WO2022229574A1 - Insertion multicopies d'un gène d'intérêt dans le génome d'un champignon - Google Patents
Insertion multicopies d'un gène d'intérêt dans le génome d'un champignon Download PDFInfo
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- WO2022229574A1 WO2022229574A1 PCT/FR2022/050826 FR2022050826W WO2022229574A1 WO 2022229574 A1 WO2022229574 A1 WO 2022229574A1 FR 2022050826 W FR2022050826 W FR 2022050826W WO 2022229574 A1 WO2022229574 A1 WO 2022229574A1
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-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
Definitions
- the present invention relates to a method for inserting several copies of a gene of interest into the genome of a fungus.
- the present invention also relates to the fungus strains thus obtained, and the different uses of such a modified fungus strain.
- the fungus strains are used for the production of proteins of interest.
- fungus strains belonging to the Trichoderma genus in particular Trichoderma reesei, are now mainly used for the production of enzymes.
- enzymes for example cellulases, are in fact used to hydrolyze cellulosic or lignocellulosic biomass into simple sugars.
- the enzymes produced by filamentous fungi are therefore useful in production chains for second-generation biofuels or even biosourced products derived from sugars originating from (ligno)cellulosic biomass.
- patent EP 448 430 B1 describes an optimized industrial production of cellulases by Trichoderma reesei.
- This production is carried out in a fed-batch protocol (feed without racking) using a feed solution containing lactose as the sugar inducing the production of proteins.
- This fermentation process comprises two stages: a first stage of growth of the fungus in the presence of an excess of carbon source and a second stage of production of enzymes thanks to the addition of an inducer in the medium with an optimized flow rate ( fed-batch mode). These steps are carried out in a liquid medium in bioreactors with stirring and in the presence of oxygen because the fungus is strictly aerobic.
- Another example of an optimized process for the production of cellulases is described in patent EP 2 744899 B1.
- Mushrooms can also be used to produce useful proteins in other types of industries, such as the food, cosmetics or pharmaceutical industry (see example Rantasalo, A., Vitikainen, M., Paasikallio, T. et al. Novel genetic tools that enable highly pure protein production in Trichoderma reesei. Soi Rep 9, 5032 (2019) or patent EP3405580).
- the TEF1 promoter is known as a strong promoter in fungi (Gao S, Zhou H, Zhou J, Chen J. Promoter-Library-Based Pathway Optimization for Efficient (2S)- Naringenin Production from p-Coumaric Acid in Saccharomyces cerevisiae. J Agric Food Chem. 2020 Jun 24;68(25):6884-6891. doi: 10.1021/acs.jafc.OcOI 130. Epub 2020 Jun 11).
- TEF1 promoter has also been described or suggested for promoting the expression of genes of interest (see for example US20080044878, US9732338, US6011147, W02018067068, US8071298, or even Kitamoto, N., Matsui, J ., Kawai, Y. et al.Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae.Appl Microbiol Biotechnol 50, 85-92 (1998);Zhang J, Cai Y, Du G, Chen J, Wang M, Kang Z.
- flanking regions are usually necessary for the transgene to be flanked by sequences upstream downstream of the target locus.
- the size (number of base pairs) of these flanking regions depends on the organisms. For those with the ability to carry out homologous recombination, these flanking sequences may be short, for example in the case of Saccharomyces cerevisiae, around twenty base pairs are sufficient to target a locus (Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M.
- the present invention is based here on the results of the inventors showing that the TEF1 promoter can be used for the multicopy insertion of a gene of interest. Each functional copy inserted leads to an increase in the expression of said gene of interest, thus increasing the production capacity of the protein of interest.
- the results of the present invention also show that it is possible to carry out successfully, in particular in Trichoderma reesei, the insertion at the target locus (the region 5' of the TEF1 promoter) by using a single flanking region of the insertion site in the expression cassette according to the invention (instead of the usual two).
- the present invention thus relates to a method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising a cassette of expression, said cassette comprising:
- TEF1 promoter identical to the endogenous TEF1 promoter of the fungus species
- the invention also relates to the use of an expression cassette for inserting several copies of a gene of interest into the genome of a fungus, said expression cassette comprising:
- TEF1 promoter identical to the endogenous TEF1 promoter of the fungus species
- the invention also relates to a method for producing a protein of interest comprising the following steps:
- the invention also relates to a fungus strain comprising in its genome several copies of a gene of interest under the control of a TEF1 promoter identical to the endogenous TEF1 promoter of the genetically modified fungus species.
- the invention relates to a method for inserting several copies of a gene of interest into the genome of a fungus, said method comprising a step of transforming said fungus using a vector comprising an expression cassette, said cassette comprising:
- TEF1 promoter identical to the endogenous TEF1 promoter of the fungus species
- the term “TEF1” refers to the translation elongation factor 1 (Translation Elongation Factor 1). It is a GTPase-like protein that is involved in protein biosynthesis.
- TEF1 protein encoded by the TEF1 gene, is represented by SEQ ID NO: 2.
- the expression of the TEF1 gene is essentially constitutive (Tiina Nakari, Edward Alatalo and Merja E. Penttila, Isolation of Trichoderma reesei genes highly expressed on glucose containing media: characterization of the TEF1 gene encoding translation elongation factor 1a, Gene, 136(1993)313-318, 1993 Elsevier Science Publishers B.V). More particularly, according to the invention, the term “TEF1” refers to “TEF1 alpha”.
- the expression "at least 60% identity with SEQ ID NO: 1" means all the values between 60% and 100%, in particular the values of 60%, 61%, 62%, 63% , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably at least 80%, at least 85%, at least 90%, at least 95%, even more particularly at least 98%, at least 99%.
- the TEF1 promoter can also mean the promoter under whose control is a gene coding for an orthologous protein or variant of the TEF1 protein represented by SEQ ID NO: 2.
- the TEF1 promoter can also to mean the promoter of a gene coding for a protein of SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO: 2, preferably at least 85%, at least 90%, at least at least 95%, at least 98%, at least 99%.
- the expression "at least 80%” represents all values between 80% and 100%, i.e. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
- a promoter represented by a sequence having at least 60% identity with SEQ ID NO: 1 preferably represents a variant promoter or an orthologous promoter in a fungus different from Trichoderma reesei.
- the expression cassette is a TEF1 promoter identical to the endogenous TEF1 promoter of the mushroom species which is used.
- the term "endogenous fungus species TEF1 promoter” also refers to the "native TEF1 promoter" of the fungus species.
- the TEF1 promoter in Trichoderma reesei being represented by SEQ ID NO: 1, then it is a promoter of SEQ ID NO: 1 which must be used in the expression cassette intended to be integrated when the host fungus is Trichoderma reesei .
- the TEF1 promoter which must be used, in the expression cassette according to the invention must be identical to the TEF1 promoter naturally present in the species of fungus which is going to be transformed.
- the TEF1 promoter to be used in the expression cassette is the endogenous/native TEF1 promoter of Saccharomyces cerevisiae.
- TEF1 promoter identical to the endogenous TEF1 promoter means that the sequence of the TEF1 promoter used is strictly identical to that of the endogenous promoter, but also that the sequence of the TEF1 promoter used may vary at the level of certain nucleotides (not essential to the function of the promoter).
- the TEF1 promoter present in a fungus species can easily be determined, for example using databases such as FungiDB, by sequencing the fungus genome or any other appropriate technique.
- the method according to the invention can thus comprise, before the transformation step, a step optional determination of the sequence of the TEF1 promoter naturally present in the fungus intended to be transformed.
- the transformation step means any technique allowing the integration of the expression cassette into the genome of the fungus.
- a technique is well known to those skilled in the art and includes, for example, biolistics (Te'o VS, Bergquist PL, Nevalainen KM., (2002). Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adapter System. J Microbiol Methods. 51(3):393-9) or the protoplast method (Penttilà M, Nevalainen H, Ràttô M, Salminen E, Knowles J., (1987). A versatile transformation System for the cellulolytic filamentous fungus Trichoderma reesei Gene 61 (2):155-64).
- an “expression cassette” comprises a DNA fragment (the gene of interest) placed under the control of the elements necessary for its expression. Said DNA fragment is thus placed under the control of the TEF1 promoter.
- the expression cassette comprises at least (1) a TEF1 promoter identical to the endogenous TEF1 promoter of the mushroom species, (2) a gene of interest, and (3) a terminator, at the exception of the endogenous TEF1 terminator of the fungus species.
- the terminator used in the expression cassette according to the invention is a different terminator from the endogenous TEF1 terminator of the fungus species (that is to say the native TEF1 terminator of the fungus species).
- a TEF1 terminator from another fungus can therefore be used, provided that it is a TEF1 terminator different from the endogenous TEF1 terminator of the transformed fungus species (this is the case if the TEF1 terminator has a percentage identity less than or equal to 75% with respect to the sequence of the endogenous TEF1 terminator of the species of transformed fungus).
- the terminator is different from an endogenous TEF1 terminator of a fungus species different from the transformed fungus species.
- the terminator is a different terminator from a TEF1 terminator. In other words, it is not a so-called TEF1 terminator in a species.
- a CBH1 terminator can be used.
- the integrative vector according to the invention (preferably a plasmid) comprises an expression cassette as mentioned above and a selection marker.
- the integrative vector according to the invention comprises an expression cassette as mentioned above, a non-fungal (for example bacterial) origin of replication and a selection marker.
- selection marker means a gene whose expression confers on the cells which contain it a characteristic making it possible to select them.
- the selection marker is a marker compatible with the host. The use of a selection marker makes it possible to identify the fungi that have integrated a genetic modification compared to those that have not integrated it. This is for example a gene for resistance to antibiotics, in particular the gene for resistance to the antibiotic hygromycin hph.
- the multicopy insertion is thus carried out in such a way that the expression of the endogenous TEF1 gene of the transformed species of fungus is maintained.
- the endogenous TEF1 coding sequence and TEF1 promoter are functional and show wild-type expression.
- the insertion is carried out upstream of the endogenous TEF1 promoter in the genome of said fungus. Indeed, as indicated below, fungi naturally possess a copy of the TEF1 gene, under the control of the TEF1 promoter.
- the multicopy insertion is therefore carried out upstream of the endogenous TEF1 promoter of the transformed fungus species.
- the insertion is thus made 5′ of the endogenous TEF1 promoter.
- the insertion is thus carried out between 750 and 1250 base pairs, preferably approximately 1000 base pairs, relative to the translation initiation site of the gene coding for TEF1.
- the insertion is thus carried out between 750 and 1250 base pairs, preferably approximately 1000 base pairs, relative to the translation initiation site of the gene coding for the protein TEF1 of SEQ ID NO: 2 or a protein having at least 80% identity with SEQ ID NO: 2.
- the endogenous TEF1 gene dependent on the endogenous TEF1 promoter are conserved in the transformed genome of said fungus.
- the gene of interest codes for an enzyme, in particular a cellulolytic enzyme such as a cellulase or a hemicellulase.
- the enzymes are cellulases.
- the term “cellulases” more particularly means enzymes chosen from endoglucanases, exoglucanases and glucosidases, and more particularly b-glucosidase.
- cellulase refers more particularly to an enzyme adapted to the hydrolysis of cellulose and allowing the microorganisms (such as Trichoderma reesei) which produce them to use cellulose as a source of carbon, by hydrolyzing this polymer into simple sugars. (glucose).
- the method according to the invention comprises a step of selecting the transformed strain, after the transformation step.
- the definitions and embodiments relating to the first aspect of the invention apply mutatis mutandis to the other aspects.
- all the definitions and preferences indicated in the first aspect of the invention above also apply to the second, third, fourth, fifth, sixth and seventh aspect of the invention described below.
- TEF1 promoter identical to the endogenous TEF1 promoter of the fungus species
- the invention also relates to a process for producing a biomass comprising the following steps:
- said process for producing a biomass comprises a step of selecting a mushroom transformed between the transformation step and the culture step.
- step (ii) a step of culturing the fungus transformed in step (i) under conditions allowing the production of the protein of interest.
- the culture step means a culture in an appropriate medium.
- This is a medium suitable for the survival of the transformed fungus and/or for the growth of the fungus and/or for the production of the protein of interest, or even its secretion. It may be a suitable medium for cultivation of the fungus, supplemented as needed to become a suitable medium for the production and/or secretion of the protein of interest.
- said method for producing a protein of interest also comprises a growth phase of a fungus strain transformed according to the invention, then a phase of growth and production of proteins of interest.
- said growth phase is carried out in the presence of a growth substrate and said phase of growth and production of proteins of interest is carried out in the presence of an inducing substrate.
- the growth substrate and the inducing substrate are preferably carbonaceous substrates.
- the carbonaceous growth substrate is preferably chosen from lactose, glucose, xylose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and/or a crude extract of water-soluble pentoses from pretreatment of cellulosic biomass.
- the inducing carbonaceous substrate is thus preferably chosen from lactose, cellobiose, sophorose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and/or a crude extract of water-soluble pentoses originating from the pretreatment of cellulosic biomass.
- the method for producing a protein of interest may comprise a step of secreting said protein of interest.
- the secretion can be consecutive or concomitant with the production of the protein of interest.
- step iii a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolyzate.
- the sugars are thus obtained at the end of the hydrolysis step.
- they are Cs-Ce sugars.
- said process for producing sugars may also comprise one or more separation steps. This thus makes it possible to separate, in particular by distillation, the various products of interest diluted in water.
- the present invention relates to a method for producing an alcohol from cellulosic or lignocellulosic substrates, comprising the following steps: - (i) a step of pretreating a cellulosic or lignocellulosic substrate in order to obtain a pretreated substrate,
- step iii a step of enzymatic hydrolysis of the pretreated substrate obtained in step i), in the presence of the cellulolytic enzymes obtained in step ii), in order to obtain a hydrolyzate
- the alcohol thus obtained is for example used as a biofuel.
- the alcohol thus obtained can also be used as a solvent or as an intermediate product in the chemical industry.
- biofuel means more particularly a second-generation biofuel, that is to say from non-food resources.
- biofuel can also be defined as being any product resulting from the transformation of biomass and which can be used for energy purposes.
- biogas products which can be incorporated (possibly after subsequent transformation) into a fuel or be a fuel in its own right, such as alcohols (the ethanol, butanol and/or isopropanol depending on the type of fermentation organism used), solvents (acetone), acids (butyric), lipids and their derivatives (short or long chain fatty acids, acid esters fatty), as well as hydrogen.
- Biofuel is thus an alcohol or a biogas.
- the alcohol is, for example, ethanol, butanol, isopropanol, 1,2-propane diol, 1,3-propane diol, 1,4-butane diol, and/or or 2,3-butane diol. More preferably, it is ethanol.
- the hydrolyzate obtained in step iii) is a hydrolyzate containing Cs-Ce sugars, in particular glucose.
- the alcoholic fermentation step of the hydrolyzate obtained is a fermentation step, in the presence of a fermentative organism, of the glucose from the hydrolyzate so as to produce a fermentation broth.
- a fermentative organism is for example a yeast.
- the separation step is a separation of the biofuel and the fermentation broth, in particular by distillation.
- the invention also relates to a strain as obtained by the method of the invention.
- the invention also relates to a fungus strain comprising in its genome several copies of a gene of interest under the control of a TEF1 promoter identical to the endogenous TEF1 promoter of the genetically modified fungus species.
- FIG. 1 represents the construction of the plasmid used in Example 1.
- FIG. 2 represents the fluorescence intensity per unit size of germinating Trichoderma reesei spores.
- Figure 2 represents the results of 8 transformants: E1, E2, E3, E4, E5, E6, E8 and E9 and those of 3 untransformed strains: CL847-1, CL847-2 and CL847-3.
- FIG. 3 represents the TEF promoter (TEF1) upstream of the TEF1 gene in the genome of a fungus.
- Fig. 4 represents the TEF promoter (TEF1) upstream of the TEF1 gene in the genome of a fungus.
- FIG. 4 represents an illustration of the integration into the E1 transformant. 7 copies of the EGFP gene of interest have been integrated. The triple indication "(1) the TEF1 promoter, (2) the EGFP gene of interest and (3) the CBH1 terminator" illustrates that the cassette has been correctly integrated and that the EGFP gene is functional.
- FIG. 5 represents an illustration of the integration into the E3 transformant. 10 copies of the EGFP gene of interest were integrated. The triple indication “(1) the TEF1 promoter, (2) the EGFP gene of interest and (3) the CBFI1 terminator” illustrates that the cases where the cassette has been correctly integrated and that the EGFP gene is functional.
- Example 1 Construction of a vector containing an expression cassette according to the invention
- a selection marker comprising a fungal “cpc” and bacterial “IM7” promoter followed by the Flygromycin (hph) resistance gene and the fungal “TrpC” terminator
- an expression cassette comprising (1) a TEF1 promoter, (2) followed by the EGFP gene expressing a green fluorescent protein and (3) the CBFI1 terminator.
- This vector can be obtained by any conventional method, well known to those skilled in the art.
- the construction of this plasmid is shown in Figure 1.
- a person skilled in the art can moreover easily obtain a vector (of plasmid type) comprising an expression cassette according to the invention and a selection marker making it possible to select the transformed fungi.
- Example 2 Transformation of a fungus with the expression vector of Example 1
- Example 1 The plasmid of Example 1 was used to transform the strain of Trichoderma reesei called CL847 (Durand H, Clanet M, Tiraby G. Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 1988, 10: 341-346; Durand et al, 1984, Proc. Colloquium SFM "Genetics of industrial microorganisms". Paris. H. FIESLOT Ed, pp 39-50).
- the transformants were selected on their ability to grow on a medium containing flygromycin.
- the subcultures revealed different macroscopies on the dish between the transformants. Only those showing, after 3 subcultures, growth at the confluence of the perimeter of the 9 cm petri dishes, and displaying a dense, green and hydrophobic spore carpet were kept.
- FIG. 2 The intensity of the fluorescence per unit size of Trichoderma reesei spores in the process of germinating is presented in FIG. 2. These measurements were carried out using a cytometer. The genomic study of four of the eight transformants studied by cytometry made it possible to highlight the site of insertion, and the number of copies of the plasmid. In the four cases studied, all the insertions were made upstream of the TEF1 promoter. The native and constitutive promoter of the TEF1 gene is conserved.
- Figure 3 illustrates the TEF1 gene under the control of the native and constitutive promoter of the TEF1 gene in the genome of the fungus.
- Figure 4 represents an illustration of the integration in the transformant E1. 7 copies of the EGFP gene of interest have been integrated
- Figure 5 represents an illustration of the integration in the transformant E3. 10 copies of the EGFP gene of interest have been integrated.
Abstract
Description
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CN202280031948.6A CN117355610A (zh) | 2021-04-30 | 2022-04-29 | 在真菌基因组中插入感兴趣的基因的多个拷贝 |
BR112023022450A BR112023022450A2 (pt) | 2021-04-30 | 2022-04-29 | Inserção de múltiplas cópias de um gene de interesse no genoma de um fungo |
EP22726786.1A EP4330401A1 (fr) | 2021-04-30 | 2022-04-29 | Insertion multicopies d'un gène d'intérêt dans le génome d'un champignon |
CA3215584A CA3215584A1 (fr) | 2021-04-30 | 2022-04-29 | Insertion multicopies d'un gene d'interet dans le genome d'un champignon |
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FR2104588A FR3122436A1 (fr) | 2021-04-30 | 2021-04-30 | Insertion multicopies d’un gène d’intérêt dans le génome d’un champignon |
FRFR2104588 | 2021-04-30 |
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- 2021-04-30 FR FR2104588A patent/FR3122436A1/fr active Pending
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- 2022-04-29 CA CA3215584A patent/CA3215584A1/fr active Pending
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