
• these chaperones are referred to as "general chaperone" in the sense that they are known to co-act to permit efficient folding of a very large number of proteins (M. mayhew et al. 1996, "Protein folding in the central cavity of the GroEL-GwES chapewnin complex” Nature 1996 Feb 1; 379 (6564): 420-6).
• the GroEL and GroES chaperones can come from any bacterium that expresses them, and, for example, E. coli (Gene ID: 948655 and 948665), S. elongatus (Gene ID: 3199735). , 199535 and 198035), S. pneumonia (GenBank accession number: AF 1 17741), S. pyogenes (GenBank accession number: SPGROELGN), S. aureus (GenBank accession number: STAHSP) or P. aeruginosa (GenBank accession number ATCC9027).
• E. coli Gene ID: 948655 and 948665
• S. elongatus Gene ID: 3199735). , 199535 and 198035
• S. pneumonia GenBank accession number: AF 1 17741
• S. pyogenes GenBank accession number: SPGROELGN
• S. aureus GenBank accession number: STAHSP
• P. aeruginosa GenBank
• sequence similarity of the chaperones is 61% between GroEL1 of S. elongatus and GroEL of E. coli; 56% between GroEL2 of S. elongatus and GroEL of E. coli; 63% between GroELl and GroEL2 of S. elongatus.
• the cells according to the invention also express the known RbcX chaperone in cyanobacteria and plants to participate in the good assembly of the RbcL and RbcS subunits of Rubisco.
• this chaperone is designated as "chaperone specific” in the sense that this protein is known to play a role in the functional association of protein complexes, as is particularly the case with Rubisco (S Saschenbrecker et al., 2007, “Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco," Cell 2007 Jun 15; 129 (6): 1189-200).
• the RbcX chaperone can come from any cyanobacterium expressing it, and, for example, from S. elongatus (SEQ ID No. 3), Synechocystis sp. (Kaneko et al., "Sequence analysis of the genome of the unicellular cyanobacterium synechocystis sp. Strain PCC6803.) Sequence determination of the resulting genome and assignment of potential protein-coding regions.” DNA Res.3 (3), 109 - 36 (1996)), Anabaena sp. (Li et al., J.
• a “chaperone activity” refers to a protein folding action and / or the functional association of protein complexes.
• the chaperones used come preferentially from two different organisms.
• the chaperones can come from one or more different bacteria.
• the three chaperones originate from at least two distant Gram negative bacterial species, at least one of which is a cyanobacterium.
• At least one of the general-interest chaperones GroES and GroEL does not come from a cyanobacterium or from another bacterium expressing an RuBisCO complex.
• the GroES and GroEL chaperones can come from the same bacterium or from two different bacteria. In a particular embodiment, the GroES and GroEL chaperones come from E. coll.
• the GroES and GroEL chaperones come from E. Coli and the chaperone RbcX comes from Synechococcus elongatus.
• the three chaperones GroES, GroEL and RbcX come from Synechococcus elongatus.
• the transformed cell can express either one or the two isoforms (GroEL1 and GroEL2) of the GroEL chaperone, preferably both isoforms.
• a protein "comes” from a given organism as soon as it has an amino acid sequence identity greater than 95% and the same function as the relevant protein of said organism.
• "GroES” and “GroEL” denote any protein exhibiting chaperone activity and having between 65 and 100% amino acid identity with GroES and GroEL of E. coli K 12, respectively.
• "GroES” and “GroEL” denote general chaperones with a lower percentage of identity, and in particular between 55 and 65%, and more particularly between 56 and 63%, such as general chaperones of S. elongatus.
• coli can be verified for example by substituting in the various examples described below, the expression cassette coding for GroES or GroEL native to E. coli by chaperone variants to be evaluated.
• the RbcX chaperone is very far from GroEL and GroES and its sequence can not be aligned with the sequences of these two chaperones.
• RbcX denotes any protein, in particular of cyanobacteria, having a chaperone activity and having more than 50% amino acid sequence identity with the RbcX chaperone encoded by the sequence SEQ ID No: 3 and retaining the chaperone activity specific for this protein.
• the specific chaperone activity can be verified in a yeast expressing the RbcL and RbcS subunits of the RuBisCO of S. elongatus, replacing the expression cassette including the sequence SEQ ID No. 3 by any other sequence to be evaluated, and in measuring by an in vitro test on cell extracts the RuBisCO activity thus obtained.
• the present invention is implemented with a chaperone RbcX whose amino acid sequence identity with chaperone RbcX encoded by SEQ ID No: 3 is greater than 80%, preferably greater than 90%, more preferably greater than at 95%, still more preferably above 99%.
• the invention can be implemented with any type of eukaryotic cell, originating from a unicellular or multicellular organism.
• the combination of chaperones according to the invention can be expressed in a yeast cell, fungus, plant, animal such as a mammal, etc.
• the present invention relates to a transformed yeast expressing a specific chaperone RbcX, a general bacterial chaperone GroES, and a generalized bacterial chaperone GroEL.
• the present invention relates to a transformed yeast, characterized in that it contains:
• the invention can be implemented with any yeast of interest.
• the yeast is chosen from Saccharomyces, Yarrowia and Pichia.
• the yeast transformed according to the invention is a Saccharomyces cerevisiae cell.
• the yeast transformed according to the invention is a Yarrowia lipolytica cell, or a Pichia pastoris cell.
• Pichia pastoris is particularly interesting for the production of recombinant proteins.
• Pichia has a eukaryotic protein expression system that is very efficient both for secretion and for intracellular expression. It is particularly suitable for large scale production of recombinant eukaryotic proteins.
• Pichia can be used for the production of high-yielding excreted proteins in order to reduce costs and production times compared to those associated with mammalian cell expression systems.
• Yarrowia lipolytica is also suitable for use in the production of recombinant proteins.
• Yarrowia has (i) high density growth, (ii) high secretion, (iii) lack of alkaline protease EPA and (iv) ability to produce S. cerevisiae invertase allows the use of sucrose as a carbon source (Nicaudet al., 1989). This last property is particularly interesting in the case of industrial production, because this strain can grow efficiently on inexpensive substrates such as molasses.
• the invention also relates to a eukaryotic cell derived from a multicellular organism, such as an animal cell and in particular a transformed mammalian cell expressing a specific RbcX chaperone, a generalized bacterial chaperone GroES, and a generalized bacterial chaperone GroEL.
• such a eukaryotic cell is transformed to contain:
• the genes encoding the GroEL, GroES and RbcX chaperones are introduced into eukaryotic cells in a form allowing their expression in said cells.
• the sequences coding for chaperones are associated with promoter sequences allowing their transcription.
• the same promoter sequence is associated with the coding sequences for the three chaperones.
• the RbcX chaperone is associated with a particular promoter different from the promoter (s) associated with the GroEL and GroES chaperones.
• each chaperone is associated with a different particular promoter.
• Promoters useful in the context of the present invention include constitutive promoters, i.e., promoters that are active in most cellular states and environmental conditions, as well as inducible promoters that are activated or repressed by exogenous physical or chemical stimuli. thus inducing a variable level of expression depending on the presence or absence of these stimuli.
• constitutive promoters are those of the genes TEF1, TDH3, PGI1, PGK, ADH1.
• Examples of inducible promoters are the tetO-2, GAL10, GAL10-CYC1, PHO5 promoters.
• the promoters used will be different from one cassette to another.
• Expression cassettes of the invention further include the usual sequences such as transcriptional terminators, and optionally other transcriptional regulatory elements.
• the expression cassettes according to the invention may be inserted into the chromosomal DNA of the host cell, and / or carried by one or more extra-chromosomal replicon (s).
• s extra-chromosomal replicon
• the relative stoichiometry of these proteins is likely to play an important role in the optimal implementation of the present invention.
• the coexpression systems described in the experimental section below are particularly relevant in this regard. The invention is however not limited to the use of these systems, and it can be implemented with any expression variant of the elements mentioned having effects at least equivalent, as they can be measured, for example, by measuring the growth of a cell transformed in a standard medium for said cell.
• the three expression cassettes form a continuous block of genetic information. It may also be advantageous for the expression cassettes of the three chaperones to be carried by a single episomal genetic element.
• a particularly interesting aspect of the present invention is therefore a unique "genetic plug-in" (continuous sequence of DNA) carried by an episomal element at the origin of centromeric replication. The transformation by this element is sufficient to introduce the properties of interest in wild yeasts or any engineering.
• the genes coding for each chaperone can be introduced in one or more copies into the cell.
• a cassette may contain several copies of a Sequence encoding GroES, GroEL or RbcX.
• the same sequence is used each time, that is to say from the same bacterium. It is otherwise possible to use sequences from different bacteria.
• the cells according to the present invention have improved properties (growth rate, resistance, production capacity, etc.). These cells are therefore particularly useful for the production of proteins or other compounds, or for the improvement of fermentation processes. Protein production
• the invention also relates to a eukaryotic cell transformed to express a combination of chaperones as described above and which furthermore comprises at least one expression cassette for a heterologous protein other than said chaperones, and / or which has has undergone sequence engineering modifying the level of expression and / or the sequence of an endogenous protein.
• the transformed eukaryotic cell is a yeast.
• the transformed eukaryotic cell is a CHO cell.
• the invention therefore also relates to a yeast or a CHO cell transformed to express a combination of chaperones as described above, and containing:
• the protein of interest is not Rubisco.
• the protein of interest is a protein other than PKR.
• the transformed eukaryotic cell expresses Rubisco and / or PKR, it advantageously expresses at least one other heterologous protein.
• the transformed cell expressing the chaplet triplet GroES, GroEL and RbcX and further modified to express and excrete a recombinant protein.
• the present invention also relates to a biotechnological process for producing at least one compound selected from chemical molecules, enzymes, hormones, antibodies and proteins, characterized in that it comprises a step of culturing a transformed cell. as described above, under conditions allowing the synthesis by said cell of this compound, and optionally a step of recovering and / or purifying said compound.
• the invention also relates to a method for producing a recombinant protein comprising (i) inserting a sequence encoding said protein into a eukaryotic cell expressing the RbcX, GroES and GroEL chaplet triplet, (ii) culturing said cell under conditions permitting the expression of said sequence and optionally (iii) the recovering and / or purifying said protein.
• said protein is an enzyme or a hormone.
• the cell according to the invention is transformed so as to produce at least one heterologous enzyme.
• the cell is transformed to produce an enzyme selected from endotoxins, such as Bacillus thuringiensis endotoxin, lipases, subtilisins, cellulases and luciferases.
• the cell according to the invention is transformed so as to produce at least one molecule of medical interest.
• the cell is transformed to produce a hormone, a growth factor, an antibody, etc.
• the molecule of medical interest is chosen from erythropoietin, type I and / or II alpha-interferons, granulocyte colony stimulating factors, insulin, growth hormones and tissue plasminogen activators. .
• the cell transformed according to the invention has an improved production of the protein or proteins of interest, compared to a recombinant cell that does not express the combination of chaperone of the invention.
• "improved" production is understood in terms of quantity and / or quality.
• the transformed cell according to the invention can produce more active and / or more stable proteins of interest, and therefore less likely to be degraded, which allows a greater accumulation of said proteins, compared to the heterologous proteins produced by a protein. recombinant cell not expressing the combination of chaperone of the invention.
• the increase in the level of expression of a recombinant protein by a eukaryotic cell transformed according to the invention is explained in particular by a greater stability and therefore a greater accumulation of said proteins in the cell and / or the medium of the invention. culture.
• the expression of the combination of chaperones by the transformed cell can advantageously increase the resistance of the recombinant cell to the recombinant proteins it expresses, thereby also contributing to the increase in production yield.
• the present invention also relates to the use of a combination of expression cassettes for the expression, in a eukaryotic cell, of the specific chaperone RbcX and the general chaperones GroES and GroEL, for improving physiology and / or performance (especially the growth rate) of said eukaryotic cell.
• the eukaryotic cell whose physiology is to be improved is a yeast.
• said eukaryotic cell has not been transformed to express a sequence coding for the RbcL subunit of a RuBisCO enzyme of form I of bacterial origin, and / or a coding sequence for the sub-unit - RbcS unit of said RuBisCO enzyme.
• a combination of expression cassettes allowing the expression of the GroES and GroEL general chaperones and the RbcX-specific chaperone is particularly useful for improving the physiology of a eukaryotic cell that has undergone sequence-altering engineering.
• the concomitant expression, in a cell, of the general-interest chaperones GroES and GroEL and of the specific chaperone RbcX makes it possible to increase the growth rate of said cell and / or to increase the resistance of said cell to an environmental stress. , in particular to stress due to the toxicity of an element present in the culture medium of the cell.
• Another advantageous application is to increase the resistance of the cell to the toxicity of a compound synthesized by itself, and thus the production of a compound of interest.
• the advantage is mainly an improvement in the result of their actions at the level of the production of a product of interest (chemical molecule).
• This effect can result from the improvement of the folding or the stability but also other phenomena for example subcellular transport, facilitated or modified formation of complexes, modulation of coupling mechanisms, etc. ;
• toxic intermediates for example drugs (hydrocortisone, artemisinic acid or even trictosinide, certain flavonoids for take developed processes) or reactive molecules such as unsaturated ketones, aldehydes (eg vanillin), etc.
• Other examples are processes producing molecules that become toxic at high concentrations, for example ethanol or other alcohols. Ethanol production for example is limited by the tolerance of yeast strains to high concentrations of alcohol.
• Other examples relate to the production of highly varied industrial chemical molecules, precursors of common products or chemical intermediates and of course biofuels.
• the use of a eukaryotic cell expressing the combination of chaperones according to the invention advantageously makes it possible to increase the resistance of the transformed cell to the toxicity induced by the recombinant proteins it expresses.
• the present invention also relates to a nucleic acid molecule comprising:
• Example 1 Materials and methods - Construction of the "CHAPERONNES plug-in” and vectors - Constructions of the different strains - Methods of culture and measurement
• pGAL10-CYCl synthetic promoter composed of the UAS of the GAL10 gene and the transcription initiation of the CYCl gene (POMPON et al, Methods Enzymol, 272, 51-64, 1996).
• the expression cassettes thus obtained are listed in Table II below.
• CEN.PK 1605 is the prototrophic version of strain 1605. It is therefore a positive control of "physiological behavior".
• Each number, compiled in the first column of Table IV below, corresponds to the association of vectors described on the corresponding line of the same table. Thus for each of the strains used, the associated reference number provides the information of the reconstructed engineering. Only CEN.PK 1605 strains have been exemplified (Table IV) for the sake of clarity, but the nomenclature is the same for the other two strains. ⁇ "
• pCM 185 Commercial Plasmid (ATCC 87659)
• PYeDP51 Plasmid "empty”, described in the following article: Urban P, C Mignote, Kazmaier M, Delorme F, Pompon D. Cloning, yeast expression, and char act of the coupling of distantly related Arabidopsis thaliana NADPH -cytochrome P450 reductases with P450 CYP73A5. J Biol Chem. 1997 Aug 1; 272 (31): 19176-86.
• Synthetic genes Synechoccocus elongatus genes encoding the RuBisCO subunits, the RuBisCO assembly specific chaperone (RbcX), the PRK and the GroES, GroEL1 and GroEL2 general chaperones were synthesized after proprietary recoding for yeast using an inhomogeneous and cloned codon bias in pCC6301 (commercial).
• the coding sequences of these proteins are presented in the appendix (SEQ ID No: 1: coding sequence of RbcS, SEQ ID No: 2: coding sequence of RbcL, SEQ ID No: 3: coding sequence of RbcX, SEQ ID No: 4: PRK coding sequence, SEQ ID No: 5: GroES coding sequence, SEQ ID No: 6: GroEL1 coding sequence, SEQ ID No: 7: GroEL2 coding sequence).
• the coding sequences of the E. coli GroES and GroEL chaperones were amplified from the bacterium, cloned into pSC-B-amp / kan (Statagene) and mounted without recoding into the expression vectors (see above). .
• the region including the three above expression cassettes (TableXI) was amplified by PCR and cloned into the commercial plasmid pYLEXl.
• plasmids were linearized and transformed individually into a Yarrowia lipolytica strain auxotrophic for leucine according to the Yeast Transcription Kit Yeastearn Biotech protocol and selected on minimal medium and glucose at 28 ° C. (YLEX Expression Kit Yeastearn Biotech (Cat #: FYY201-1KT)).
• Yarrowia lipolytica POlf (ATCC®MYA2613 TM)
• Genotype MATA ura3-302 leu2-270 xpr2-322 axp2-deltaNU49 XPR2 :: SUC2 Evaluation of the impact of chaperones was performed on a culture of strains POlf_01 and POlf_02 in synthetic medium without leucine at 28 ° C for 72H . 1.6. CHO cell construction
• lentiviral transduction system To ensure easy, versatile handling and a successful transfer of the "Chaperones" plug-in to all higher eukaryotic cells, a fourth-generation lentiviral transduction system was chosen. These lentiviral particles allow to transfer indifferently plug-in immortalized or transformed primary cells from different species of higher eukaryotes such as human or murine cells for example.
• the region including the RbcX open reading phase and the two CAS55 and CAS56 expression cassettes below were PCR amplified and XhoI-KpnI cloned into the pLVX-Puro commercial plasmid from Clontech (Catalog No. 632164).
• Table XV expression vector
• the plasmids pLVX-Puro or pCB 10 were transformed using the Lenti-X packaging system (Clontech) in Lenti-X 293T cells (Clontech) according to the kit protocol.
• the supernatant containing the viral particles was filtered and added diluted to 1 / 5th or 1/2 on CHO cells in culture in 10cm petri dishes for a final volume of 5mL medium.
• the cells are washed with PBS and fresh culture medium supplemented with 2 ⁇ g / ml of puromycin is added for selection over 48 hours at 37 ° C.
• the cell line thus established is maintained at a concentration of 0 ⁇ g / ml of puromycin in the culture medium.
• the transformed cells are cultured at 30 ° C. in ambient air on YNB medium (yeast without nitrogen base) supplemented with ammonium sulfate 6.7 gL -1 , glucose 20 gL -1 , agar 20 gL -1 for the agar plates. complemented with a commercial medium CSM (MP Biomedicals) adapted to the selection markers of the plasmids used for the transformation (-ura, -leu, -trp) and in the presence of doxycycline 2 ⁇ / ⁇ 1 The cultures are stopped by cooling to 4 ° C.
• YNB medium yeast without nitrogen base
• ammonium sulfate 6.7 gL -1
• glucose 20 gL -1 glucose 20 gL -1
• agar 20 gL -1 for the agar plates.
• CSM commercial medium CSM (MP Biomedicals) adapted to the selection markers of the plasmids used for the transformation (-ura, -leu, -
• spheroplasts are prepared by enzymatic digestion of the cell walls with a zymolyase-cytohelicase mixture in hypernic sorbitol medium (1.2M sorbitol) .
• the spheroplases are washed in medium hypertonic sorbitol in the presence of saturating concentrations of PMSF and EDTA (protease inhibitors), then broken by repeated pipetting and light sonication in isotonic medium (0.6M) sorbitol.
• low-speed centrifugation (1500 rpm) to remove large debris and then at medium speed (4000rpm) to recover intermediate size debris and mitochondria, the supernatant is recovered and the protein concentration is quantified by the Bradford method.
• the precultures were carried out on a chemically defined medium. After thawing, 1 mL of a stock tube (-80 ° C) was taken to inoculate a penicillin flask (100 mL) containing 10 mL of culture medium, incubated 18 hours at 30 ° C and 120 rpm. The precultures were carried out anaerobically (flasks previously flushed with nitrogen) and in the presence of doxycycline in order to avoid the toxicity problems observed in the presence of the PRK gene.
• the precultures were then washed three times (centrifugation, resuspension, vortex 15 s) with physiological water (NaCl, 9 g L "1 ), then the cell pellet was resuspended in culture medium without doxycycline.
• the starting volume of the cultures was 50 ml aerobically (flasks of Erlenmeyer flasks 250 mL) or anaerobic 35 mL (penicillin flasks of 100 mL).
• Glucose, formic acid and major metabolites were measured by high performance liquid chromatography (HPLC).
• HPLC high performance liquid chromatography
• the apparatus used was a chromatograph (Waters, Alliance 2690) equipped with an Aminex HPX 87-H + column (300 mm x 7.8 mm). Detection of the molecules was provided by a refractive index detector (Waters 2414 refractometer).
• the eluent was 4 to 8 mM H 2 SO 4 at a flow rate of 0.5 mL min -1 , and the column temperature was set at 50 ° C. In anaerobiosis, this analysis was performed on a single flask. In this case, the calculation of the standard deviation was performed on the loss of mass and then applied to the metabolites. .
• the Calvin cycle allows plants and cyanobacteria to make glucose from carbon dioxide.
• the critical step is the fixation of CO2 on ribulose-1,5-bisphosphate (RuBP), a molecule with 5 carbons.
• RuBP ribulose-1,5-bisphosphate
• This step requires an enzyme called RuBisCO (for Ribulose-1, 5-Biphosphate Carboxylase / Oxygenase).
• RuBisCO for Ribulose-1, 5-Biphosphate Carboxylase / Oxygenase.
• This enzyme allows the formation of an unstable molecule with 6 carbons which rapidly gives two molecules of 3-phosphoglycerate with 3 carbons.
• RuBisCO There are several forms of RuBisCO. Form I consists of two types of subunits: large subunits (RbcL) and small subunits (RbcS), whose correct assembly also requires the intervention of at least one specific chaperone: RbcX.
• RuBP substrate of RuBisCO, is
• an artificial Calvin cycle is reconstituted by the co-transformation of the yeast strain CEN.PK 1605 by the combinations of vectors No. 3 and 4 of Table IV above, which allow the simultaneous expression of the RbcS and RbcL units of RuBisCO, RbcX-specific chaperone and Synechoccocus elongatus PRK enzyme, with (combination 3 and 101) or without (combination 4) the general Groel and GroES chaperones of E. coli or synechoccocus according to the number of the combination.
• RuBisCO activity tests on three independent experiments A, B and C are made from protein extracts derived from yeast culture on glucose (protocol detailed above, point 1.3); their yield is evaluated by measuring the phosphoglyceric acid production as a function of time. The results are shown in Table XVII below.
• Table XVII In vitro activity tests of RuBisCO made from extracts of CEN-PK strains grown on glucose and containing the engineering indicated in the first column. The tests are carried out for 80 min of incubation with 0.01-0.02 mg of yeast soluble extract protein in a reaction volume of 200 microL containing 2 mM ribulose diphosphate at room temperature. The activities are given in nmoles of 3-phosphoglycerate formed / min / mg of total protein in the extract.
• experiment C shows that the RuBisCO activity drops dramatically by more than 90% when the RbcL / RbcS subunits of Synechoccocus elongatus are associated with homologous RbcX, GroES GroEL2 chaperones from the same organism. This illustrates in a striking way the interest of an association of heterologous chaperonines.
• Example 3 Protective effect of the combination of chaperones against the toxicity of recombinant proteins
• Example 1 The methods and analyzes used are described in Example 1 above.
• the expression of ribulokinase alone in yeast (strain 18b) results in a long latency phase (of more than 50 hours) and a drastic fall in its maximum growth rate (from 70% aerobically and 82% anaerobically) relative to the wild-type strain (WT) (Table XVIII).
• Table XVIII Genotype, maximum growth rate and growth retardation of strains WT, lb, 18b, 102, 15, 14b.
• This PRK-induced toxicity can be partially overcome by coexpression in strain 102 of GroES / GroEL chaperones of E. coli. coli (removal of 26% anaerobiosis and 42% aerobic aerobic toxicity) or RbcX chaperone from Synechococcus elongatus in strain 14b (release of 34% anaerobiosis 10% aerobic).
• Example 4 Exemplification of the general effect on the growth of a transformed cell
• Example 1 The methods and analyzes used are described in Example 1 above. The results are shown in Table XIX below.
• Table XIX Genotype, maximum growth rate and growth retardation of strains WT, lb, 103, 13b.
• strain 13b is equal to 96 and 86% of the growth rate of the wild-type (WT) strain CEN.PK 113-7D which is not transformed and therefore not stressed, aerobically and anaerobically respectively (Table XVIII). ).
• the two strains were seeded at the same cell concentration evaluated on a 100H fermentation, the maximum mu of the strain calculated on the exponential phase of the growth curve for the strain PPGC115_02 a proliferative advantage of the order of 30% by compared to that of the control strain PPGC115_01.
• the CHO-01 and CHO-02 lines are seeded at the same density (ie 2.10 6 cells per 10 cm dish and the growth is evaluated over 4 days)
• the cells are detached and individualized by treatment trypsin and counted daily using a meter automatic.
• the growth rate of the cells of the CHO-02 line is higher than that of the CHO-01 control line of the order of 25%.
• the combination of chaperones has an effect on the cell doubling time.
• Example 5 Exemplification of the Effect on the Production of Recombinant Proteins 5.1. Production of human growth hormone in Saccharomyces cerevisiae
• the human growth hormone gene (GenBank: K02382.1) was synthesized and cloned downstream of the constitutive promoter TEF1 according to the cassette below.
• the strains 200, 230 and 231 were cultured in a synthetic medium (-leu-ura) up to a OD 600 of 0.7.
• the cells were harvested and washed once with 1 ml of cold lysis buffer (PBS IX pH 7.4, 1 mM PMSF), then resuspended in 0.3 ml of Laemli buffer and incubated for 5 min at 98 ° C. .
• Serial dilution (1:10) is performed and the same volume of each sample is deposited on a 4% -20% SDS gradient page gel, transferred to a nitrocellulose membrane.
• hGH The expression of hGH is evaluated with the Abcam [GH-1] antibody (ab9821) and standardized with respect to the expression of the ubiquitous GAPDH gene (Ab9485) from Abcam. Moreover, the quantification of the expression of hGH is carried out by Elisa assay with and according to the protocol of the Growth Hormone kit ELISA Kit, Human Catalog number: EHGH1 from thermoscientific
• the amount of hGH protein produced evaluated in strain 230 is 40% higher than that obtained in strain 211.
• the firefly luciferase gene was amplified from the pGL4 vector (Promega) and cloned downstream of the constitutive promoter TEF1 according to the cassette below.
• Table XXIII expression cassette
• Table XXV plasmids and strains
• the strains 200, 210 and 211 were cultured in a synthetic medium (-leu-ura) up to a OD 600 of 0.7.
• the cells were harvested and washed once with 1 ml of cold lysis buffer (pH 7.4 PBS, 1 mM PMSF), and resuspended in 0.3 ml of the same buffer.
• the cells in suspension were lysed with glass beads (fast prep).
• the concentration of crude lysates was determined by the Bradford method (BioRad) and diluted to 0.5 mg / mL, and Luciferase activities were determined using 5 ⁇ L of lysate / sample using the Luciferase Assay System (Promega) and the luminescence evaluated on a luminometer.
• the activity is standardized with respect to the amount of total protein.
• the luciferase activity evaluated in strain 210 is 60% higher than that evaluated in strain 211 5.3. Evaluation of the activity of a recombinant cellulase in Saccharomyces cerevisiae
• Chaperonnes engineering is associated with engineering to express Talaromyces emersonii cellulase, cellobiohydrolase 1 (CBH1) (GenBank accession No. AAL89553) under TEF2 promoter.
• CBH1 cellobiohydrolase 1
• Cellulase activity analysis was conducted as described in Y. Ito et al. 2015 (Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System, PLoS One 2015 Dec 21; 10 (12)).
• the activity recorded for the strain coexpressing Cellulase engineering in the presence of Chaperones exhibits a 37% higher activity yield than the strain expressing only Cellulase engineering alone.
• chaperones to improve the production of proteins described above for Saccharomyces can easily be implemented in any eukaryotic cell of interest and in particular in Pichia and Yarrowia, so as to optimize the yield and / or the activity of proteins endogenous or heterogeneous.
• Those skilled in the art may in particular refer to the publications below to produce by yeast expressing the combination of chaperones according to the invention various proteins of interest for the food industry, the pharmaceutical field, the hydrolysis of biomass , energy, etc. :
• Zinjarde SS Zinjarde SS. Food-related applications of Yarrowia lipolytica. Food Chem.

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