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    • 专利摘要:
    The invention relates to yeast cells transformed to express a functional Form I Rubisco enzyme, and a Class II phosphoribulokinase. The expression of these enzymes reconstitutes a Calvin cycle in said yeasts to enable them to use carbon dioxide.
    公开号:FR3016371A1
    申请号:FR1450349
    申请日:2014-01-16
    公开日:2015-07-17
    发明作者:Denis Pompon；Frederic Paques；Julie Lessage；Stephane Guillouet；Florence Bonnot
    申请人:INST NAT SCIENCES APPLIQ；Centre National de la Recherche Scientifique CNRS；Institut National des Sciences Appliquees de Toulouse；Institut National de la Recherche Agronomique INRA；
    IPC主号:
    专利说明:

    [0001] MODIFIED YEASTS FOR USING CARBON DIOXIDE. The invention relates to the creation of modified yeast strains in order to enable them to use carbon dioxide as a carbon source.
    [0002] Human and industrial activity generates increasing amounts of carbon dioxide, which is one of the main factors of the greenhouse effect, responsible for a global warming of the planet likely to induce major climatic changes.
    [0003] The capture of carbon dioxide and its conversion into organic compounds are naturally carried out by certain organisms, particularly in the context of photosynthesis. Photosynthesis comprises two phases: during the first phase, there are photochemical reactions at the end of which the light energy is converted into chemical energy in the form of ATP and NADPH / NADH; during the second phase, called the Calvin cycle, this chemical energy is used to incorporate carbon from carbon dioxide into organic molecules. The key enzyme in the Calvin cycle is the Rubisco complex (Ribulose 1,5 bisphosphate carboxylase / oxygenase) that converts ribulose 1,5 diphosphate into 2 molecules of 3-phosphoglycerate by capturing a molecule of carbon dioxide. There are several forms of Rubisco (TABITA et al., J Exp Bot, 59, 1515 - 24, 2008), of which the most represented are Form I and Form II. Form I consists of two types of subunits: large subunits (RbcL) and small subunits (RbcS). The functional enzymatic complex is a hexadecamer consisting of eight L subunits and eight S subunits. The correct assembly of these subunits also requires the intervention of at least one specific chaperone: RbcX (LIU et al. , Nature, 463, 197-202, 2010). Form II is much simpler: it is a dimer formed of two identical RbcL subunits. Ribulose 1.5 diphosphate, substrate of Rubisco, is formed by reaction of ribulose 5-phosphate with ATP; this reaction is catalyzed by a phosphoribulokinase (PRK). There are two classes of PRKs: the class I enzymes found in the proteobacteria are octamers, while the class II enzymes found in the cyanobacteria and in the plants are tetramers or dimers. Non-photosynthetic organisms, such as yeasts, do not possess either Rubisco or phosphoribulokinase, but instead contain the other enzymes of the Calvin cycle, since they also play a role in the general metabolism of pentoses. It has been proposed to introduce Rubisco and PRK in yeasts to reconstitute a Calvin cycle, and to allow them to use carbon dioxide. Thus, GUADALUPE-MEDINA et al. (Biotechnology for Biofuels, 6, 125, 2013) report that the expression in Saccharomyces cerevisiae of the Rubisco enzyme of form II of Thiobacillus denitrificans, and PRK of Spinacia oleracea allows to improve ethanol production by decreasing the glycerol formation. However, so far, it has not been possible to express in the yeast a form I Rubisco enzyme of bacterial origin. Indeed, because of the complexity of this form, the reconstitution of a functional enzyme requires coexpression in a suitable stoichiometry, RbcL and RbcS subunits and the chaperone RbcX, and the correct association of these subunits. units in the enzyme complex. However, the transposition in a eukaryotic organism of the stoichiometry of expression which in prokaryotes is provided by the organization of genes in operons, poses problems. In addition, the differences between eukaryotes and prokaryotes in the intracellular environment can be reflected in particular by posttranslational modifications interfering with the folding of the peptide chains constituting the enzymatic subunits and / or with the assembly of these subunits. . The inventors have, however, succeeded in expressing in the yeast the different subunits of the Rubisco enzyme of Form I of Synechococcus elongatus and in obtaining the assembly of these subunits to reconstitute the enzyme complex, by co-expressing these sub-units. units with the RbcX specific chaperone, and with the general bacterial chaperones GroES and GroEL. The present invention thus relates to a yeast cell, preferably a transformed Saccharomyces cerevisiae cell, characterized in that it contains a) an expression cassette containing a sequence coding for the Rbol subunit, a form I Rubisco enzyme of bacterial origin, under transcriptional control of a suitable promoter; b) an expression cassette containing a sequence encoding the RbcS subunit of said Rubisco enzyme, under transcriptional control of a suitable promoter; c) an expression cassette containing a sequence encoding the specific RbcX chaperone of said Rubisco enzyme, under transcriptional control of a suitable promoter; d) an expression cassette containing a sequence coding for a GroES bacterial chaperone, under transcriptional control of an appropriate promoter; e) an expression cassette containing a coding sequence for a GroEL bacterial chaperone, under transcriptional control of a suitable promoter. Preferably, the Rubisco form I enzyme of bacterial origin is a cyanobacterium Rubisco enzyme, preferably a cyanobacterium of the genus Synechococcus and most preferably Synechococcus elongatus. The general bacterial chaperones GroES and GroEL can come from the same bacterial species as that from which the Rubisco enzyme is derived. They may also come from a bacterium of a different genus and species, for example E. coli. It may also be general chaperone of mitochondrial origin. According to a preferred embodiment of the present invention, said cell further contains an expression cassette f) containing a sequence coding for a PRK, preferably a Class II PRK, for example a PRK of Spinnacia oleacera, Euglena gracilis, or Synechococcus elongatus, under transcriptional control of a suitable promoter. A wide variety of tools (promoters, expression vector cassettes, transformation methods) that can be used for the expression of genes of interest in yeast cells is available in the art (for a review see, for example, "Methods in Yeast Genetics. D. Amberg, D. Burke and J. Strathem, Cold Spring Harbor Laboratory Press, 2005). 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.
    [0004] For the expression cassettes a) to e) constitutive promoters, such as for example TEF1, TDH3, PGI1, PGK, ADH1, will preferably be used. Preferably, these promoters will be different from one cassette to another. For the expression cassette f) of the PRK, an inducible promoter will preferably be used. By way of example, mention may be made of the tetO-7 promoter, the expression of which is repressed by doxicycline, and thus induced by the absence thereof. Other inducible promoters that can be used in the context of the present invention are in particular the tetO-2, GAL10, GAL10-cycl and PHO5 promoters. The expression cassettes of the invention further include the usual sequences in this type of construct, such as transcriptional terminators, and optionally other transcriptional regulatory elements such as amplifiers. 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 extrachromosomal replicon (s). The yeast strains according to the invention can be cultured under the usual conditions of culture of strains of the same species. Advantageously, these cultures will be carried out under an atmosphere containing at least 90% of carbon dioxide. The present invention will be better understood with the aid of the additional description which follows, which refers to non-limiting examples describing the expression of a Rubisco complex and a PRK in Saccharomyces cerevisiae. EXAMPLE 1 EXPRESSION AND ASSEMBLY OF THE RUBISCO COMPLEX OF SYNECHOCCOCUS E1ONGATVS IN YEAST SACCHAROMYCES CEREVISIAE Synthetic genes encoding the RbcS and RbcL subunits and the RbcX specific Chaperone of Synechoccocus elongatus pCC6301 Rubisco, and optimized for yeast expression, were prepared and cloned into the plasmid pBSII (Genecust) . Variants in which an HA tag has been added at the 3 'end of the coding sequence have also been constructed. The sequences of these synthetic genes (without the HA tag) are respectively indicated in the attached sequence listing under the numbers SEQ ID NO: 1 to 25 SEQ ID NO: 3 The sequences coding for the GroES and GroEL chaperones of E. coli were amplified from E. coli cultures. coli and cloned into the plasmid pSC-B-amp / kan (Stratagene). The sequences recovered from the cloning vectors were introduced into yeast expression vectors. These host vectors are listed in Table I below.
    [0005] Table I Names Origin replication Transcription cassette tag (promoter-terminator) Replicon E. coli yeast selection pFPP5 2u URA3 pGAL10-CYC1-tPGK Yes (AmpR) pFPP10 2u URA3 pTDH3- -tADH Yes (AmpR) pFPP11 2u URA3 pTDH3- - tCYC1 Yes (AmpR) pFPP12 2u URA3 pTGI1- -tCYC1 Yes (AmpR) pFPP13 ARS-CEN6 LEU2 pTEF1-tPGK Yes (AmpR) Note: pGAL10-CYC1: synthetic promoter composed of the UAS of the GAL10 gene and the initiation of transcription of the CYC1 gene (POMPON et al, Methods Enzymol, 272, 51-64, 1996).
    [0006] The resulting xpression cassettes are listed in Table II below. Table II Names Promoter Open reading phase Label Terminator CAS1 TEF1p RbcL-HA PGK CAS2 TEF1p RbcS-HA PGK CAS3 TEF1p RbcX-HA PGK CAS4 PGI1p RbcX without CYC1 CAS5 TDH3p RbcL- HA ADH1 CAS6 TDH3p RbcL without ADH1 CAS16 TEF1 RbcS Without PGK CAS17 TDH3 RbcL-HA PGK CAS18 TDH3 RbcL Without ADH CAS19 TEF1p RbcX Without PGK CAS20 PGIl RbcX-HA CYC1 CAS21 PGI1p GroES Without CYC1 CAS22 TDH3 GroEL Without ADH In some vectors, 2 or 3 cassettes were inserted. For this, the plasmids were amplified in the Escherichia DH5a bacterium and prepared by maxiprep, and then digested with appropriate restriction enzymes. Finally, the fragments are integrated into the host vectors by ligating with T4 ligase (FERMENTAS). The list of vectors constructed is shown in Table III below.
    [0007] Table III Names Type Origin Cassette 1 Cassette2 Cassette 3 markers Host vector pFPP6 2u CAS1 without without URA3 pFPP5 pFPP7 2u CAS2 without without URA3 pFPP5 pFPP18 2u CAS2 * CAS6 without URA3 pFPP5 / pFPP10 pFPP19 2u CAS2 CAS6 without URA3 pFPP5 / pFPP10 pFPP23 ARS416-CEN6 CAS3 without CAS2 without CAS2 CAS6 CAS6 CAS16 without URA3 pFPP10 pFPP45 2u CAS6 without without URA3 pFPP5 pFPP49 2u CAS19 without without LEU2 pFPP12 / pFPP13 pFPP55 ARS415-CEN6 CAS19 CAS21 CAS22 LEU2 pFPP13 pFPP56 ARS415-CEN6 CAS19 CAS21 CAS22 * LEU2 pFPP13 * Reverse orientation Different vectors or vector combinations were used to transform S. cerevisiae yeast cells (strain W303.1B). These vectors and vector combinations are shown in Table IV below. Table IV Transformed strain Parent strain Vector 1 Vector 2 Vector 3 Proteins expressed (§ indicates C-terminal fusion with HA tag) RbcS RbcL RbcX GroES GroEL 11.19 W303 pCM185 pFPP23 pFPP19 X§ XX§ 18.3 W303 pFPP45 pFPP49 XXX 22.2 W303 pFPP45 pFPP56 XXXXX 22.3 W303 pFPP45 pFPP55 XXXXX 30.1 W303 pCM185 11.5 W303 pCM185 pFL36 pFPP5 11.7 W303 pCM185 pFL36 pFPP18 X§ X 11.9 W303 pCM185 pFL36 pFPP19 X§ X 11.15 W303 pCM185 pFPP23 pFPP5 X§ 11.17 W303 pCM185 pFPP23 pFPP18 X§ XX§ 14.5 W303 pFPP6 X § 14.12 W303 pFPP40 X§ 14.6 W303 pFPP7 X§ 14.7 W303 pFPP23 X§ 16.3 W303 pFPP48 X§ 16.5 W303 pFPP43 pFPP23 X§ n X§ 16.6 W303 pFPP43 X§ X§ yFB3 CEN.PK pFPP45 pFPP20 pFPP56 XXXXX Notes: pCM185: plasmid ATCC 87659; pFL36: plasmid ATCC 77202 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) supplemented with a commercial medium CSM (MP Biomedicals) adapted to the markers of selection of the plasmids used for the transformation. The cultures are stopped by cooling to 4 ° C one generation before the end of the exponential phase. An aliquot is taken from each culture and the cells are lysed with sodium hydroxide in the presence of SDS for the analysis of total proteins on denaturing SDS gel. The rest of the cultures are centrifuged, then spheroplasts are prepared by enzymatic digestion of the cell walls with a zymolyase-cytohelicase mixture in hypertonic sorbitol medium (1.2M sorbitol). Spheroplases are washed in hypertonic sorbitol medium 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. After centrifugation at low speed (1500 rpm) to remove the large debris and then at medium speed (4000rpm) to recover the debris of intermediate sizes and mitochondria, the supernatant is recovered and the proteins are precipitated at 80% saturation of sulfate. ammonium with pH maintained at 6.5-7.0. The precipitate is redissolved and dialysed in the presence of protease inhibitors and then fractionated by molecular sieving on a column of Sephacryl S300 (GE Healthcare). The eluted fractions are pooled for gel analysis. The total lysate and the fractions sorted by molecular weight (native globular protein range of 104 to 1.5 × 10 6 daltons) are analyzed on SDS-PAGE denaturing gel and non-denaturing gel (coomassie blue-PAGE). The gels are stained with Coomassie Blue and Ponceau Red for total protein analysis. The RbcL RbcS and RbcX proteins are detected after electro-transfer to loaded nylon by immuno-detection. In the case of RbcL the detection can be carried out directly using an antiRbcL antibody, and in the case of RbcS and RbcX, indirectly via an anti-tag HA antibody. The different experiments were repeated alternating the coexpression of labeled and non-labeled proteins to verify that the presence of the labels did not affect the folding or assembly of the complexes. Figure 1 represents the analysis of total lysates of transformed strains. LA: SDS-PAGE analysis of a total lysate of strains 11.5, 11.15, 11.7, 11.17, 11.9, 11.19, 11.5, 11.19. ; IB: immunodetection analysis of a total lysate of strains 14.5, 14.12, 14.6, 14.7, 16.3, 16.5, 16.6. The 2 subunits are expressed in yeast. RbcL is expressed at a high level (visible in non-specific staining of the total proteins of an extract). The level of expression of RbcS has not been quantified but seems similar to that of RbcL based on immunodetections.
    [0008] Both proteins show no signs of degradation (absence of fuzzy or multiple bands) suggesting good quality of folding and endogenous protease resistance. The RbcX chaperone is well expressed and shows no signs of degradation. The plasmid coexpression systems of the 3 components are operational and do not show any significant interference between the expression of the different components. Figure 2 shows the immunodetection analysis of a total lysate (box on the left of the Figure), and fractions sorted by molecular weight, strain 16.5, which coexpresses RbcL, RbcS, and RbcX and its control, strain 16.3 which expresses RbcX. A mono-modal distribution of the subunit RbCL is observed within complexes of size greater than or equal to 500 kD while the mass of the isolated subunit is 55 kD. The distribution of RbcS and RbcX is on the contrary bimodal, one mode being similar in size to that observed for RbcL, the other corresponding to small sizes, close to those of isolated RbcS and RbcX proteins. The native Rubisco complex is not visibly visible in native gel and non-specific staining at the expected size (about 500 kD) under these conditions. Nevertheless, a very large complex is detectable at about 750-1000 kD (larger than the predicted size) by immunodetection of RbcL. Figure 3 shows the results of the non-denaturing gel assay, followed by immunodetection with anti-RbcL antibody, total extracts of strains 11.9, 18.3 and 22.2, and fractions sorted by weight. strain 22.2, which coexpresses RbcL, RbcS, and RbcX, S. Elongatus and E. neck chaperones. Then, in parallel, the fractions sorted by molecular weight of strains 18.3 (left) and 22.2 (right). These results show that co-expression with GroES and GroEL chaperones induced a reduction in the size of the high molecular weight complex (about 7501000 kD) that was detected in the absence of these chaperones; in cells co-expressing RbcL, RbcS, RbcX, GroES and GroEL, a well-defined band corresponding to the expected size (approximately 500kD) is observed for the native Rubisco complex.
    [0009] These results show that a Form I-type Rubisco complex of prokaryotic origin can be expressed and assemble correctly in S. cerevisiae cells, this assembly being improved by the presence of general-interest chaperones GroES and GroEL.
    [0010] For the analysis of the Rubisco activity in vitro, the extraction of the soluble proteins of the yFB3 strain is carried out. The 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 agar plates. ) with a CSM (MP Biomedicals) commercial medium, and adapted to the selection markers of the plasmids used (medium without Leucine, without Uracil and without Tryptophan for yFB3). The cultures are stopped by cooling to 4 ° C one generation before the end of the exponential phase. The cultures are centrifuged, then spheroplasts are prepared by enzymatic digestion of the cell walls with a zymolyase-cytohelicase mixture in hypertonic sorbitol medium (1.2M sorbitol). The spheroplasts are washed in hypertonic medium sorbitol in the presence of 1 mM PMSF and EDTA (protease inhibitors), then broken by repeated pipetting and light sonication in isotonic medium (0.6M) sorbitol. After centrifugation at low speed (200 g for 5 min) to remove large debris and then at medium speed (1500 g for 10 min) to recover the debris of intermediate sizes and mitochondria, the supernatant is recovered. The activity tests on the protein extracts are carried out in 50 mM TRIS / HCl (pH 7.5), 60 mM NaHCO 3 (13 C or 35 C) 10 mM MgCl 2 in the presence of 2 mM ribulose diphosphate (RiDP) and 0.5 mg / ml of total protein from yFB3 extracts. At t = 10 min and t = 60 min, 100 μl of the reaction mixture are removed, the reaction is stopped by the addition of 2 μl of HCl, and the sample is centrifuged for 10 min at 9300 g and then analyzed by HPLM / MS. (Reverse phase 018 ion pairing with a tributylamine acetatelOmM / acetonitrile gradient pH 6.0). Metabolites are detected by electrospray negative ionization mass spectrometry, and identified on the basis of their m / e ratios and elution times, compared to those of standard compounds. The results are shown in Figure 4. This figure represents the amount of 3-phosphoglycerate detected (m / e 185 and 186 ie the unlabeled 3-phosphoglycerate and 130-labeled 3-phosphoglycerate ions). a carbon at ES1 obtained at different reaction times (0.10 and 60 min) The amount of 3-phosphoglycerate detected on the extracts alone or the RiDP alone at these times is less than 20 AU (arbitrary units). The experiments carried out in the presence of 13002 and on the right those carried out in the presence of 1: 2. In the presence of 13002, the labeling rate of the 3-phosphoglycerate formed at 60 min is 52% as expected. At the bottom of Figure 8, the reaction catalyzed by Rubisco is the formation of 2 molecules of 3-phosphoglycerate from a molecule of CO2 and a molecule of RiDP In the presence of 12002, the 3-phosphoglycerate is formed but only in its form e not marked.
    [0011] The Rubisco present in the extracts is therefore capable of incorporating CO2 carbon to produce 3-phosphoglycerate. EXAMPLE 2 EXPRESSION OF PHOSPHORIBULOKINASES IN YEAST SACCHAROMYCES CEREVISIAE
    [0012] Synthetic genes encoding PRKs of different origin: Synechococcus elongatus (Syn), Rhodobacter sphaeroides (Rsph), Rhodopseudomonas palustris (Rpal), Spinnacia oleracea (Sole), Euglena gracilis (Egra) and optimized for expression in yeast , and flanked by a C-terminal HA tag, were prepared. The sequences of these synthetic genes (without the HA tag) are respectively indicated in the attached sequence listing under the numbers SEQ ID NO: 4 to SEQ ID NO: 8.
    [0013] The PRKs Rsph and Rpal are PRKs of class I existing in native form in octamer for Rsph and hexamer for Rpa. The Sole, Egra and Syn kinases are class II kinases whose native form is a dimer for the first two, and a tetramer for the third. The sequences of Rhodobacter sphaeroids (Rsph), Rhodopseudomonas palustris (Rpal), Spinacia oleracea (Sole) and Euglena gracilis (Egra) were synthesized by Genecust and delivered in a plasmid pBlueScript II +. The plasmids were amplified in the bacterium Escherichia coli DH5a. Extraction by maxiprep is performed for each of the plasmids. They are then digested with the enzymes BamHI and PstI and the product of the digestion is then deposited on a 0.8% agarose gel containing SYBER Safe. The migration is carried out in 1x TAE buffer at 50V for 30 minutes. The bands corresponding to the open reading phases (972 bp for PRK from Rpal, 966 bp for Rsph, 1461 bp for Egra and 1277 for Sole) are cut out of the gel and the DNA is extracted with the extraction kit for gel. the FERMENTAS company.
    [0014] Finally, the fragments are integrated into the vectors pCM185, pCM188-2 and pCM188-7 by ligating with T4 ligase (FERMENTAS) under the control of the repressible tet0 promoter by doxicycline, to give, respectively, the expression vectors pFPP20, pJLP1, pJLP2, pJLP3, pJLP4.
    [0015] The cassettes and expression vectors thus obtained are listed in Table V below. Table V Names Type Origin Promoter Terminator Open reading phase Markers HostVector pCM185 ARS416-CEN4 TetO7 CYC1 0 TRPI 0 pCM188-2ARS416-CEN4 TetO7 CYC1 0 URA3 0 pCM188-7ARS416-CEN4 TetO7 CYC1 0 URA3 0 pFPP20 ARS416-CEN4 TetO7 CYC1 PRK S. elongatus tag HA TRPI pCM185 pFPP21 ARS416-CEN4 TetO2 CYC1 PRK S. elongatus tag HA URA3 pCM188-2 pFPP22 ARS416-CEN4 TetO7 CYC1 PRK S. elongatus tag HA URA3 pCM188-7 pJLP1 ARS416-CEN4 TetO7 CYC1 PRK E. gracilis tag HA TRP1 pCM185 pJLP2 ARS416-CEN4 TetO7 CYC1 PRK R. sphaeroides tag HA TRP1 pCM185 pJLP3 ARS416-CEN4 TetO7 CYC1 PRK R. palustris tag HA TRPI pCM185 pJLP4 ARS416-CEN4 TetO7 CYC1 PRK S. oleracea tag HA TRP1 pCM185 These vectors were used to transform S. cerevisiae cells of strains W303.1B and CNPK. The first of these strains is a typical laboratory strain, the second is a semi-industrial strain. The transformation was carried out according to the protocol of CHEN et al (Curr Genet, 1992, 21, 83-4), maintaining at all stages of transformation and subcloning a doxicycline concentration of 2 μg / ml, adapted to the suppression of the tetO promoter. The transformants were stored in a glycerol medium (50% gycerol) at -80 ° C. in the presence of 2 μg / ml doxicycline.
    [0016] The transformed strains obtained are listed in Table VI below. Table VI Transformed strain Parent strain Vector 1 Vector 2 Vector 3 PRK expressed 11.5 W303 pCM185 pFL36 pFPP5 30.2 W303 pFPP20 PRKsyn 11.6 W303 pFPP20 pFL36 pFPP5 PRKsyn yJL1 W303 pJLP1 PRK Egra yJL2 W303 pJLP2 PRK Rsph yJL3 W303 pJLP3 PRK Rpal yJL4 W303 pJLP4 PRK Sole yJL5 CENPK pFPP20 PRKsyn yJL6 CENPK pJLP1 PRK Egra yJL7 CENPK pJLP2 PRK Rsph yJL8 CENPK pJLP3 PRK Rpal yJL9 CENPK pJLP4 PRK Sole Transformed cells are precultured from the stock in YNB medium (yeast without nitrogen base supplemented with ammonium sulfate 6 , 7 gL-1, glucose 20 gL-1, agar 20 gL-1 for agar plates) with commercial medium CSM (HP Biomedicals adapted to maintain the selection of the plasmid and containing a concentration of 2 μg / ml doxicycline suitable for suppression The influence of the expression of the different PRKs on the viability of the cells was evaluated on agar medium in the presence or absence of doxicycline: Each strain It is grown in liquid on selective CSM medium with doxicycline at 2 μg / mL. An equivalent of 2 OD (OD 600nm) is recovered and then washed twice to remove doxicycline. Tenth dilutions are made. 10 μL of the dilutions are deposited in the form of drops (series of serial dilutions) of the cell suspensions, on plates of agar media (containing or not 2 μg / mL doxicycline) and incubated at 28 ° C. in a normal atmosphere or alternatively in closed bag whose atmosphere contains at least 90:10 of carbon dioxide / air (vol / vol).
    [0017] The results in a normal atmosphere are illustrated in FIG. 4. The results in a CO2-rich atmosphere are illustrated in FIG. 5. It is found that all the PRKs are more or less toxic in the high-expression strain W3031B (induced). . Nevertheless, the toxicity appears much weaker in the CENPK strain, or only the PRK of Syn is toxic in the induced state. Other experiments show that in strain W303.1B, toxicity is greatly attenuated in a low oxygen and carbon dioxide rich atmosphere. The influence of the expression of the different PRKs on the growth of the cells was evaluated on cultures in liquid medium: The strains are cultured in CSM selective medium in closed tubes (containing or not 2 μg / mL doxicycline) . Growth is monitored by measuring the optical density at 600 nm until entering the stationary phase. For each strain, the ratio between the maximum growth rate p (growth of the population per unit of time) of the strain and that of the control strain (strain + empty plasmid) is made. The maximum growth rate ratios (blmax) for each strain are shown in Figure 6. * Dark bars: induced state (high level expression) of the tetO promoter (medium containing no doxicycline); clear bars: (partially) repressed (low level expression) state of the tetO promoter (medium containing 2 μg / mL of doxicycline). These results confirm the lower toxicity of kinases in the context of strain CNPK 113-7D than in that of strain W303.1B. A dose (induction level) - response (growth rate) toxicity effect is observed only for Sole kinase and W303.1B.
    [0018] In W303.1B, there is significant toxicity of Rpal, Rsph, Syn kinases weakly and strongly expressed. Toxicity appears lower for the kinase Egra.
    [0019] For the analysis of the ribulose-1,5-bisphosphate metabolite of the central carbon cycle, the cells are washed to remove doxicycline and placed in liquid culture at 30 ° C. on YNB (yeast without nitrogen base) supplemented with sodium sulfate. ammonium 6,7 gL-1, glucose 20 gL-1, (agar 20 gL-1 for agar plates) supplemented with commercial medium CSM (MP Biomedicals), adapted according to the selection marker of the plasmid used. The cultures are carried out in a closed tube without oxygen supply in excess of 3-10 volumes of air (not renewed) per volume of culture medium.
    [0020] The carbon dioxide resulting from the culture is thus maintained in the volume of the culture tube. This procedure limits the toxicity of the expression. The metabolism is blocked by dilution of the culture in methanol-water 60:40 (v / v) at -80 ° C (mixture maintained at -40 ° C in a dry ice / acetonitrile bath), followed by rapid centrifugation (maintained temperature less than -20 ° C.) and lysing the cells in a methanol-water mixture (60:40 v / v) containing 0.3M sodium hydroxide and then freezing at -80 ° C. according to the protocol described by LUO et al. (J.
    [0021] Chromatography A 1147: 153-164, 2007). After thawing an aliquot is neutralized with glacial acetic acid, centrifuged and the supernatant analyzed by HPLM / MS (C18 reverse phase ion pairing with a gradient tributylamine acetate / acetonitrile pH 6.0).
    [0022] The metabolites are detected by negative ion electrospray mass spectrometry and identified on the basis of their m / e mass ratios and elution times compared to those of standard compounds. The results are shown in Figure 7.
    [0023] This figure represents the detection of ribulose 1,5-disphosphate (molar mass 309 g / mol) as a function of the elution time in the various extracts. The left panel shows the chromatograms of strain W303-1B grown in closed tubes containing selective CSM medium; the center panel shows the chromatograms of the CENPK strain grown in closed tubes and selection medium (CSM medium), the right panel represents the chromatograms obtained for the CENPK strain grown in closed tubes and minimum medium. The level of activity (not standardized for the level of expression) estimated by the level of accumulation of ribulose 1,5 diphosphate product of the reaction appears: very high for PRK Syn even in repressed condition (50% of induced level ). This activity is accompanied by toxicity with a significant drop in the level of intracellular ATP; - detectable but lower for PRKs Egra 15 and Sole in medium minimum; undetectable under conditions used for PRKs RspH and Rpai; - Dependent on the culture medium, with the PRK Syn a level of accumulation of ribulose 1.5 diphosphate 20 much higher in poor than rich medium. All these observations indicate that only class II kinases lead to the accumulation of high levels of ribulose diphosphate in S. cerevisiae.
    权利要求:
    Claims (8)
    [0001]
    CLAIMS1) Transformed yeast cell, characterized in that it contains: a) an expression cassette containing a coding sequence for the RbcL subunit of a Rubisco enzyme of form I of bacterial origin, under the transcriptional control of an appropriate promoter; b) an expression cassette containing a sequence encoding the RbcS subunit of said Rubisco enzyme, under transcriptional control of a suitable promoter; c) an expression cassette containing a sequence encoding the specific RbcX chaperone of said Rubisco enzyme, under transcriptional control of a suitable promoter; d) an expression cassette containing a sequence encoding a general Groes bacterial chaperone, under the transcriptional control of an appropriate promoter; E) an expression cassette containing a coding sequence for a generalized bacterial chaperone GroEL, under transcriptional control of a suitable promoter.
    [0002]
    2) yeast cell according to claim 1, characterized in that said yeast belongs to the species Saccharomyces cerevisiae.
    [0003]
    3) A yeast cell according to any one of claims 1 or 2, characterized in that the Rubisco enzyme of form I of bacterial origin is a Rubisco enzyme of cyanobacteria.
    [0004]
    4) yeast cell according to any one of claims 1 to 3 characterized in that said cyanobacterium belongs to the genus Synechococcus.
    [0005]
    5) A yeast cell according to any one of claims 1 to 4, characterized in that it further contains an expression cassette containing a coding sequence for a phosphoribulokinase (PRK) under transcriptional control of a suitable promoter.
    [0006]
    6) yeast cell according to claim 5, characterized in that said PRK is a PRK class II.
    [0007]
    7) yeast cell according to claim 6, characterized in that said class II PRK is selected from PRK of Spinnacia oleacera, Euglena gracilis, or Synechococcus elongatus.
    [0008]
    8) A yeast cell according to any one of claims 5 to 7, characterized in that the promoter controlling the transcription of the sequence coding for PRK is an inducible promoter.
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    同族专利:
    公开号 | 公开日
    BR112016016524A2|2018-01-23|
    WO2015107496A1|2015-07-23|
    CA2937079A1|2015-07-23|
    AU2015207758A1|2016-08-11|
    FR3016371B1|2018-02-02|
    EP3094717A1|2016-11-23|
    KR20160122735A|2016-10-24|
    JP2017502682A|2017-01-26|
    US20170002368A1|2017-01-05|
    US10066234B2|2018-09-04|
    EP3094717B1|2019-01-16|
    CN106103694A|2016-11-09|
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    FR3062394B1|2017-01-27|2021-04-16|Enobraq|GENETICALLY OPTIMIZED MICROORGANISM FOR THE PRODUCTION OF MOLECULES OF INTEREST|
    CN108624516A|2017-03-20|2018-10-09|华东理工大学|A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items|
    WO2018219995A1|2017-05-31|2018-12-06|Universität Für Bodenkultur Wien|Yeast expressing a synthetic calvin cycle|
    CA3064143A1|2017-06-13|2018-12-20|Dsm Ip Assets B.V.|Recombinant yeast cell|
    FR3079525A1|2018-03-30|2019-10-04|Enobraq|MODIFIED MICROORGANISM WITH IMPROVED RUBISCO ACTIVITY|
    法律状态:
    2015-10-29| PLFP| Fee payment|Year of fee payment: 3 |
    2017-01-10| PLFP| Fee payment|Year of fee payment: 4 |
    2018-01-22| PLFP| Fee payment|Year of fee payment: 5 |
    2019-01-30| PLFP| Fee payment|Year of fee payment: 6 |
    2020-10-16| ST| Notification of lapse|Effective date: 20200910 |
    2020-12-11| CD| Change of name or company name|Owner name: INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE T, FR Effective date: 20201103 Owner name: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, FR Effective date: 20201103 Owner name: INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULT, FR Effective date: 20201103 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1450349|2014-01-16|
    FR1450349A|FR3016371B1|2014-01-16|2014-01-16|MODIFIED YEASTS TO USE CARBON DIOXIDE|FR1450349A| FR3016371B1|2014-01-16|2014-01-16|MODIFIED YEASTS TO USE CARBON DIOXIDE|
    EP15707418.8A| EP3094717B1|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
    PCT/IB2015/050346| WO2015107496A1|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
    BR112016016524-1A| BR112016016524A2|2014-01-16|2015-01-16|transformed yeast cell|
    US15/111,410| US10066234B2|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
    AU2015207758A| AU2015207758A1|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
    JP2016546974A| JP2017502682A|2014-01-16|2015-01-16|Yeast modified to use carbon dioxide|
    CN201580004952.3A| CN106103694A|2014-01-16|2015-01-16|It is modified to utilize the yeast of carbon dioxide|
    KR1020167022332A| KR20160122735A|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
    CA2937079A| CA2937079A1|2014-01-16|2015-01-16|Yeasts modified to use carbon dioxide|
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