Fusion protein and use for biovconverting molecules

Abstract

The present invention relates to a fusion protein, to a nucleic acid coding for the protein, to a vector comprising the nucleic acid, to a host cell comprising the nucleic acid and / or vector, to a process for producing a fusion protein and to a process for bioconverting a substrate. The fusion protein of the present invention successively comprises (i) at least one polypeptide for targeting, and attaching to, the bacterial membrane, (ii) at least one polypeptide corresponding to the hydrophilic domain of a plant P450 cytochrome, (iii) at least one binding polypeptide comprising at least 47 amino acids, and (iv) at least one polypeptide corresponding to the hydrophilic domain of a plant NADPH-dependent cytochrome P450 reductase.

Description

[0001] Fusion protein and use for bioconversion of molecules

[0002] Technical field of the invention

[0003] The present invention relates to a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, (ii) at least one polypeptide corresponding to the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide, and (iv) at least one polypeptide corresponding to the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

[0004] The present invention also relates to the nucleic acid encoding the fusion protein, to the vector comprising said nucleic acid, to the host cell comprising said nucleic acid and / or vector and to the method of producing said fusion protein.

[0005] The present invention also relates to a method of bioconversion of a substrate comprising the use of a fusion protein.

[0006] The present invention finds an application, in particular in the field of the production of proteins and / or polypeptides, of the synthesis of molecules, for example of bioconversion, and in the biological and / or medical field.

[0007] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text.

[0008] Prior art

[0009] The specialized metabolism of plants is a metabolism that allows plants to adapt to changing environmental conditions. Each plant has developed an arsenal of molecules over the course of its evolution, enabling it to respond to its specific living conditions. The diversity of molecules thus produced is virtually inexhaustible. These molecules, which can be very complex, have been widely used by humans, particularly for health applications.

[0010] The synthesis of these molecules is carried out through complex biosynthetic pathways involving numerous steps catalyzed by specific enzymes. Cytochromes P450s (P450s) are among these enzymes and can be considered as very high-precision tools for producing high-value molecules.

[0011] Cytochromes P450s are enzymes involved in many processes related to the adaptation of plants to their environment. They are at the origin of part of the great diversity of molecules with remarkable physicochemical properties in a physiological context but also for applications in humans in various fields including in the medical, cosmetic, pharmaceutical and agronomic fields. Molecular data concerning P450s have increased through the use of high-throughput sequencing methods.

[0012] The data made available for many plants, particularly those known as medicinal plants, open up prospects for targeted production of molecules already identified or not identified / characterized.

[0013] The functional study of cytochromes P450 is however complex insofar as they are 1) membrane and intracellular proteins, 2) relatively fragile proteins, 3) proteins which, to be active, need to function in tandem with an NADPH P450 reductase which provides electrons necessary for oxidation reactions.

[0014] To achieve a functional characterization of these enzymes, tools / methods have therefore been developed. Since cytochrome P450s function with NADPH-P450 reductases, these two enzymes must be in close interaction to allow a transfer of electrons from the reductase to the P450 in order to allow the reaction, namely an oxidation reaction, to take place. In plants, both enzymes are anchored in the membrane of the endoplasmic reticulum and are therefore intracellular. A known method for the functional characterization of P450s includes, in particular, a heterologous production of said P450 in yeast. From this production, a bioconversion approach has been considered. To do this, a potential substrate of the P450 is added to the culture medium, said substrate penetrates the yeast and the product obtained can be stored in the yeast.Furthermore, when the obtained product is stored in yeast, additional steps of yeast lysis and purification for product recovery are necessary.

[0015] However, the implementation of this process is not possible, especially when the substrate is hydrophobic. Indeed, when it is hydrophobic, the substrate cannot enter the yeast (pass the membrane) used.

[0016] There is therefore a real need to find a means and / or process allowing the production and / or functional characterization of cytochromes P450 and / or allowing the bioconversion of a hydrophobic molecule or substrate by cytochromes P450.

[0017] Another known method for functional characterization of P450s or conversion of molecules / substrates by P450s further comprises cell wall removal and production of membrane extracts, namely microsomes. Said microsomes are incubated in the presence of NADPH and potential substrates. This method notably comprises complex steps of protein extraction, notably P450s. Furthermore, this method involves, due to the numerous steps of cell wall removal and / or protein extraction, notably degradation of P450s thus preventing multiple uses of P450s.

[0018] There is therefore a real need to find a means and / or process allowing the production and / or functional characterization of cytochromes P450 while allowing the conservation of active cytochromes P450. There is also a real need to find a means and / or process for bioconversion that is reusable and / or does not include a protein extraction step.

[0019] Statement of the invention

[0020] The present invention aims precisely to meet these needs by providing a fusion protein successively comprising (i) at least one polypeptide for addressing and anchoring to the bacterial membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

[0021] The inventors have surprisingly and unexpectedly demonstrated that the fusion protein according to the invention can be advantageously addressed to the plasma membrane of bacteria and / or to the external membrane of bacteria, advantageously via its addressing sequence, for example in the form of a beta barrel. Furthermore, the inventors have surprisingly demonstrated that the fusion protein according to the invention is addressed to the surface of said membranes, and advantageously the hydrophilic part is on the external surface of said membrane.

[0022] Advantageously, the inventors have demonstrated that once at the bacterial membrane, the portion of the fusion protein comprising a polypeptide comprising the hydrophilic domain of a plant cytochrome P450, a linker polypeptide and a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase is located outside the bacterial cell or bacterium and faces the external environment of the bacterium.

[0023] The inventors have also surprisingly demonstrated that the fusion protein according to the invention, advantageously when present on the external surface of the cell membrane, can be used in substrate bioconversion processes.

[0024] The inventors have also surprisingly demonstrated that when the fusion protein according to the invention is used in a bioconversion process, it advantageously allows bioconversion of the substrate directly in the medium, advantageously outside the bacterium, advantageously allowing bioconversion of the substrate, advantageously by avoiding membrane protein extraction steps, advantageously making it possible to limit the risks of protein degradation during the protein purification steps and to carry out the production of molecules of interest directly in the culture medium, thus reducing the purification steps. In the present invention, membrane means a bacterial membrane. It may be, for example, any membrane on the surface of the bacterium. It may be, for example, the external membrane of a gram-negative bacterium, the plasma membrane of a gram-positive bacterium.Advantageously the bacterial membrane is the outer membrane of gram negative bacteria.

[0025] In the present invention, by polypeptide for addressing and anchoring to the bacterial membrane is meant any polypeptide known to a person skilled in the art suitable for addressing and anchoring said polypeptide to the bacterial membrane. It may be, for example, a polypeptide for addressing and anchoring to the plasma membrane of Gram-positive bacteria. It may be, for example, a polypeptide for addressing and anchoring to the outer membrane of Gram-negative bacteria. It may be, for example, a polypeptide described in the document Jarmander, J., Gustavsson, M., Do, TH. et al. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. Microb Cell Fact 11, 118 (2012). https: / / doi.org / 10.1186 / 1475-2859-11-118

[0014] , For example, it could be a polypeptide whose quaternary structure forms a beta barrel.It may be for example a polypeptide having a percentage identity of at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with the polypeptide of sequence.

[0026] MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAWSTIGNAFAVDHHH HHHLEALFQGPGTQKQRTELENLYFQGEQKLISEEDLSRVNNNGSIVINNSIIN GNITNDADLSFGTAKLLSATVNGSLVNNKNIILNPTKESAAAIGNTLTVSNYTGT PGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGINIISV EGNSDAEFSLKNRWAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYR PENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSG KLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTS NKAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLE EEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDT HQEDNGTWQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHN THEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSN AISGALGIKYSF (SEQ ID NO 1 ).<h2 style=";text-align:left;direction:ltr">Avantageusement, the polypeptide d'address and d'ancrage of the membrane is a polypeptide of the sequence MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAWSTIGNAFAVDHHH HHHLEALFQGPGTQKQRTELENLYFQGEQKLISEEDLSRVNNNGSIVINNSIIN GNITNDADLSFGTAKLLSATVNGSLVNNKNIILNPTKESAAAIGNTLTVSNYTGT PGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGINIISV EGNSDAEFSLKNRWAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYR PENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSG KLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTS NKAARNTLDGYSVGVYGTWYQNGENATGLFAETWMQYNWFNASVKGDGLE EEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDT HQEDNGTWQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWIHN THEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSN AISGALGIKYSF (SEQ ID NO 1 ).Advantageously, the inventors have demonstrated that the membrane-directing and anchoring peptide effectively addresses and anchors the fusion protein to the outer membrane of the bacterium and also advantageously allows transport of the fusion protein from the cytosolic space to the outer space of the bacterial cell. In addition, the membrane-directing and anchoring peptide advantageously allows membrane passage by the fusion protein, advantageously allowing the portion of the fusion protein comprising a polypeptide comprising the hydrophilic domain of a plant cytochrome P450, a binding polypeptide, and a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase to be located outside the bacterial cell or bacterium and facing the external environment of the bacterium.In this context, by cytochrome P450 is meant proteins with mono-oxygenase activity capable of oxidizing substrates using molecular oxygen dissolved in the cytoplasm or in the medium, as well as reducing equivalents provided by NADPH-cytochrome P450-reductase. (Guengerich and Macdonald, “Mechanisms of cytochrome P450 catalysis”, FASEB J. 1990, 4, pp 2453-2459 [1]). It may be, for example, any plant cytochrome P450 known to those skilled in the art. It may be, for example, plant cytochromes P450 as described in Xu Jun et al. "The cytochrome P450 superfamily: Key players in plant development and defense" Journal of Integrative Agriculture 2015, 14(9): 1673-1686 [2], Examples include plant cytochromes P450 belonging to the families CYP51, CYP71, CYP72, CYP74, CYP85, CYP86, CYP97, CYP710, CYP711 and CYP727.Examples include plant cytochrome P450 belonging to the family CYP51, CYP71, CYP73, CYP75, CYP76, CYP77, CYP78, CYP79, CYP80, CYP81, CYP82, CYP83, CYP84, CYP89, CYP92, CYP93, CYP98, CYP99, CYP701, CYP703, CYP705, CYP706, CYP712, CYP719, CYP723, CYP726, CYP736, CYP72, CYP709, CYP714, CYP715, CYP721, CYP734, CYP735, CYP749, CYP74, CYP85, CYP87, CYP88, CYP90, CYP702, CYP707, CYP708, CYP716, CYP718, CYP720, CYP724, CYP725, CYP728, CYP729, CYP733, CYP86, CYP94, CYP96, CYP704, CYP730, CYP731, CYP732, CYP97, CYP710, CYP711 or CYP727. This could be, for example, a cytochrome P450 belonging to the CYP76 or CYP73 family. This could be, for example, cytochrome P450 CYP76F112 or CYP73A1.

[0027] In the present invention, by hydrophilic domain of a plant cytochrome P450 is meant the polypeptide sequence of the cytochrome P450 comprising the enzymatic domain and the biological activity, advantageously the enzymatic activity, of the cytochrome P450. A person skilled in the art, using this general knowledge, knows how to identify the enzymatic domain of the cytochrome P450. It may be, for example, a polypeptide isolated from a cytochrome P450.It may be, for example, a polypeptide having a percentage identity of at least 25%, for example 28%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% with a polypeptide chosen from the group comprising HRNLTDLAKRFGEILLLRMGQRNLWVSSPELAKEVLHTQGVEFGSRTRNVVF DIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRYGWEAEAAAW DDVKKNPAAATEGIVIRRRLQLMMYNNMFRIMFDRRFESEDDPLFLKLKALNG ERSRLAQSFEYNYGDFIPILRPFLRNYLKLCKEVKDKRIQLFKDYFVDERKKIGS TKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINVAAIETTLWSIEWGIAEL (SEQ ID NO 2),.

[0028] IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMGQRNLVWSSPELAKEVL HTQGVEFGSRTRNWFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKW QQYRYGWEAEAAAWDDVKKNPAAATEGIVIRRRLQLMMYNNMFRIMFDRRF ESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPILRPFLRNYLKLCKEVKDKRI QLFKDYFVDERKKIGSTKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINV AAIETTLWSIEWGIAELVNHPEIQAKLRHELDTKLGPGVQITEPDVQNLPYLQAV VKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPDQWKKP EEFRPERFLEEEAKVEANGNDFRYLPFGVGRRSCPGIILALPILGITIGRLVQNF ELLPPPGQSKIDTDEKGGQFSLHILKHSTIVAKPRSF (SEQ ID NO 3), MDIFTSLLYLALILFFSLQVFRSFAFPKHKRLPPGPKPRPIIGSLLELGDQPHRSL ARLSESYGPFMHLKLGQVTTWISSTTMAKEVLQANSQVVSSRTITDASRAHR HSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDGNMELRNKKVQELLNDVHK SVQAGEAVEIASLSFRATLNLLSTTFFSMDMADDTNSVTLKELKEAMSHMMEE LGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLFGRIIDQRLKVREASGSLKD DDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFSAGTETTSTTLEWAMAELVK APEIMSKARAELDQVIGKGNQVKESDVSRLPYLQAIVKETFRMHPTAPLLIPRK ADSDIEISDYIIPKDAQ (SEQ ID NO 4),

[0029] KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTWISSTTMAKEVLQ ANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDG NMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFFSMDMADDT NSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLF GRIIDQRLKVREASGSLKDDDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFS AGTETTSTTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESDVSRLPYLQ AIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQ (SEQ ID NO 5), KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTVVISSTTMAKEVLQ ANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDG NMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFFSMDMADDT NSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLF GRIIDQRLKVREASGSLKDDDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFS AGTETTSTTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESDVSRLPYLQ AIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQVIVNVWAIGRDSSTWENPD KFIPERFLDIDIDVGGRDFKLIPFGAGRRICPGFPLAMRMLHLMLGSLLHSFDW KLEDGVRPDALNMDEKFGLTLQMAQPLRAIPVPTKH (SEQ ID NO 70).

[0030] Advantageously, it may be a polypeptide isolated from a plant cytochrome P450 comprising the hydrophilic domain of said plant cytochrome P450 free of transmembrane domain. It may be, for example, a polypeptide having a percentage identity of at least 25%, for example 28%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% with a polypeptide chosen from the group comprising IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMGQRNLVWSSPELAKEVL HTQGVEFGSRTRNWFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKW QQYRYGWEAEAAAWDDVKKNPAAATEGIVIRRRLQLMMYNNMFRIMFDRRF ESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPILRPFLRNYLKLCKEVKDKRI QLFKDYFVDERKKIGSTKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINV ELLPPPGQSKIDTDEKGGQFSLHILKHSTIVAKPRSF (SEQ) ID NO. 3),KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTWISSTTMAKEVLQ ANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDG NMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLSTTFSMDMADDT NSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLF GRIIDQRLKVREASGSLKDDDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFS AGTETTSTTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESDVSRLPYLQ AIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQ (SEQ ID NO 5),and KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTWISSTTMAKEVLQ ANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDG NMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFFSMDMADDT NSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLF GRIIDQRLKVREASGSLKDDDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFS AGTETTSTTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESDVSRLPYLQ AIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQVIVNVWAIGRDSSTWENPD KFIPERFLDIDIDVGGRDFKLIPFGAGRRICPGFPLAMRMLHLMLGSLLHSFDW KLEDGVRPDALNMDEKFGLTLQMAQPLRAIPVPTKH (SEQ ID NO 70). it may be a polypeptide isolated from a plant cytochrome P450 comprising the hydrophilic domain of said plant cytochrome P450 selected from the group comprising the polypeptide,

[0031] IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMGQRNLVWSSPELAKEVL HTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKW QQYRYGWEAEAAAWDDVKKNPAAATEGIVIRRRLQLMMYNNMFRIMFDRRF ESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPILRPFLRNYLKLCKEVKDKRI QLFKDYFVDERKKIGSTKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINV AAIETTLWSIEWGIAELVNHPEIQAKLRHELDTKLGPGVQITEPDVQNLPYLQAV VKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPDQWKKP EEFRPERFLEEEAKVEANGNDFRYLPFGVGRRSCPGIILALPILGITIGRLVQNF ELLPPPGQSKIDTDEKGGQFSLHILKHSTIVAKPRSF (SEQ ID NO 3) et KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTWISSTTMAKEVLQ ANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSSKALDG NMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFFSMDMADDT NSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVTFRKLINLF GRIIDQRLKVREASGSLKDDDILDTLINMMWDQEKKEDQLDKTIIEHFLLDLFS AGTETTSTTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESDVSRLPYLQ AIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQVIVNVWAIGRDSSTWENPD KFIPERFLDIDIDVGGRDFKLIPFGAGRRICPGFPLAMRMLHLMLGSLLHSFDW KLEDGVRPDALNMDEKFGLTLQMAQPLRAIPVPTKH (SEQ ID NO 70).In the present invention, by a binding polypeptide is meant any suitable binding peptide known to those skilled in the art. It may be, for example, a binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids. It may be, for example, a polypeptide of sequence PGGSGGGSGGGGSGGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGSP (SEQ ID NO 6).

[0032] In this context, plant cytochrome P450 NADPH P450 reductase means proteins with oxidoreductase activity that catalyze the reaction

[0033] NADPH + H + + n oxidized hemoprotein NADP + + n reduced hemoprotein. This may be, for example, any plant cytochrome P450 NADPH P450 reductase known to those skilled in the art. This may be, for example, plant cytochrome P450 NADPH P450 reductase described in Kenneth Jensen et al., “Plant NADPH-cytochrome P450 oxidoreductases,” Phytochemistry 2010, Volume 71, 2-3, Pages 132-141 [3],

[0034] As used herein, by hydrophilic domain of plant cytochrome P450 NADPH P450 reductase is meant the polypeptide sequence of plant cytochrome P450 NADPH P450 reductase comprising the reductase domain and the biological activity, advantageously the reducing activity, of the plant cytochrome P450 NADPH P450 reductase. The term "reductase domain", as used herein, refers to an amino acid sequence that functions as an electron donor. In particular, it serves as an electron donor for the oxygenase portion of a cytochrome P450. A person skilled in the art, by this general knowledge, knows how to identify the catalytic domain, in particular the reductase domain, of a plant cytochrome P450 NADPH P450 reductase. For example, it may be a polypeptide isolated from a plant cytochrome P450 NADPH P450 reductase. For example, it may be a polypeptide having a percentage identity of at least 90%, for example 91%,<h2 style=";text-align:left;direction:ltr">92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with a polypeptide chosen in the group comprising TRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLK KETLAF FCVATYG DG E PTD N AARF YKWFTE ENE RD I KLQQ LAYG VF ALG N RQ YEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLL KDEDDKSVATPYTAVIPEYRWTHDPRFTTQKSMESNVANGNTTIDIHHPCRV DVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKL LGHSLLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSAL VALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPL GVFFAAIAAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTW SEQ ID NO. 7),YEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFYKW FTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLG DDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRWTHDPRF TTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGI TYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPF PGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEY SQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRV HVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPS NPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIY EDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLY VCGDAKGMARDVHRTLHTIVQEQEGVSSSE (SEQ ID NO 8).,

[0035] Advantageously, it may be a polypeptide isolated from a cytochrome P450 NADPH P450 reductase comprising the hydrophilic domain of said cytochrome P450 NADPH P450 reductase free of transmembrane domain.<h2 style=";text-align:left;direction:ltr">It can be used by an example of a polypeptide that contains a percentage of the identity of the moons: 90%, by example of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with the polypeptide sequence. TRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLK KETLAF FCVATYG DG E PTD N AARF YKWFTE ENE RD I KLQQ LAYG VF ALG N RQ YEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLL KDEDDKSVATPYTAVIPEYRWTHDPRFTTQKSMESNVANGNTTIDIHHPCRV DVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKL LGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSAL VALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPL G VF FAAI AP RLQ P RYYS ISSSPRLAPS RVH VTS ALVYG PTPTG RIH KG VCSTW MKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQE RMALKEDGEELGSSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREG AQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQ EGVSSSEAEAIVKKLQTEGRYLRDVW (SEQ ID NO 7).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0036] Advantageously, the inventors have demonstrated that the linker polypeptide makes it possible to obtain a fusion protein with a quaternary structure allowing a functional enzymatic activity of the hydrophilic domain of a plant cytochrome P450 and the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase advantageously allowing the effective bioconversion of substrates of different structures and sizes.

[0037] Herein, the fusion protein may comprise one or more unnatural amino acids, e.g., one or more D-amino acids and / or chemically modified amino acids.

[0038] Unnatural amino acids can be left-handed (L-), right-handed (D-), or mixtures thereof. Unnatural amino acids are those amino acids that are not generally synthesized in the normal metabolic processes of living organisms and are not naturally present in proteins. In addition, unnatural amino acids are also not recognized by common proteases. The unnatural amino acid can be present at any position in the fusion protein. For example, the unnatural amino acid can be at the N-terminus, the C-terminus, or any position between the N-terminus and the C-terminus.

[0039] Unnaturally occurring amino acids may, for example, be chemically modified amino acids and may, for example, include alkyl, aryl, or alkylaryl groups not found in naturally occurring amino acids. Some examples of unnaturally occurring alkylated amino acids include α-aminobutyric acid, β-aminobutyric acid, γ-aminovaleric acid, and ε-aminocaproic acid. Some examples of unnaturally occurring aryl amino acids include ortho-, meta-, and para-aminobenzoic acid. Some examples of unnaturally occurring alkylaryl amino acids include ortho-, meta-, and para-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid. Unnaturally occurring amino acids include derivatives of naturally occurring amino acids. Derivatives of naturally occurring amino acids may, for example, include the addition of one or more chemical groups to the naturally occurring amino acid.For example, one or more chemical groups may be added to one or more of the 2', 3', 4', 5', or 6' positions of the aromatic ring of a phenylalanine or tyrosine residue, or to the 4', 5', 6', or 7' position of the benzo ring of a tryptophan residue. The group may be any chemical group that can be added to an aromatic ring. Some examples of such groups include branched or unbranched C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C1-C4 alkyloxy (i.e., alkoxy), amino, C1-C4 alkylamino, and C1-C4 dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples of unnatural derivatives of naturally occurring amino acids include norvaline (Nva) and norleucine (Nie).

[0040] The fusion protein according to the invention can be produced and / or synthesized by any suitable method known to those skilled in the art.

[0041] As used herein, the terms "polypeptide", "peptide" and their grammatical equivalents refer to a polymer of amino acid residues.

[0042] As used herein, a "functional protein" is a protein that is biologically active.

[0043] Herein, the percentage of sequence identity may be determined by any method known to those skilled in the art. It may be determined for example by the use of BLASTP and BLASTN, for example using default parameters.

[0044] As used herein, the term "percent identity" means the percentage determined by direct comparison of two sequences (nucleic or protein), determining the number of nucleotides or amino acid residues common to both sequences, then dividing it by the number of nucleotides or amino acid residues in the longer of the two sequences and multiplying the result by 100.

[0045] As used herein, the word "includes" and its variations, such as "includes" and "comprising", shall be interpreted in an open and inclusive sense, i.e., as "including, but not limited to".

[0046] As used herein, "consisting of" or "constituted" means including, and limited to, what follows the expression "consisting of" or "constituted." Thus, the expression "consisting of" or "constituted" indicates that the elements listed are required or mandatory, and that no other elements may be present.

[0047] Another subject of the invention relates to a polynucleotide or nucleic acid encoding a fusion protein according to the invention.

[0048] It may be, for example, a polynucleotide or nucleic acid coding for a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

[0049] It may be, for example, a polynucleotide or nucleic acid coding for a fusion protein according to the invention in which the polynucleotide or nucleic acid sequence coding for a polypeptide for targeting and anchoring to the bacterial membrane, advantageously to the external membrane of Gram-negative bacteria, is chosen from the group comprising the nucleic acid of sequence: accatgggcaataaggcctacagtatcatttggagccactccagacaggcctggattgtggcctcagagttag ccagaggacatggttttgtccttgcaaaaaatacactgctggtattggcggttgtttccacaatcggaaatgcattt gcagtcgaccaccatcaccatcaccatctggaagcgctgttccagggtccgggtaccgctacagtgaatggta gtcttgttaataacaaaaatatcattcttaatcctacaaaagaaagtgcggccgctataggtaatactcttaccgt gtcaaattatactgggacaccgggaagtgttatttctcttggtggtgtgcttgaaggagataattcacttacggacc gtctggtggtgaaaggtaatacctctggtcaaagtgacatcgtttatgtcaatgaagatggcagtggtggtcaga cgagagatggtattaatattatttctgtagagggaaattctgatgcagaattctctctgaagaaccgcgtagttgccggagcttatgattacacactgcagaaaggaaacgagagtgggacagataataagggatggtatttaaccagt catcttcccacatctgatacccggcaatacagaccggagaacggaagttatgctaccaatatggcactggcta actcactgttcctcatggatttgaatgagcgtaagcaattcagggccatgagtgataatacacagcctgagtctg catccgtgtggatgaagatcactggaggaataagctctggtaagctgaatgacgggcaaaataaaacaaca accaatcagtttatcaatcagctcgggggggatatttataaattccatgctgaacaactgggtgattttaccttagg gattatgggaggatacgcgaatgcaaaaggtaaaacgataaattacacgagcaacaaagctgccagaaac acactggatggttattctgtcggggtatacggtacgtggtatcagaatggggaaaatgcaacagggctctttgct gaaacttggatgcaatataactggtttaatgcatcagtgaaaggtgacggactggaagaagaaaaatataatc tgaatggtttaaccgcttctgcaggtgggggatataacctgaatgtgcacacatggacatcacctgaaggaata acaggtgaattctggttacagcctcatttgcaggctgtctggatgggggttacaccggatacacatcaggagga taacggaacggtggtgcagggagcagggaaaaataatattcagacaaaagcaggtattcgtgcatcctgga aggtgaaaagcaccctggataaggataccgggcggaggttccgtccgtatatagaggcaaactggatccat aacactcatgaatttggtgttaaaatgagtgatgacagccagttgttgtcaggtagccgaaatcagggagagataaagacaggtattgaaggggtgattactcaaaacttgtcagtgaatggcggagtcgcatatcaggcaggcagg tcacgggagcaatgccatctccggagcactggggataaatacagcttctgataatga (SEQ ID NO 9), atgaataaggcctacagtatcatttggagccactccattccagcggcgtcggcgagca gacatggttttgtccttgcaaaaatacactgctggtattggcggttttccacaatcggaaatgcatttgcagtc gaccaccatcaccatcaccatggaagcgctgttccagggtccgggtacccagaaacagcgtaccgagctc gaaaacctgtacttccagggtgaaaaaatactgactgatactgagatgagatc agcattgtcattaataacagcattataaacgggaatacgaatgatgctgacttaagttttggtacagcaaagc tgctctctgctacagtgaatggtagtcttgttaataaaaaatatcattctttaatcctacaaaagaaagtgcggc cgctataggtaatactcttaccgtgtcaaattatactgggacaccgggaagttatttcttggtggtgcttga aggagataattcacttacggaccgtctggtggtgaaagtaatacctctggtcaaagtgacatcgtttatgtcaat gaagatggcagtggtggtcagacgagagatggtattaatattattctgtagggaaattctgatgcagaattct ctctgaagaacccgtagttgccggagcttatgacacactgcagaaaggaaacgagagtgggacagat aataagggatttaaccagtcatcttcccacatctgacccgaccgaccgacacgattctatgctaccaatatggcactggctaactcactgttcctcatggatttgaatgagcgtaagcaattcagggccatga gtgataatacacagcctgagtctgcatccgtgtggatgaagatcactggaggaataagctctggtaagctgaat gacgggcaaaataaaacaaccaatcagtttatcaatcagctcgggggggatatttaataattccatgctg aaactgggtgattttaccttagggattatgggaggatacgcgaatgcaaaaggtaaaacgataaattacac gagcaacaaagctgccagaacacactggatggttattctgtcggggtatacggtacgtggtatcagaatggg gaaaatgcaagggctctttgctgaaacttggatgcaatataactggtttaatgcatcagtgaaaggtgacgg actggaagaagaaaaataatctgaatggttaaccctgctgctgaggagcacct catggacatcacctgaaggaataacaggtgaattctggttacagcctcatttgcaggctgtctgggggtta caccggatacacatcaggataacggaacggtggtgcaggagcagggaaaatatattcagacaa aagcaggtattcgtgcatcctggaaggtgaaagcaccctcggtagggggttcgg atatagaggcaaactggatccataacactcatgaatttggtgttaaaatgagtgatgacagccagttgttgtcag gtagccgaaatcagggagagataaagacaggtattgaaggggtgattactcaaaacttgtcagtgaatggcg gagtcgcatatcaggcaggcaggtcacgggagcaatgccactcctgactgactcactag gataatga (SEQ ID NO 10).

[0050] It may be, for example, a polynucleotide or nucleic acid coding for a fusion protein according to the invention in which the polynucleotide or nucleic acid sequence coding for a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 is chosen from the group comprising the nucleic acid of sequence: cctggcccaatcccggttccaattttcggcaactggctacaagttggcgatgatttgaaccaccggaacttaacc gatctggctaagaggtttggtgagatcttgctgctacgcatggggcagaggaatctggtagttgtgtcttcgcctg agcttgctaaagaggtgttgcatacacaaggagtggagtttggttcgagaacaaggaatgttgtgttcgatattttt actgggaagggtcaggatatggtgtttacggtttatggtgagcattggaggaagatgaggaggatcatgaccgt accctttttcaccaacaaagttgttcagcaatacaggtatgggtgggaggctgaggccgcggcggttgtggacg atgtgaagaagaatccggctgcagcaactgaaggaatcgtgatccgaagacggttacaactcatgatgtata acaacatgttcagaatcatgttcgacagacgattcgaaagtgaagatgatcccttgtttttgaaactcaaggcgtt gaacggtgagaggagtcgattggcgcagagctttgagtacaactatggcgatttcatccctattttgcggccgtttttgagaaattatttgaagttgtgcaaggaagttaaagataaaaggattcagctcttcaaggattacttcgttgacg aaaggaagaagattggaagcactaagaaaatggacaacaatcagttgaaatgtgccattgatcacattcttg aagctaaagagaagggtgagatcaatgaagacaatgttctttacattgttgaaaacatcaatgttgcagcaatc gagacaactctatggtcgatcgaatggggaattgcggagctagttaaccatcccgagatccaagccaaactc aggcacgagctcgacaccaagctcgggcccggtgtccagatcaccgagcccgacgtccaaaacctccctta cctccaagccgtggtcaaggaaaccctccgtctccgtatggcgatcccgcttctagtcccacacatgaacctcc atgacgctaagctcggcgggtttgacatcccggccgaaagcaagatcttggtcaacgcgtggtggttagcaaa caaccccgaccaatggaagaaacccgaggagtttaggccagagaggtttttggaagaggaagcgaaggtt gaggctaacgggaatgattttaggtacttgccgtttggagtcgggagaaggagttgccccgggattattcttgcat tgccgatacttggtattacaatcgggcgtttggtgcagaatttcgagctgttgcctccaccgggacagtctaagat cgataccgatgagaagggtgggcagtttagtttgcatatcttgaagcactctactatcgtagctaaacctaggtc attt (SEQ ID NO 11 ), atggacctcctcctcatagaaaaaaccctcgtcgccttattcgccgccattatcggcgcaatactaatctccaaa ctccgcggtaaaaaattcaagctcccacctggcccaatcccggttccaattttcggcaactggctacaagttggcgatgatttgaaccaccggaacttaaccgatctggctaagaggtttggtgagatcttgctgctacgcatggggca gaggaatctggtagttgtgtcttcgcctgagcttgctaaagaggtgttgcatacacaaggagtggagtttggttcg agaacaaggaatgttgtgttcgatatttttactgggaagggtcaggatatggtgtttacggtttatggtgagcattgg aggaagatgaggaggatcatgaccgtaccctttttcaccaacaaagttgttcagcaatacaggtatgggtggg aggctgaggccgcggcggttgtggacgatgtgaagaagaatccggctgcagcaactgaaggaatcgtgatc cgaagacggttacaactcatgatgtataacaacatgttcagaatcatgttcgacagacgattcgaaagtgaag atgatcccttgtttttgaaactcaaggcgttgaacggtgagaggagtcgattggcgcagagctttgagtacaact atggcgatttcatccctattttgcggccgtttttgagaaattatttgaagttgtgcaaggaagttaaagataaaagg attcagctcttcaaggattacttcgttgacgaaaggaagaagattggaagcactaagaaaatggacaacaatc agttgaaatgtgccattgatcacattcttgaagctaaagagaagggtgagatcaatgaagacaatgttctttaca ttgttgaaaacatcaatgttgcagcaatcgagacaactctatggtcgatcgaatggggaattgcggagctagtta accatcccgagatccaagccaaactcaggcacgagctcgacaccaagctcgggcccggtgtccagatcac cgagcccgacgtccaaaacctcccttacctccaagccgtggtcaaggaaaccctccgtctccgtatggcgatcccgcttctagtcccacacatgaacctccatgacgctaagctcggcgggtttgacatcccggccgaaagcaag atcttggtcaacgcgtggtggttagcaaaccccgaccaatggaagaaacccgaggagtttaggccaga gaggtttttggagagaggcgaaggttgaggctaaccgggacgcgaggtcgaggtt gaaggagttgccccgggattattcttgcattgccgatacttggtattacaatcgggcgtttggtgcagaatttcgag ctgttgcctccaccgggacagtctcgataccgatcgatgaagggtgggcagtttagtttgcatatcttgaag cactctactatcgtagctaacctaaactcat (IDSEQ 12), atggatattttcacctccttactgtatctttgctctcattctttctttttcttctaaaca caaaaggcttccacctggtccaaaacctcgtcccatcggaagcctcttggagctcggcgaccaacccca caggtccttggccaggctttccgagtcttacggcccgttatgcatttgaagctcggccaagtcacgacggttgtc atttcctccaccaccatggctaaagagtcctccaggcaaacagccaagtcgtctccagccggacaatcacc gacgcaagccgcgcccacacacaggattttagcatggttatgttgcccgtatcccctgtggcgaaacct tcggaaaataagcaactcacacttgctttcctccaagctcttgatggcaacatggagctgagaaaaaaag gtgcaagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcggtggagatcgcgagccttctttcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatgacacaaattccgtcactcta aaagagctcaaggaggctatgtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtt tctacaaaagattgacccccaaggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttg ggcgtatcatcgaccaaagattgaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacact cttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaaaccataattgaacatttttt actggatttattttcagcggggactgaaacgacttcaaccacgttggagtgggcaatggctgagctagtaaaag cgccagagattatgtcaaaagcccgagcagagctagatcaagttataggcaaaggaaaccaagtgaagga atcggacgtatctcgactcccttacttacaagccattgttaaagaaaccttccgcatgcaccctacagctccatta ttgattcctcgcaaagccgacagtgacatcgaaatctccgactatatcatcccgaaggatgctcaggtgattgtc aatgtatgggccattggtagagactcaagcacatgggaaaatcccgacaagtttataccggagaggtttttgga catcgatatagatgtcggaggccgggattttaagctcattccgttcggtgctggtcggagaatatgtcccggattc ccattggcgatgcgaatgttgcacttgatgttggggtctttgcttcactcgtttgattggaagttggaagatggggtta gacctgatgctctaaacatggatgaaaagtttggcctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcattag (SEQ ID NO 13), atcatcggaagcctcttggagctcggcgaccaacccacaggtccttggccaggctttcgagtcggcgg gtttatgcatttgaagctcggccaagtcacgacggttgtcattcctccaccatgctaaagaagtcctccag gcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcccacacacagcgatttta gcatggttatgcccgtatccccctgtggcgaaaccttcggaaaataagcaactcacacttgctttcctccaa ggctcttgatggcaacatggagctgagaaaaaaaggtgcaagagctctaaatgatgtccacaaaagcg tccaggccggggaggcggtggagatcgcgagcctttttcagagctactctgaatctcttgtccaccacatttttc tccatggacatggcggatgacaaattccgtcactctaaagagctcaaggaggctatgtcgcacatgatgg aagagttggggaagcctaacttggccgattatttcccgtctacaaaagattgacccccaaggcattaggcgg cgcaacacggttactttccggaactgatcaacttgtttgggcatcgacgatgacgacgacgac agcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaa agaggatcagcttgacaaaccataattgaacattttactggatttatttcagggggactgaaacgacttca accacgttggagtgggcaatgggactgaaacgacttca accacgttggagtgggcaatgctgagctagcagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccatt gttaaagaaaccttccgcatgcaccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaat ctccgactatatcatcccgaaggatgctcaggtgattgtcaatgtatgggccattggtagagactcaagcacatg ggaaaatcccgacaagtttataccggagaggtttttggacatcgatatagatgtcggaggccgggattttaagct cattccgttcggtgctggtcggagaatatgtcccggattcccattggcgatgcgaatgttgcacttgatgttggggt ctttgcttcactcgtttgattggaagttggaagatggggttagacctgatgctctaaacatggatgaaaagtttggc ctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcattag (SEQ ID NO 14), caagtcacgacggttgtcatttcctccaccaccatggctaaagaagtcctccaggcaaacagccaagtcgtct ccagccggacaatcaccgacgcaagccgcgcccacagacacagcgattttagcatggttatgttgcccgtat cccctctgtggcgaaaccttcggaaaataagcaactcacacttgctttcctccaaggctcttgatggcaacatgg agctgagaaacaaaaaggtgcaagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcgg tggagatcgcgagcctttctttcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatg acacaaattccgtcactctaaaagagctcaaggaggctatgtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgacccccaaggcattaggcggcgcaacacggttactttccg gaaactgatcaacttgtttgggcgtatcatcgaccaaagattgaaagtgagagaagcgagtggttctttgaaag atgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaa accataattgaacattttttactggatttattttcagcggggactgaaacgacttcaaccacgttggagtgggcaat ggctgagctagtaaaagcgccagagattatgtcaaaagcccgagcagagctagatcaagttataggcaaag gaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaaccttccgcatg caccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaatctccgactatatcatcccgaa ggatgctcag (SEQ ID NO 45), aaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttccg agtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaa agaagtcctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacag acacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaataagcaactcaca cttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgcaagagctcctaaatgatg tccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctttcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctat gtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgaccccca aggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagatt gaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggtggat caggagaagaaagaggatcagcttgacaaaaccataattgaacattttttactggatttattttcagcggggact gaaacgacttcaaccacgttggagtgggcaatggctgagctagtaaaagcgccagagattatgtcaaaagc ccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactccctta cttacaagccattgttaaagaaaccttccgcatgcaccctacagctccattattgattcctcgcaaagccgacag tgacatcgaaatctccgactatatcatcccgaaggatgctcaggtgattgtcaatgtatgggccattggtagaga ctcaagcacatgggaaaatcccgacaagtttataccggagaggtttttggacatcgatatagatgtcggaggc cgggattttaagctcattccgttcggtgctggtcggagaatatgtcccggattcccattggcgatgcgaatgttgca cttgatgttggggtctttgcttcactcgtttgattggaagttggaagatggggttagacctgatgctctaaacatggat gaaaagtttggcctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcat (SEQ ID NO 69), etaaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttccg agtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaa agaagtcctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacag acacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaataagcaactcaca cttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgcaagagctcctaaatgatg tccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctttcagagctactctgaatctcttg tccaccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctat gtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgaccccca aggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagatt gaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggtggat caggagaagaaagaggatcagcttgacaaaaccataattgaacattttttactggatttattttcagcggggact gaaacgacttcaaccacgttggagtgggcaatggctgagctagtaaaagcgccagagattatgtcaaaagc ccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaaccttccgcatgcaccctacagctccattattgattccgcaaagccgacag tgacatcgaaatctccgactatatatcatcccgaaggatgctcaggtgattgtcaatgtatgggccattgggaga ctcaagcacatgggaaaatcccgacaagttttac cgggattttaagctcattccgttcggtgctggtcggaatatgtcccggatcccattggcgatgcgaatgttgca cttgatgttggggtctttgctcctcgtgattggaagttggaagatggggttagacctgctctaaacatggat gaaaagtttggcctcaccttgcaaatggctcagcctttggagctatccccgtgccgacaaagcat (SEQ ID 71 ) of sequence preference SEQ ID NO 11 or SEQ ID NO 71

[0051] It may be, for example, a polynucleotide or nucleic acid coding for a fusion protein according to the invention in which the polynucleotide sequence or nucleic acid coding for a binding polypeptide is chosen from the group comprising the nucleic acid of sequence: ccgggcggttctggtggcggtagcggcggtggcggttctggcggtggcggtagcggcggtggcggttctggcg gtggcggtagcggcggtggcggttctggcggtggcggtagcggcggtggcggttctggtggcggtagcggcg gttctccg (SEQ ID NO 15).

[0052] It may be, for example, a polynucleotide or nucleic acid coding for a fusion protein according to the invention in which the polynucleotide or nucleic acid sequence coding for a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase is chosen from the group comprising the nucleic acid of sequence: atgacttctgctttgtatgcttccgatttgtttaagcagctcaagtcaattatggggacagattcgttatccgacgatg ttgtacttgtgattgcaacgacgtctttggcactagtagctggatttgtggtgttgttatggaagaaaacgacggcg gatcggagcggggagctgaagcctttgatgatccctaagtctcttatggctaaggacgaggatgatgatttggat ttgggatccgggaagactagagtctctatcttcttcggtacgcagactggaacagctgagggatttgctaaggc attatccgaagaaatcaaagcgagatatgaaaaagcagcagtcaaagtcattgacttggatgactatgctgcc gatgatgaccagtatgaagagaaattgaagaaggaaactttggcatttttctgtgttgctacttatggagatggag agcctactgacaatgctgccagattttacaaatggtttacggaggaaaatgaacgggatataaagcttcaaca actagcatatggtgtgtttgctcttggtaatcgccaatatgaacattttaataagatcgggatagttcttgatgaagagttatgtaagaaaggtgcaaagcgtcttattgaagtcggtctaggagatgatgatcagagcattgaggatgatttt aatgcctggaaagaatcactatggtctgagctagacaagctcctcaaagacgaggatgataaaagtgtggca actccttatacagctgttattcctgaataccgggtggtgactcatgatcctcggtttacaactcaaaaatcaatgga atcaaatgtggccaatggaaatactactattgacattcatcatccctgcagagttgatgttgctgtgcagaagga gcttcacacacatgaatctgatcggtcttgcattcatctcgagttcgacatatccaggacgggtattacatatgaa acaggtgaccatgtaggtgtatatgctgaaaatcatgttgaaatagttgaagaagctggaaaattgcttggcca ctctttagatttagtattttccatacatgctgacaaggaagatggctccccattggaaagcgcagtgccgcctcctt tccctggtccatgcacacttgggactggtttggcaagatacgcagaccttttgaaccctcctcgaaagtctgcgtt agttgccttggcggcctatgccactgaaccaagtgaagccgagaaacttaagcacctgacatcacctgatgg aaaggatgagtactcacaatggattgttgcaagtcagagaagtcttttagaggtgatggctgcttttccatctgca aaacccccactaggtgtattttttgctgcaatagctcctcgtctacaacctcgttactactccatctcatcctcgcca agattggcgccaagtagagttcatgttacatccgcactagtatatggtccaactcctactggtagaatccacaag ggtgtgtgttctacgtggatgaagaatgcagttcctgcggagaaaagtcatgaatgtagtggagccccaatctttattcgagcatctaatttcaagttaccatccaacccttcaactccaatcgttatggtgggacctgggactgggctgg caccttttagaggttttctgcaggaaaggatggcactaaaagagagaagaactaggttcatctttgctctt ctttgggtgtagaaatcgacagatggactttactacgattgattgatacgatacgattcgaatcgaattagg ctgagctcatcatggcattctcccgtgaaggagctcagaaggagtatgttcaacataagatgagaaggc agcacaagttgggatctaaaggaagaaggatatctctatgtatgcggtgatgctaagggcatggcgagg gacgtccaccgaactctacacaccattgttcccgtgaggtcgtcgaggcgaggc gttaagaaacttcaaaccgaaggagatacctcagagatgtcttcttcggtacgcagactggaacagctgagggatttgctaaggcattatccgaagaaatc aaagcgagatatgaaaaagcagcagtcaaagtgactgactgactgagatgcgattggattc aagagaaattgaagaaggaaactttggcatttttctgtgttgctacttatggagatggagagcctactgacaatg ctgccagattttacaaatggttacggaggaaaatgaacgggatataaagcttcaaactagcatatggtgtgt ttgctcttggtaatcgccaatatgaacattggtgtgtc gcaaagcgtcttattgaagtcggtctaggagatgatgatcagagcattgaggatgatttaatgcctggaaagaatcactatggtctgagctagacaagctcctcaaagacgaggatgataaaagtgtggcaactccttatacagctg ttattcctgaataccgggtggtgactcatgatcctcggtttacaactcaaaaatcaatggaatcaaatgtggccaa tggaaatactattgacattcatcatccctgcagagttgatgttgctgtgcagaaggagcttcacacacatgaa tctgatcggtctcttgcattcatctcgagttcgacatatccaggacgggtattacatatgaaacaggtgaccatgtag gtgtatatgctgaaaatcatgttgaaatagttgaagaagctggaaaattgcttggccactctttagatttagtattttc catacatgctgacaaggaagatggctccccattggaaagcgcagtgccgcctcctttccctggtccatgcaca cttgggactggtttggcaagatacgcagaccttttgaaccctcctcgaaagtctgcgttagttgccttggcggcct atgccactgaaccaagtgaagccgagaaacttaagcacctgacatcacctgatggaaaggatgagtactca caatggattgttgcaagtcagagaagtcttttagaggtgatggctgcttttccatctgcaaaacccccactaggtgt attttttgctgcaatagctcctcgtctacaacctcgttactactccatctcatcctcgccaagattggcgccaagtag agttcatgttacatccgcactagtatatggtccaactcctactggtagaatccacaagggtgtgtgttctacgtgga tgaagaatgcagttcctgcggagaaaagtcatgaatgtagtggagccccaatctttattcgagcatctaatttca agttaccatccaacccttcaactccaatcgttatggtgggacctgggactgggctggcaccttttagaggttttctgcaggaaaggatggcactaaaagagatggagaactaggttcatctttgctcttcttgggtgtagaaatcgaggatgagctcaataatttgtgatcaaggcgtaatatctgagctcatcatgcat tctcccgtgaaggagctcagaaggagtatgttcaacacagagagagattc aataaaggaagaaggatatctctatgtatgcggtgatgctaagggcatggcgagggacgtccaccgaactt acacaccattgttcaggagcaggaaggtgagttcgtcagaggcagaggctatagttaagaaacttcaaac cgaaggaagatacctcagagatgtctgg (SEQ ID NO 17), de férence ID NO séquence SEQ 17.

[0053] In this document, the terms "polynucleotide(s)", "oligonucleotide(s)", "nucleic acid(s)", "polynucleic acid(s)", or any grammatical equivalent as used herein, refer to a polymeric form of nucleotides or nucleic acids of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, triplex DNA, as well as double- and single-stranded RNA. It also includes modified, for example by methylation and / or capping, and unmodified forms of the polynucleotide. The term also encompasses molecules that include non-naturally occurring or synthetic nucleotides as well as nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein may be introduced into a cell by, for example, transfection, transformation, or transduction.

[0054] Herein, the nucleic acid or polynucleotide may be produced and / or obtained by any suitable method known to those skilled in the art.

[0055] Another subject of the invention relates to a vector comprising a nucleic acid coding for a fusion protein according to the invention. It may be, for example, a vector comprising a polynucleotide or nucleic acid coding for a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

[0056] In the present invention, the vector may be any vector known to those skilled in the art suitable for the expression of a nucleic acid. It may be, for example, any vector replicated in low copy number in bacteria known to those skilled in the art and / or commercially available. It may be, for example, the vector described in the document Rosano GL and Ceccarelli EA “Recombinant protein expression in Escherichia coli: advances and challenges.” Front. Microbiol. 2014, 5:172. doi: 10.3389 / fmicb.2014.00172

[0015] , for example replicated in about ten copies per bacteria. This may be, for example, any vector chosen from the vectors listed in the catalog https: / / blog.addgene.org / plasmid-101-origin-of-replication [4] or https: / / www.qiagen.com / cn / resources / faq?id=1f42840e-fbd7-4734-b0cd- e17372a9e5a4&lang=en [5]. This may be, for example, the expression vector described in WO 83 / 004261 [7],

[0057] The vector may be any suitable plasmid known to those skilled in the art and / or commercially available. It may be, for example, a plasmid chosen from the group comprising pACYC, pSC101, SuperCos, pWE15, pGEX, pColEI, pR-K, pAIDAI, preferably pAIDAI.

[0058] It may be, for example, a modified plasmid, for example the plasmid pAIDAI, comprising a tetracycline resistance gene and a cloning cassette comprising the T7 RNA polymerase promoter and the T7 RNA polymerase terminator. This may be, for example, the plasmid pAIDAI in which the cmIA gene is replaced by the tetracycline resistance gene of sequence ttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggc accgtgtatgaaatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggctt ggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacagcatcgccagtcactatggcgtgct gctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgc ccagtcctgctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggatc ctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgac atcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcagg ccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaactacttactgggctgcttcctaatgcaggagtcgcataagggagagcgcgaccgatgcccttgagagc cttcaacccagtcagctccttcggtgggcgcggggcatgactatcgtcgcgcacttatgactgtcttctttatca tgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggcgctttcgctggagcgcgac gatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccacc aaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgctgggctacgtcttgctggcgt tcgcgcgaggctggatggccttccccattatgattcttcgcttcggcggcatcgggatgcccgcgttgc aggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggcttacca gcctaacttcgatcattggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttg gcatggattgtaggcgccgccctataccttgtctgcctccccgttgcgtcgcggtgcatggagccgggccac ctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaattct tgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatcgcgtccgccatctccagcagcc gcacgggcgcatctcgggcagcgt (SEQ ID NO 18) and in which the AIDA cassette is removedgagatataccatgggcaataaggcctacagtatcatttggagccactccagacaggcctggattgtggcctca gagttagccagaggacatggttttgtccttgcaaaaaatacactgctggtattggcggttgtttccacaatcggaa atgcatttgcagtcgaccaccatcaccatcaccatctggaagcgctgttccagggtccgggtacccagaaaca gcgtaccgagctcgaaaacctgtacttccagggtgaacagaaactgattagcgaagaagatctgtctagagt gaataacaatggaagcattgtcattaataacagcattataaacgggaatattacgaatgatgctgacttaagtttt ggtacagcaaagctgctctctgctacagtgaatggtagtcttgttaataacaaaaatatcattcttaatcctacaa aagaaagtgcggccgctataggtaatactcttaccgtgtcaaattatactgggacaccgggaagtgttatttctct tggtggtgtgcttgaaggagataattcacttacggaccgtctggtggtgaaaggtaatacctctggtcaaagtga catcgtttatgtcaatgaagatggcagtggtggtcagacgagagatggtattaatattatttctgtagagggaaatt ctgatgcagaattctctctgaagaaccgcgtagttgccggagcttatgattacacactgcagaaaggaaacga gagtgggacagataataagggatggtatttaaccagtcatcttcccacatctgatacccggcaatacagaccg gagaacggaagttatgctaccaatatggcactggctaactcactgttcctcatggatttgaatgagcgtaagca attcagggccatgagtgataatacacagcctgagtctgcatccgtgtggatgaagatcactggaggaataagctctggtaagctgaatgacgggcaaaataaaaacaaccaatcagtttatcaatcgctcgggggggatattt aaattccatgctgaacaactgggtgatttaccttagggattatgggaggatacgcgaatgcaaaaggtaaa acgataaattacacgagcaacaaagctgccagaaacacactggatggttattctgtcggggtatacggtacgt ggtatcagaatggggaaaatgcaacagggctttgctgaaacttggatgcaatataactggtttaatgcatcag tgaaaggtgacggactggaagaagaaaaataattactcttacttacggcgcggg cctgaatgtgcacacatggacatcacctgaaggaataacaggtgaattctggttacagcctcatttgcaggctgt ctggatgggggttacaccggatacacatcagagataacggaacggtggtgcaggagcagggaaaat aatattcagacaaaagcaggtattcgtgcatcctgagggacgacctactactacg gaggttccgtccgtatagaggcaactggatccataacactcatgaatttggtgttaaaatgagtgatgacag ccagttgttgtcaggtagccgaaatcagggagagataaagacaggtattgaaggggtgattactcaaaacttg tcagtgaatggcggagtcgcatatcaggcaggtcgcggcgcactcgcgg aaaatacagcttctgataatgacagatccggctgctaaaaagcccgaaggaagctgagttggctgctgcc accgctgagcaataactagcataaccccttggggctctaaacgggtcttgaggggttttttg (SEQ ID NO 44) is replaced by a cassette ofcloning comprising the promoter sequence taatacgactcactata (SEQ ID NO 19) and the terminator sequence tagcataaccccttggggcctctaaacgggtcttgaggggttttttg (SEQ ID NO 20) of the T7 RNA polymerase gene.

[0059] The vector may be any suitable yeast artificial chromosome known to those skilled in the art. It may be, for example, a Yeast Artificial Chromosome selected from the group comprising pYAC-RC, pYAC3(+).

[0060] The vector may be any suitable bacterial artificial chromosome known to those skilled in the art. It may be, for example, a bacterial artificial chromosome selected from the group comprising pUvBBAC, pCCI BAC, pBAC 108L.

[0061] The expression vector of a fusion protein according to the invention may comprise the following operably linked elements: a) a promoter b) a sequence coding for a fusion protein according to the invention c) transcription termination signals.

[0062] As used herein, "promoter" means a cis-acting DNA sequence located 5' to the transcription initiation site of the polypeptide coding sequence to which a DNA sequence of an RNA polymerase can bind and initiate correct transcription, and optionally comprising activators. This may be, for example, any suitable promoter known to those skilled in the art. This may be, for example, a constitutive promoter, a viral promoter, a bacterial promoter. This may be, for example, the T7 promoter, the bacteriophage T7 promoter, preferably the T7 promoter.

[0063] As used herein, "transcription termination signals" means a DNA sequence suitable for transcription termination. This may be any transcription termination signal known to those skilled in the art. For example, it may be the T7 terminator of sequence tagcataaccccttggggcctctaaacgggtcttgaggggttttttg (SEQ ID NO 20).

[0064] The vector can be chosen according to the selected host cell. A person skilled in the art, taking into account his technical knowledge, will adapt the vector according to the host cell.

[0065] The host cell may be any cell suitable for the expression of a nucleic acid or a vector according to the invention. It may be, for example, bacteria, for example gram-negative bacteria or gram-positive bacteria. It may be, for example, Escherichia coli, Pischia pastoris, Saccharomyces cerevisiae. Preferably, the host cell is Escherichia coli, even more preferably, the host cell may be the bacterial strain Escherichia coli BL21 (DE3) pLysE.

[0066] Advantageously, the inventors have demonstrated that the expression of the fusion protein in host cells, for example bacterial cells, advantageously allows production of the fusion protein and localization on the surface of the membrane of the host cell. For example, when the host cell is a gram-negative bacterium, the expression of the fusion protein advantageously allows production of the fusion protein and localization on the surface of the outer membrane of the bacterium. In other words, the inventors have demonstrated that the expression of the fusion protein in host cells advantageously allows production of the fusion protein, localization on the surface of the host cell and advantageously anchoring of the fusion protein to the host cell via its membrane. The present invention also relates to a host cell comprising a nucleic acid according to the invention and / or a vector according to the invention.

[0067] The host cell may be as defined above. Preferably the host cell is a bacterial cell, for example a gram negative bacterium or a gram positive bacterium, preferably Escherichia coli, even more preferably the host cell may be the bacterial strain Escherichia coli (BL21 (DE3) pLysE).

[0068] The present invention also relates to a method for producing a fusion protein according to the invention comprising the culture of a host cell according to the invention under conditions suitable for the expression of the fusion protein.

[0069] The culture of the host cell according to the invention can be carried out in any suitable culture medium known to those skilled in the art. This may be, for example, any suitable rich culture medium known to those skilled in the art. Those skilled in the art, based on this general knowledge, will adapt and / or choose the culture medium according to the host cell. For example, when the host cell is a bacterial cell, the culture medium may be an SOC medium, a lysogenic broth (LB) medium, preferably an SOC medium.

[0070] The culture of the host cell according to the invention can be carried out at any temperature suitable for the host cell known to those skilled in the art. For example, the culture can be carried out at a temperature of 20 to 30°C, for example at a temperature of 26°C.

[0071] The culture time may be any time adapted to the host cell known to those skilled in the art. For example, the culture time of the host cell according to the invention may be from 24 to 72 hours, for example 53 hours.

[0072] Advantageously, the inventors have demonstrated that the fusion protein according to the invention is present on the surface of the host cell in which it is expressed and is a functional protein. In particular, the inventors have surprisingly demonstrated that the fusion protein exhibits biological activity, in particular enzymatic activity of plant cytochrome P450 and reductase activity of plant cytochrome P450 NADPH P450 reductase. In other words, the fusion protein advantageously simultaneously possesses the biological activity of plant cytochrome P450 and plant cytochrome P450 NADPH P450 reductase.The inventors have also demonstrated in a surprising and unexpected manner that the fusion protein according to the invention, being a functional protein and exhibiting the biological activity of plant cytochrome P450 and a reductase activity of plant cytochrome P450 NADPH P450 reductase, allows bioconversion of substrates present in the culture medium.

[0073] The present invention also relates to a method for bioconversion of a substrate by a fusion protein according to the invention comprising the steps:

[0074] - introduction into a culture medium comprising a host cell according to the invention, of the substrate,

[0075] - incubation of said culture medium for a time sufficient for the bioconversion of said substrate by said fusion protein, and

[0076] - optionally recovery of metabolites resulting from the bioconversion of said substrate.

[0077] The culture medium is as defined above.

[0078] The host cell is as defined above.

[0079] In the present invention, the substrate may be any suitable substrate known to those skilled in the art. It may be, for example, a compound or substrate, for example natural or obtained by synthesis or chemical semi-synthesis. It may be, for example, any known compound or substrate, for example natural or obtained by synthesis or chemical semi-synthesis, known to those skilled in the art. It may be, for example, cinnamic acid, demethylsuberosin. It may also be any new compound which it is sought to demonstrate is a substrate for a plant cytochrome P450. In the present invention, the concentration of the substrate in the culture medium comprising a host cell may be from 100 pM to 250 pM, for example equal to 200 pM.

[0080] In the present invention, the bioconversion process may further comprise, prior to the incubation step, a step of introducing nicotinamide adenine dinucleotide phosphate (NADPH) into the culture medium.

[0081] Herein, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) in the culture medium comprising a host cell may be from 200 pM to 450 pM, for example equal to 400 pM.

[0082] The step of incubating the host cell according to the invention can be carried out at any temperature suitable for the host cell known to those skilled in the art. For example, the incubation can be carried out at a temperature of 15 to 30°C, for example at a temperature of 20°C.

[0083] The incubation time may be any time suitable for the host cell known to those skilled in the art. For example, the incubation time of the host cell according to the invention may be from 30 minutes to 72 hours, for example 1 hour.

[0084] In the present invention, the recovery of the metabolites may be carried out by any suitable method known to those skilled in the art. This may be, for example, a technique chosen from ultrafiltration, membrane or gel filtration, ion exchange, elution on hydroxyapatite, separation by hydrophobic interactions, by chromatography, for example by liquid chromatography or any other known means.

[0085] In the present invention, the metabolites obtained may be any plant metabolite. For example, they may be metabolites belonging to the family of polyphenols, alkaloids and / or terpenes. Advantageously, they may be metabolites belonging to the family of polyphenols.

[0086] The metabolites obtained can provide access to new products that can be food, cosmetic, pharmaceutical and parapharmaceutical active ingredients that can be used in the agri-food, cosmetic, pharmaceutical and parapharmaceutical sectors. These new products can also be active or non-active products but with a neutrality and / or stability that is very interesting for use in each of these sectors.

[0087] Other advantages will become apparent in the light of the following examples, given for illustrative and non-limiting purposes, with reference to the attached figures.

[0088] Brief description of the figures

[0089] Figure 1 represents a diagram of the fusion protein successively comprising an outer membrane addressing and anchoring polypeptide (C), a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 (B), a linker polypeptide comprising at least 47 amino acids (L) and a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase (A), M corresponds to the bacterial membrane.

[0090] Figure 2 shows a schematic representation of the plasmid pAIDAI - TetR-lacIQ.

[0091] Figure 3 shows a schematic representation of plasmid pAIDA1-T7. Figure 4 shows a schematic representation of plasmid pAIDA1-T7. Figure 5 shows a schematic representation of plasmid pAIDA1-T7-CYP73A1-ATR1.

[0092] Figure 6 represents a diagram of different elements of an example of a fusion protein according to the invention. In the figure AIDAI -barrel-beta AIDAI - 01 and AIDAI linker respectively represent an outer membrane targeting and anchoring polypeptide, a linker polypeptide, P450 73A1 a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 (P450 CYP73A1), flexible linker: a linker polypeptide and ATR1:P450 Reductase a polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

[0093] Figure 7 represents chromatograms corresponding to the analysis carried out by high-performance liquid chromatography (HPLC) of culture medium from a bioconversion experiment in the presence of cinnamic acid. The abscissa corresponds to the elution time in minutes and the ordinate corresponds to arbitrary units and allows us to see the efficiency of the metabolism. Figure 7A represents a diagram concerning the metabolism of cinnamic acid by bioconversion, in this figure Figure 7A represents the chromatograms obtained after culture of the strain E. Coli BL21 (DE3) pLysE transformed with the plasmid pAIDA1 -T7-CYP73A1 -ATR1 and Figure 7B represents the diagram after culture of the strain E. Coli BL21 (DE3) pLysE transformed with the plasmid pAIDA1 -T7 in Figure 4. In Figure 7A we see the appearance of para-coumaric acid which is the product of metabolism of cinnamic acid. In Figure 7B, cinnamic acid is not metabolized.

[0094] Figure 8 represents an alignment of peptide sequences carried out with the Basic Local Alignment Search Tool (BLAST). The sequences correspond to cytochromes P450 CYP76F112 and P450 CYP73A1

[0095] Figure 9 represents the chemical reaction corresponding to the transformation of cinnamic acid into para-coumaric acid by cytochrome P450 CYP73A1 (CYP73A1) and the chemical reaction corresponding to the transformation of demethylsuberosin into Marmesin by cytochrome P450 CYP76F112 (CYP76F112).

[0096] EXAMPLES

[0097] Example 1: Method for preparing a fusion protein and biotransformation method using said protein

[0098] 1) Construction of a generic expression plasmid pAIDA-T7

[0099] The basis of the constructed expression plasmid is based on a commercial plasmid pAIDAI (https: / / www.addgene.org / 79180 /

[0011] ) which is a low copy plasmid.

[0100] Replacement of the selection gene The plasmid pAIDAI was first modified by replacing the gene conferring resistance to chloramphenicol with a gene conferring resistance to Tetracycline cloned from the plasmid pBR322 marketed by Fisher Scientific (https: / / www.fishersci.fr / shop / products / fermentas-pbr322-dna / 10191220

[0011] ). For this, the plasmid pAIDAI was the template which was amplified by polymerase chain reaction (PCR) using the enzyme PrimeSTAR Max polymerase marketed by Takara Bio Inc, according to the method Protocol 1: PrimeSTAR Max polymerase Protocol as described in (https: / / www.takarabio.com / documents / User%20Manual / R045A_e.v2102Da.pdf [6]). The amplification was carried out using the primers LAEWXpr17-laclQ (tggcgacaccatcgaatggtgc (SEQ ID NO: 21) and LAEWXprO2: Reverse: tttagcttccttagctcctg (SEQ ID NO: 22).This amplification made it possible to copy the plasmid in its entirety with the exception of the chloramphenicol resistance gene. The same process was implemented and made it possible to amplify the coding sequence of the Tetracycline resistance gene. The amplification was carried out this time from the plasmid pBR322 using the primers LAEWXprO3 (gctaaggaagctaaaatgaaatctaacaatgcgct (SEQ ID NO 41)) and LAEWXprO4 (tcgatggtgtcgccacgctgcccgagatgc (SEQ ID NO 42)). The two PCR products obtained, namely the plasmid pAIDAI without the chloramphenicol resistance gene and the coding sequence of the tetracycline resistance gene, were fused using the In-Fusion kit marketed by Takara Bio Inc according to the Protocol 2: In-Fusion Protocol method described in the document https: / / www.takarabio.com / documents / User%20Manual / ln / ln-Fusion%20Snap%20Assembly%20User%20Manual_071320.pdf

[0016] .The resulting recombinant plasmid was introduced into chemocompetent Escherichia coli TQP10 bacteria marketed by Life Technologies Corporation according to the TQP10 transformation protocol described in https: / / assets.thermofisher.com / TFS-.

[0101] Assets / LSG / manuals / oneshottop10_man.pdf

[0017] ). The transformed bacteria were plated on LB (lysogenic broth) culture medium (10g peptone, 5g yeast extract, 5g NaCl) containing tetracycline. The insertion of the gene coding for tetracycline resistance was verified by PCR using primers LAEWXpr17 (SEQ ID NO 21) and LAEWXprO2 (SEQ ID NO 22).

[0102] The plasmid pAIDA1 -TetR-lacl Q, shown in Figure 2, recombinant thus constructed was used for the construction of the other plasmids. To simplify the nomenclature, the plasmid is also designated pAIDAI. This plasmid was amplified and purified from a positive colony according to the method Protocol 3: the plasmid purification protocol described in the document https: / / www.mn-net.com / media / pdf / 45 / 51 / 02 / lnstruction-NucleoSpin-Plasmid.pdf

[0018] . a) Promoter and terminator

[0103] The promoter and terminator of the AIDA cassette of the plasmid pAIDAI were replaced by two cloning cassettes containing the promoter and terminator of T7 RNA Polymerase. To carry out this step, different clonings were carried out

[0104] (i) pAIDAI was amplified by PCR using primers LAEWXpr30 (tcatcatcatgcctaatgagtgagaattcc (SEQ ID NO 23)) and LAEWXpr31 (ttggtgcgcaaactattaactgg (SEQ ID NO 24)). This amplification step included the DNA fragment contained between the pAIDAI promoter and the pAIDAI terminator. The amplification product therefore corresponded to the plasmid without promoter and terminator.

[0105] (ii) In parallel, the T7 terminator was amplified by PCR from plasmid pET28b-2 using primers LAEWXpr32-1-fr (ctcattaggcatgatgatgaaaggaagggaagaaagcgaaagg (SEQ ID NO

[0106] 25)) and LAEWXpr32-2-rv

[0107] (agtactcctaggactagtggtaccagatccggctgctaacaaagc (SEQ ID NO

[0108] 26)). Finally, the T7 promoter was amplified by PCR from plasmid pET28b-2 using primers LAEWXpr33-1 -rv (gttaatagtttgcgcaccaaatcggtgatgtcggcgatatagg (SEQ ID NO 27)) and LAEWXpr33-2-fr (ggtaccactagtcctaggagtactatggctgctgcccatggtata (SEQ ID NO 28)) according to Protocol 1 as mentioned above, (iii) The three fragments were fused using the In-Fusion kit according to Protocol 2 as mentioned above. This ligation product generated plasmid pAIDA1-T7 shown in Figure 3. b) Insertion of the AIDA1 cassette

[0109] The AIDA sequence of the original plasmid pAIDAI was amplified by PCR using primers LAEWXpr36-AIDA-fr

[0110] (aactttaagaaggagatataccatgggcaataaggcctacagtatcatttgg (SEQ ID NO 29)) and LAEWXpr37-AIDA-rv (tttgttagcagccggatctgtcattatcagaagctgtattttatc (SEQ ID NO 30)) according to Protocol 1 as mentioned above.

[0111] In parallel, plasmid pAIDA1-T7 was digested with restriction enzymes A / col and Kpn\ according to Protocol 4: Digestion protocol as described in http: / / assets.thermofisher.com / TFS-Assets / BID / Reference-Materials / fastdigest-restriction-enzymes-labaid.pdf

[0019] . Linearized plasmid pAIDA1-T7- Nco\-Kpn\ and AIDAI amplicon were fused by In-Fusion according to Protocol 2 mentioned above. Generic recombinant plasmid pAIDAI-T7 complete with AIDAI was amplified by transformation of bacteria. The obtained generic plasmid pAIDA1-T7 complete with AIDAI is shown in Figure 4.

[0112] 2) Construction of recombinant plasmid for the expression of a P450-ATR fusion protein a) The plasmid pAIDA1-T7 was digested with restriction enzymes Kpn\ and Sacl according to Protocol 4 as mentioned above. b) The sequence coding for the extra-membrane part of the Arabidopsis thaliana NADPH P450 reductase 1 (ATR1 ) as described in Urban et al, (1997) J Biol Chem 272(31 ):19176-86

[0020] ) was amplified by PCR from a plasmid (pCR8_ATR1) using the primers LAEWXprOô Forward (agcgctgttccagggtccgggtaccactagagtctctatcttc (SEQ ID NO 31 )) and LAEWXprO6 Reverse (ccgccaccagaaccgcccggccagacatctctgaggtatc (SEQ ID NO 32)) according to Protocol 1 as mentioned above).c) The linker (rich in GC pair) was amplified from a synthetic sequence using the PrimeSTAR GXL DNA Polymerase enzyme marketed by Takara Bio Inc using the primers LAEWX15-FlexL-fr (ccgggcggttctggtggcgg (SEQ ID NO 33)) and LAEWX16-FlexL-rvs (cggagaaccgccgctaccgc (SEQ ID NO 34)) according to the Protocol 5 method described in PrimeSTAR GXL DNA Polymerase Manual, https: / / www.takara.co.kr / file / manual / pdf / R050A_e.v1906Da.pdf

[0021] ). d) The DNA sequence coding for the extra-membrane part of P450, CYP73A1 (Urban et al, (1997) J Biol Chem 272(31 ):19176-86

[0020] ) was amplified by PCR from a plasmid pYeDP60-CYP73A1 using the primers LAEWXprO7 Forward (gcggtagcggcggttctccgcctggcccaatcccggttcc (SEQ ID NO 35)) and LAEWXprO8 Reverse (gaagtacaggttttcgagctcaaatgacctaggtttagc (SEQ ID NO 36)) according to Protocol 1 mentioned above. e) The 4 DNA fragments generated by PCR amplification, namely SEQ ID NO.

[0113] 17, SEQ ID NO 15, SEQ ID NO 11 and plasmid pAIDAI -T7 was digested by Kpn\ and Sacl restriction enzymes according to Protocol 4 as mentioned above. The sequence of the digested plasmid obtained corresponds to the sequence: aaggaagggaagaaagcgaaaggacgggctagggcgctggcaagtgtagcggtcacgctgcg cgtaaccaccacacccgcgcgcgcgcgcgcgcccccgccgc gttcctcctttcagcaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctca gcggtggcagcaactcagcttccttcgggctttgttagcagccggatgtctcattatcagaagctgta tttatccccagtgctccggagatgcattgctcccgtgacctctctgcctgatatgcgactccgccattcactg acaagttttgagtaatcaccccttcaatacctctctctctccctgatttcggctacctgacaacaactggct gtcatcactcattttaacaccaaattcgagtcgtcctc gccggtatccttatccagggtgcttttcaccttccaggatgcacgaatacctgctttgtctgaatattattttc cctgctccctgcaccaccgttccgtctctctctgtgtatccggtgtaacccccatccagacagcctgca aatgaggctgtaaccagaattcacctgttattccttcaggtgatgtccatgtgtgcacattcaggttatatcccccacctgcagaagcggttaaaccattcagattatatttttcttcttccagtccgtcacctttcactgatgcattaaa ccagttatattgcatccaagtttcagcaaagagccctgttgcattttccccattctgataccacgtaccgtata ccccgacagaataaccatccagtgtgtttctggcagctttgttgctcgtgtaatttatcgttttaccttttgcattcg cgtatcctcccataatccctaaggtaaaatcacccagttgttcagcatggaatttataaatatcccccccga gctgattgataaactgattggttgttgttttattttgcccgtcattcagcttaccagagcttattcctccagtgatctt catccacacggatgcagactcaggctgtgtattatcactcatggccctgaattgcttacgctcattcaaatcc atgaggaacagtgagttagccagtgccatattggtagcataacttccgttctccggtctgtattgccgggtat cagatgtgggaagatgactggttaaataccatcccttattatctgtcccactctcgtttcctttctgcagtgtgta atcataagctccggcaactacgcggttcttcagagagaattctgcatcagaatttccctctacagaaataat attaataccatctctcgtctgaccaccactgccatcttcattgacataaacgatgtcactttgaccagaggtat tacctttcaccaccagacggtccgtaagtgaattatctccttcaagcacaccaccaagagaaataacactt cccggtgtcccagtataatttgacacggtaagagtattacctatagcggccgcactttcttttgtaggattaag aatgatatttttgttattaacaagactaccattcactgtagcagagagcagctttgctgtaccaaaacttaagtcagcatcattcgtaatattcccgtttataatgctgttattaatgacaatgcttccattgttattcactctagacaga tcttcttcgctaatcagtttctgttcaccctggaagtacaggttttcgagctccggaccctggaacagcgcttcc agatggtgatggtgatggtggtcgactgcaaatgcatttccgattgtggaaacaaccgccaataccagca gtgtattttttgcaaggacaaaaccatgtcctctggctaactctgaggccacaatccaggcctgtctggagtg gctccaaatgatactgtaggccttattgcccatggtatatctccttcttaaagttaaacaaaattatttctagag gggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctcta cgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacat caccgatttggtgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactg gatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgt atcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagata ggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttc atttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccttaataagatgatcttcttgagatcgttttggtctgcgcgtaatctcttgctct gaaaacgaaaaaaccgccttgcagggcggtttttcgaaggttctctgagctaccaactctttgaaccgag gtaactggcttggaggagcgcagtcaccaaaacttgtcctttcagtttagccttaaccggcgcatgacttca agactaactcctctaaatcaattaccagtggctgctgccagtggtgcttttgcatgtctttccgggttggactca agacgatagttaccggataaggcgcagcggtcggactgaacggggggttcgtgcatacagtccagcttg gagcgaactgcctacccggaactgagtgtcaggcgtggaatgagacaaacgcggccataacagcgg aatgacaccggtaaaccgaaaggcaggaacaggagagcgcacgagggagccgccagggggaaa cgcctggtatctttatagtcctgtcgggtttcgccaccactgatttgagcgtcagatttcgtgatgcttgtcaggg gggcggagcctatggaaaaacggctttgccgcggccctctcacttccctgttaagtatcttcctggcatcttc caggaaatctccgccccgttcgtaagccatttccgctcgccgcagtcgaacgaccgagcgtagcgagtc agtgagcgaggaagcggaatatatcctgtatcacatattctgctgacgcaccggtgcagccttttttctcctg ccacatgaagcacttcactgacaccctcatcagtgccaacatagtaagccagtatacactccgctagcgc tgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgcc acggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaa caaagccacgttgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaata aaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcg agtatccgctcatgagattatcaaaaaggatcttcacctagatccttttgtaagttctcatgtttgacagcttatc atcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatcta acaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtac tgccgggcctcttgcgggatatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgctat atgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcct gctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggatcctcta cgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacat caccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggca ggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaccgatgcc cttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatga ctgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgcttt cgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttc gtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgc gctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccg gcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacag cttcaaggatcgctcgcggctcttaccagcctaacttcgatcattggaccgctgatcgtcacggcgatttatg ccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctcccc gcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaac ggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaaccctt ggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgtggcg acaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccg cgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcgga gctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgcc acctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgg gtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaat cttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaa gctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctccc atgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttag cgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaat tcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgct gaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgc cattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacag ctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggacc gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagct ggcacgacaggtttcccgactggaaagcgggcaagtgagtggataaccgtattaccgcctttgagtgag ctgataccgggaattctcactcattaggcatgatgatga (SEQ ID NO 43). Then, the sequences SEQ ID NO 17, SEQ ID NO 15, SEQ ID NO 11 were fused by In-Fusion according to the protocol Protocol 2 mentioned above. The ligation product was introduced into E. coli bacteria and directly cultured without going through a selection phase on selective medium. The presence of the constituent elements of the plasmid was verified by PCR using the primers LAEWXpr35-upstream-T7 (tccatccagtctattaattgttgc (SEQ ID NO 37)), LAEWXpr22-ATR1-rv (ccagacatctctgaggtatcttcc (SEQ ID NO 38)), LAEWXpr24-2kbATR1-fr (ggagcaggaaggtgtgagttcgtc (SEQ ID NO 39)) and LAEWXpr25-P540-rv (accctggaagtacaggttttcg (SEQ ID NO 40)). All steps a to ea allowed the construction of a plasmid pAIDA1-T7-CYP73A1-ATR1 shown in Figure 5.

[0114] 3) Fusion protein

[0115] The resulting fusion protein contains from the N-terminus to the C-terminus:

[0116] - a tag of 6 Histidines, allowing immunodetection of proteins with anti His antibodies

[0117] - a cleavage sequence to remove the tag,

[0118] - a c-MYC sequence, allowing immunodetection of proteins with anti-c-MYC antibodies

[0119] - an AIDAI binding peptide

[0120] - an external membrane addressing and anchoring polypeptide: the beta Barel anchoring sequence of AIDAI, namely the sequence SEQ ID NO 1

[0121] - a polypeptide sequence of cytochrome P450 CYP73A1 (cinnamate hydroxylase from Helianthus tuberosus, NCBI ID: Sequence ID: Q04468.1, UniProtKB / Swiss-Prot: Q04468.1) in which the membrane anchor has been removed from the coding sequence, namely the sequence SEQ ID NO 3),

[0122] - a binding peptide of 51 amino acids PGGSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGSGGSP (SEQ ID NO 6), and

[0123] - a polypeptide sequence of Arabidopsis NADPH P450 reductase (Arabidopsis thaliana P450 reductase 1 (ATR1), mRNA - Sequence ID: NM_118585.4, NCBI Reference Sequence: NM_118585.4) in which the membrane anchor has been removed, namely the sequence SEQ ID NO 7. The coding nucleic acid sequences corresponding to the different elements of the fusion protein described above were used and assembled successively from 5' to 3'. In particular, these were:

[0124] - the sequence SEQ ID NO 44 coding for an AIDAI linker,

[0125] - the sequence SEQ ID NO 10 coding for a polypeptide for targeting and anchoring to the external membrane: the Beta Barrel anchor sequence

[0126] - the sequence SEQ ID NO 11 coding for the cytochrome P450 CYP73A1 cinnamate hydroxylase of Helianthus tuberosus, NCBI ID: Sequence ID: Q04468.1, UniProtKB / Swiss-Prot: Q04468.1)

[0127] - the sequence SEQ ID NO 15 coding for a linker peptide of 51 amino acids, and

[0128] - the sequence SEQ ID NO 17 coding for the Arabidopsis NADPH P450 reductase (Arabidopsis thaliana P450 reductase 1 (ATR1), mRNA - Sequence ID: NM_118585.4, NCBI Reference Sequence: NM_118585.4)

[0129] Figure 6 is a schematic representation of the resulting fusion protein.

[0130] 4) Expression of recombinant proteins in E. coli bacteria a) The expression system used

[0131] The pAIDAI plasmid and recombinant plasmids containing the genes encoding the fusion protein were introduced into E. coli BL21 (DE3) plysE bacteria (Novagen's® pET Systems). BL21 (DE3) pLysE bacteria are adapted for the production of proteins placed under the control of the T7 promoter. BL21 (DE3) pLysE bacteria carry the lambda DE3 lysogen and contain the pLysE plasmid, which constitutively expresses T7 lysozyme. T7 lysozyme reduces the basal expression of target genes by inhibiting T7 RNA polymerase. The BL21 (DE3) pLysE strain therefore allows tighter control of T7 RNA polymerase. b) Preparation of an example fusion protein The fusion protein was expressed from the plasmid whose construction was described previously. After transformation of bacteria by the ligation product, there is no selection step on solid medium.The transformed bacteria were immediately cultured in 50 ml of SOC medium (Dextrose, 3.603 g / L, KCl, 0.186 g / L, MgSO4, 4.8 g / L, Tryptone, 20 g / L, Yeast extract 5 g / L) including Tetracycline (20 pg / ml) and Chloramphenicol (20 pg / ml), in a sterile 250 mL Erlenmeyer flask. The culture was carried out for 53 h at 26°C with shaking at 180 rpm. The cultures were then cooled on ice for 10 minutes. The optical density of the culture was adjusted to OD 550nm = 0.3 by dilution in SOC medium (4°C). The volume of the culture medium was measured and the antibiotics were re-added to a final concentration of 20 pg / ml Tetracycline and 20 pg / ml Chloramphenicol. Production of the fusion protein was induced by addition of isopropyl 0-D-1-thiogalactopyranoside (IPTG) to a final concentration of 20 pM for 24 h at 7°C, with shaking at 180 rpm.After 24 h, the optical density was adjusted to 0.3 by dilution in SOC medium (4°C). Bacteria present in 1.5 mL were harvested by two successive low-speed centrifugations (4°C, 20 min, 1000 xg and 4°C, 10 min, 4000 xg).

[0132] The bacteria were then suspended in KPi buffer (88 mM with additives of 1 mM KCl, 4 mM MgSO4, 5% v / v glycerol, 5% w / v glucose, at 4°C (products ordered from Sigma Aldrich)).

[0133] Figure 1 is a schematic representation of the resulting fusion protein, said fusion protein being anchored in the bacterial membrane.

[0134] 5) Bioconversion

[0135] A study of the bioconversion of cinnamic acid was carried out. Figure 9 (CYP73A1) represents the corresponding bioconversion reaction. For this, cinnamic acid (200 pM) as substrate and nicotinamide adenine dinucleotide phosphate (NADPH) (400 pM) were added to the medium comprising the resuspended E. co t bacteria obtained in point 4 above. The bioconversion process was carried out at 20°C for 1 h and with stirring at 180 rpm. The reaction was stopped by extraction with 1 volume of Ethyl Acetate. The media were mixed by vortexing for 1 minute followed by centrifugation at 10,000xg to separate the organic and aqueous phases. The upper organic phase was recovered and evaporated by Vivaspin. The obtained powder was suspended in 150 μL of methanol. The extract thus obtained was analyzed by ultra-high performance liquid chromatography coupled with mass spectra (UHPLC / MS / MS).

[0136] The results obtained are shown in Figure 7. Figure 7A represents a chromatogram obtained at 300 nm and showing 2 peaks. The majority peak corresponds to the substrate, i.e. cinnamate. The minority peak corresponds to p-coumarate or p-couramic acid formed by bioconversion in the culture medium. This chromatogram was obtained from the culture medium in which the recombinant bacteria transformed with the plasmids pAIDA1 -T7-CYP73A1 -ATR1 were cultivated according to point 4 above. The chromatogram in Figure 7B corresponds to an analysis of the culture medium in which recombinant bacteria transformed with the plasmid pAIDA1 -T7 in Figure 4 were cultivated. This plasmid cannot produce fusion protein and represents a negative control. Analysis of the chromatogram indicates the presence of cinnamate which was added to the culture medium. No metabolism of p-coumarate could be demonstrated.

[0137] This example therefore clearly demonstrates that an example of a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, advantageously to the external membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase allows substrate bioconversion. This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously be used in a substrate bioconversion process.

[0138] This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously allow bioconversion of substrates in the culture medium of said cell.

[0139] Example 2: Fusion protein and bioconversion

[0140] In this example, the cytochrome P450 is cytochrome CYP76F112 (marmesin synthase, Ficus carica cytochrome P450 CYP76F112 mRNA, complete cds Sequence ID: MW348922.1 , GenBank: MW348922.1 ).

[0141] In this example the process for obtaining the fusion protein is identical to the process described in Example 1 except for the following elements:

[0142] - La séquence codant pour le cytochrome P450 CYP73A1 est remplacée par la séquence codant pour le cytochrome P450 CYP76F112 de séquence caagtcacgacggttgtcatttcctccaccaccatggctaaagaagtcctccaggcaaacagccaagtcgtct ccagccggacaatcaccgacgcaagccgcgcccacagacacagcgattttagcatggttatgttgcccgtat cccctctgtggcgaaaccttcggaaaataagcaactcacacttgctttcctccaaggctcttgatggcaacatgg agctgagaaacaaaaaggtgcaagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcgg tggagatcgcgagcctttctttcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatg acacaaattccgtcactctaaaagagctcaaggaggctatgtcgcacatgatggaagagttggggaagccta acttggccgattatttcccgtttctacaaaagattgacccccaaggcattaggcggcgcaacacggttactttccg gaaactgatcaacttgtttgggcgtatcatcgaccaaagattgaaagtgagagaagcgagtggttctttgaaag atgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaa accataattgaacattttttactggatttattttcagcggggactgaaacgacttcaaccacgttggagtgggcaat ggctgagctagtaaaagcgccagagattatgtcaaaagcccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaaccttccgcatg caccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaatctccgactatatcatcccgaa ggatgctcag (SEQ ID NO 45) ou aaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttccg agtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaa agaagtcctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacag acacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaataagcaactcaca cttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgcaagagctcctaaatgatg tccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctttcagagctactctgaatctcttg tccaccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctat gtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgaccccca aggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagatt gaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaaaccataattgaacatttttactggatttattcgcggggact gaaacgacttcaaccacgttggagtgggcaatggctgagctagtaaaagcgccagagattatgtcaaaagc ccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactccctta cttacaagccattgttaaagaaaccttccgcatgcaccctacagctccattattgattccgcaaagccgacag tgacatcgaaatctccgactatatcccgaaggatgctcaggtgattgtcaatgtatgggccattggtagaga ctcaagcacatgggaaaatcccgacaagtttataccggaggttttggacatcgatatagatgtcggaggc cttgatgttggggtctttgcttcgtcgtgattggaagttggaagatggggttagacctgctctaaacatggat gaaaagttttggcctcaccttgcaaatggctcagcctttgcgagctatcccgtgccgacaaagcat (SEQ ID NO 71 )

[0143] - The substrate cinnamic acid is replaced by demethylsuberosine. Bioconversion of the substrate generates marmesine as shown in Figure 9 (CYP76F112).

[0144] A comparison of the peptide sequences of cytochromes P450 CYP76F112 and P450 CYP73A1 by peptide sequence alignment performed with the Basic Local Alignment Search Tool (BLAST) is shown in Figure 8. The result obtained demonstrates a percentage identity of 28.7%.

[0145] In this example, the bioconversion is carried out according to the method described in Example 1 above in which the substrate used is demethylsuberosin. This example clearly demonstrates that an example of a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one linker polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase allows the substrate bioconversion.

[0146] This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously be used in a process for the bioconversion of substrates.

[0147] This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously allow bioconversion of substrates in the culture medium of said cell.

[0148] Example 3: Method for preparing a fusion protein and biotransformation method using said protein

[0149] Dans cet exemple, le cytochrome P450 est le cytochrome CYP76F112 (marmesin synthase, Ficus carica cytochrome P450 CYP76F112 mRNA, complete cds Sequence ID: MW348922.1 , GenBank: MW348922.1 ) dont la séquence est aaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttccg agtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaa agaagtcctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacag acacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaataagcaactcaca cttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgcaagagctcctaaatgatg tccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctttcagagctactctgaatctcttg tccaccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctat gtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgaccccca aggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagatt gaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaaccataattgaacatttttactggatttattcagcggggact gaaacgacttcaaccacgttggagtgggcaatggctgagctagtaaaagcgccagagattatgtcaaaagc ccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactccctta cttacaagccattgttaaagaaaccttccgcatgcaccctacagctccattattgattccgcaaagccgacag tgacatcgaaatctccgactatatcatcccgaaggatgctcaggtgattgtcaatgtatgggccattggtagaga ctcaagcacatgggaaaatcccgacaagtttataccggaggttttggacatcgatatagatgtcggaggc cgggattttaagctcattccgttcggtgctggtcggaatcggcatcgcatcgattcgcgc cttgatgttggggtctttgcttcgtttgattggaagttggaagatggggttagacctgctctaaacatggat gaaaagttttggcctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcat (SEQ ID NO 71 ) (nucleic acid encoding the hydrophile domain of the P450 phospholipid CYP76F112).

[0150] A comparison of the peptide sequences of cytochromes P450 CYP76F1 12 and P450 CYP73A1 by peptide sequence alignment was performed with the Basic Local Alignment Search Tool (BLAST) and is shown in Figure 8. The result obtained demonstrates a percentage identity of 28.7%.

[0151] 1) Construction of a generic expression plasmid pAIDA-T7

[0152] The basis of the constructed expression plasmid is based on a commercial plasmid pAIDAI (https: / / www.addgene.org / 79180 /

[0011] ) which is a low copy plasmid.

[0153] Replacement of the selection gene

[0154] The plasmid pAIDAI was first modified by replacing the gene conferring resistance to chloramphenicol with a gene conferring resistance to Tetracycline cloned from the plasmid pBR322 marketed by Fisher Scientific (https: / / www.fishersci.fr / shop / products / fermentas-pbr322-dna / 10191220

[0011] ) as described in Example 1 above. For this, the plasmid pAIDAI was the template that was amplified by polymerase chain reaction (PCR) using the enzyme PrimeSTAR Max polymerase marketed by Takara Bio Inc, according to the method Protocol 1: PrimeSTAR Max polymerase Protocol as described in (https: / / www.takarabio.com / documents / User%20Manual / R045A_e.v2102Da.pdf [6]). The amplification was carried out using the primers LAEWXpr17-laclQ (tggcgacaccatcgaatggtgc (SEQ ID NO: 21) and LAEWXprO2: Reverse: tttagcttccttagctcctg (SEQ ID NO: 22).This amplification made it possible to copy the plasmid in its entirety with the exception of the chloramphenicol resistance gene. The same process was implemented and made it possible to amplify the coding sequence of the Tetracycline resistance gene. The amplification was carried out this time from the plasmid pBR322 using the primers LAEWXprO3 (gctaaggaagctaaaatgaaatctaacaatgcgct (SEQ ID NO 41)) and LAEWXprO4 (tcgatggtgtcgccacgctgcccgagatgc (SEQ ID NO 42)). The two PCR products obtained, namely the plasmid pAIDAI without the chloramphenicol resistance gene and the coding sequence of the tetracycline resistance gene, were fused using the In-Fusion kit marketed by Takara Bio Inc according to the Protocol 2: In-Fusion Protocol method described in the document https: / / www.takarabio.com / documents / User%20Manual / ln / ln-Fusion%20Snap%20Assembly%20User%20Manual_071320.pdf

[0016] .The resulting recombinant plasmid was introduced into chemocompetent Escherichia coli TQP10 bacteria marketed by Life Technologies Corporation according to the TQP10 transformation protocol described in https: / / assets.thermofisher.com / TFS-.

[0155] Assets / LSG / manuals / oneshottop10_man.pdf

[0017] ). The transformed bacteria were plated on LB (lysogenic broth) culture medium (10g peptone, 5g yeast extract, 5g NaCl) containing tetracycline. The insertion of the gene coding for tetracycline resistance was verified by PCR using primers LAEWXpr17 (SEQ ID NO 21) and LAEWXprO2 (SEQ ID NO 22).

[0156] The plasmid pAIDA1 -TetR-lacl Q, shown in Figure 2, recombinant thus constructed was used for the construction of the other plasmids. To simplify the nomenclature, the plasmid is also designated pAIDAI. This plasmid was amplified and purified from a positive colony according to the method Protocol 3: the plasmid purification protocol described in the document https: / / www.mn-net.com / media / pdf / 45 / 51 / 02 / lnstruction-NucleoSpin-Plasmid.pdf

[0018] . a) Promoter and terminator

[0157] The promoter and terminator of the AIDA cassette of the plasmid pAIDAI were replaced by two cloning cassettes containing the promoter and terminator of T7 RNA Polymerase as described in Example 1 above. To carry out this step, different clonings were carried out

[0158] (i) pAIDAI was amplified by PCR using primers LAEWXpr30 (tcatcatcatgcctaatgagtgagaattcc (SEQ ID NO 23)) and LAEWXpr31 (ttggtgcgcaaactattaactgg (SEQ ID NO 24)). This amplification step included the DNA fragment contained between the pAIDAI promoter and the pAIDAI terminator. The amplification product therefore corresponded to the plasmid without promoter and terminator.

[0159] (ii) In parallel, the T7 terminator was amplified by PCR from plasmid pET28b-2 using primers LAEWXpr32-1 -fr (ctcattaggcatgatgatgaaaggaagggaagaaagcgaaagg (SEQ ID NO 25)) and LAEWXpr32-2-rv (agtactcctaggactagtggtaccagatccggctgctaacaaagc (SEQ ID NO 26)). Finally, the T7 promoter was amplified by PCR from plasmid pET28b-2 using primers LAEWXpr33-1 -rv (gttaatagtttgcgcaccaaatcggtgatgtcggcgatatagg (SEQ ID NO 27)) and LAEWXpr33-2-fr (ggtaccactagtcctaggagtactatggctgctgcccatggtata (SEQ ID NO 28)) according to Protocol 1 as mentioned above.

[0160] (iii) The three fragments were fused using the In-Fusion kit according to Protocol 2 as mentioned above. This ligation product generated the plasmid pAIDAI-T7 shown in Figure 3. b) Insertion of the AIDAI cassette The AIDA sequence of the original plasmid pAIDAI was amplified by PCR using the primers LAEWXpr36-AIDA-fr

[0161] (aactttaagaaggagatataccatgggcaataaggcctacagtatcatttgg (SEQ ID NO 29)) and LAEWXpr37-AIDA-rv (tttgttagcagccggatctgtcattatcagaagctgtattttatc (SEQ ID NO 30)) according to Protocol 1 as mentioned above.

[0162] In parallel, plasmid pAIDA1-T7 was digested with restriction enzymes A / col and Kpn\ according to Protocol 4: Digestion protocol as described in http: / / assets.thermofisher.com / TFS-Assets / BID / Reference-Materials / fastdigest-restriction-enzymes-labaid.pdf

[0019] . Linearized plasmid pAIDAI-T7-Nco\-Kpn\ and AIDAI amplicon were fused by In-Fusion according to Protocol 2 mentioned above. Generic recombinant plasmid pAIDA1-T7 complete with AIDAI was amplified by transformation of bacteria. The obtained generic plasmid pAIDA1-T7 complete with AIDAI is shown in Figure 4.

[0163] 2) Construction of recombinant plasmid for the expression of a P450-ATR fusion protein a) The plasmid pAIDA1-T7 was linearized and amplified by PCR using the primers Vector 5 ATR1 -FL-76F112 fwd (cgacaaagcatgagctcgaaaacctgtacttcc (SEQ ID NO 48)) and New Vector 5 rvs (agactctagtggtacccggaccctggaaca (SEQ ID NO 49)) according to protocol 1 as mentioned above. The linearized plasmid sequence obtained corresponds to the sequence agactctagtggtacccggaccctggaacagcgcttccagatggtgatggtgatggtggtcgactgcaaa tgcatttccgattgtggaaacaaccgccaataccagcagtgtattttttgcaaggacaaaaccatgtcctctg gctaactctgaggccacaatccaggcctgtctggagtggctccaaatgatactgtaggccttattgcccatg gtatatctccttcttaaagttaaacaaaattatttctagaggggaattgttatccgctcacaattcccctatagtg agtcgtattaatttcgcgggatcgagatctcgatcctctacgccggacgcatcgtggccggcatcaccggc gccacaggtgcggttgctggcgcctatatcgccgacatcaccgatttggtgcgcaaactattaactggcga actacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtat cattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcag accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatccttt ttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccttaataagatga tcttcttgagatcgttttggtctgcgcgtaatctcttgctctgaaaacgaaaaaaccgccttgcagggcggtttt tcgaaggttctctgagctaccaactctttgaaccgaggtaactggcttggaggagcgcagtcaccaaaact tgtcctttcagtttagccttaaccggcgcatgacttcaagactaactcctctaaatcaattaccagtggctgct gccagtggtgcttttgcatgtctttccgggttggactcaagacgatagttaccggataaggcgcagcggtcg gactgaacggggggttcgtgcatacagtccagcttggagcgaactgcctacccggaactgagtgtcagg cgtggaatgagacaaacgcggccataacagcggaatgacaccggtaaaccgaaaggcaggaacag gagagcgcacgagggagccgccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacc actgatttgagcgtcagatttcgtgatgcttgtcaggggggcggagcctatggaaaaacggctttgccgcggccctctcacttccctgttaagtatcttcctggcatcttccaggaaatctccgccccgttcgtaagccatttccg ctcgccgcagtcgaacgaccgagcgtagcgagtcagtgagcgaggaagcggaatatatcctgtatcac atattctgctgacgcaccggtgcagccttttttctcctgccacatgaagcacttcactgacaccctcatcagtg ccaacatagtaagccagtatacactccgctagcgctgaggtctgcctcgtgaagaaggtgttgctgactca taccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgt aggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatc tgatccttcaactcagcaaaagttcgatttattcaacaaagccacgttgtgtctcaaaatctctgatgttacatt gcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtg ttatgagccatattcaacgggaaacgtcttgctcgagtatccgctcatgagattatcaaaaaggatcttcac ctagatccttttgtaagttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaat tgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctcggcaccgtcaccct ggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacag catcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgactacgcg atcatggcgaccacacccgtcctgtggatcctctacgccggacgcatcgtggccggcatcaccggcgcc acaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcggg ctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctc cttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgca ggagtcgcataagggagagcgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtg ggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgcc ggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttg cggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgag aagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcga ggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgc tgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaa cttcgatcattggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccac ctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatca attcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccgccatctcca gcagccgcacgcggcgcatctcgggcagcgtggcgacaccatcgaatggtgcaaaacctttcgcggta tggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgt cgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcga aaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaac aactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgca aattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacga agcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcatta actatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgat gtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagca tctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgt ctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggtt gccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggat atctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaac aggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaa gggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccg cctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggca agtgagtggataaccgtattaccgcctttgagtgagctgataccgggaattctcactcattaggcatgatgat gaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctg cgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccaatccggata tagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctc agcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatctgtcattatcagaagctgt attttatccccagtgctccggagatggcattgctcccgtgacctcctgcctgatatgcgactccgccattcact gacaagttttgagtaatcaccccttcaatacctgtctttatctctccctgatttcggctacctgacaacaactggctgtcatcactcattttaacaccaaattcatgagtgttatggatccagtttgcctctatatacggacggaacctc cgcccggtatccttatccagggtgcttttcaccttccaggatgcacgaatacctgcttttgtctgaatattattttt ccctgctccctgcaccaccgttccgttatcctcctgatgtgtatccggtgtaacccccatccagacagcctgc aaatgaggctgtaaccagaattcacctgttattccttcaggtgatgtccatgtgtgcacattcaggttatatcc cccacctgcagaagcggttaaaccattcagattatatttttcttcttccagtccgtcacctttcactgatgcatta aaccagttatattgcatccaagtttcagcaaagagccctgttgcattttccccattctgataccacgtaccgta taccccgacagaataaccatccagtgtgtttctggcagctttgttgctcgtgtaatttatcgttttaccttttgcatt cgcgtatcctcccataatccctaaggtaaaatcacccagttgttcagcatggaatttataaatatcccccccg agctgattgataaactgattggttgttgttttattttgcccgtcattcagcttaccagagcttattcctccagtgatc ttcatccacacggatgcagactcaggctgtgtattatcactcatggccctgaattgcttacgctcattcaaatc catgaggaacagtgagttagccagtgccatattggtagcataacttccgttctccggtctgtattgccgggta tcagatgtgggaagatgactggttaaataccatcccttattatctgtcccactctcgtttcctttctgcagtgtgta atcataagctccggcaactacgcggttcttcagagagaattctgcatcagaatttccctctacagaaataatattaataccatctctcgtctgaccaccactgccatcttcattgacataaacgatgtcactttgaccagaggtat tacctttcaccaccagacggtccgtaagtgaattatctccttcaagcacaccaccaagagaaataacactt cccggtgtcccagtataatttgacacggtaagagtattacctatagcggccgcactttcttttgtaggattaag aatgatatttttgttattaacaagactaccattcactgtagcagagcagctttgctgtaccaaaacttaagt cagcatcattcgtaatattcccgtttataatgctgttattaatgacaatgcttccattgttattcactctagacaga tcttcttcgctaatcagtttctgttcaccctggaagtacaggttttcgagctcatgctttgtcg (SEQ ID NO 50). b) The sequence coding for the extramembrane part of Arabidopsis thaliana NADPH P450 reductase 1 (ATR1) as described in Urban et al, (1997) J Biol Chem 272(31):19176-86

[0020] ) was amplified by PCR from a plasmid (pCR8_ATR1) using primers ATR1 fwd 16123 (tccgggtaccactagagtctctatcttcttcggt (SEQ ID NO 51)) and ATR1 rvs redo (ccgggagctcccagacatctctgaggtatcttc (SEQ ID NO 52)) according to Protocol 1 as mentioned above). c) The linker (GC-rich) was amplified atfrom a synthetic sequence using the PrimeSTAR GXL DNA Polymerase enzyme marketed by Takara Bio Inc using the primers FLfwd redo(agatgtctgggagctcccgggcggttctggt (SEQ ID NO 53)) and FLrvs 76F112 (ggacgaggtttctcgagcggagaaccgccgct (SEQ ID NO 54) according to the two-step Protocol 5 method described in PrimeSTAR GXL DNA Polymerase Manual, https: / / www.takara.co.kr / file / manual / pdf / R050A_e.v1906Da.pdf

[0021] , d) The DNA sequence coding for the hydrophilic extra-membrane part (SEQ ID 71) of P450, CYP76F112 (Villard et al, (2021) New Phytologist 231 (5): 1923-1939

[0022] ) was amplified by PCR from a plasmid pcr8- CYP76F112 described in Villard et al, (2021 ) New Phytologist 231 (5): 1923-1939

[0022] ) using primers 76F112 fwd (tccgctcgagaaacctcgtcccatcatcgga (SEQ ID NO 55)) and 76F112 rvs (tttcgagctcatgctttgtcggcacgggga (SEQ ID NO 56)) according to Protocol 1 mentioned above. The coding sequence e) The 3 DNA fragmentsfrom the sequences SEQ ID NO 71, SEQ ID NO 15, SEQ ID NO 17 were fused by PCR fusion. PCR fusion consists of using as a template for a PCR reaction the 3 DNA fragments at equimolar concentration to hybridize the 3 sequences into one via their homologous parts allowing to amplify by PCR reaction, according to Protocol 4 mentioned above, a sequence corresponding to the sequence tccgggtaccactagagtctctatcttcttcggtacgcagactggaacagctgagggatttgctaaggcatt atccgaagaaatcaaagcgagatatgaaaaagcagcagtcaaagtcattgacttggatgactatgctgc cgatgatgaccagtatgaagagaaattgaagaaggaaactttggcatttttctgtgttgctacttatggagat ggagagcctactgacaatgctgccagattttacaaatggtttacggaggaaaatgaacgggatataaag cttcaacaactagcatatggtgtgtttgctcttggtaatcgccaatatgaacattttaataagatcgggatagtt cttgatgaagagttatgtaagaaaggtgcaaagcgtcttattgaagtcggtctaggagatgatgatcagag cattgaggatgattttaatgcctggaaagaatcactatggtctgagctagacaagctcctcaaagacgagg atgataaaagtgtggcaactccttatacagctgttattcctgaataccgggtggtgactcatgatcctcggtttacaactcaaaaatcaatggaatcaaatgtggccaatggaaatactactattgacattcatcatccctgcag agttgatgttgctgtgcagaaggagcttcacacacatgaatctgatcggtcttgcattcatctcgagttcgac atatccaggacgggtattacatatgaaacaggtgaccatgtaggtgtatatgctgaaaatcatgttgaaata gttgaagaagctggaaaattgcttggccactctttagatttagtattttccatacatgctgacaaggaagatg gctccccattggaaagcgcagtgccgcctcctttccctggtccatgcacacttgggactggtttggcaagat acgcagaccttttgaaccctcctcgaaagtctgcgttagttgccttggcggcctatgccactgaaccaagtg aagccgagaaacttaagcacctgacatcacctgatggaaaggatgagtactcacaatggattgttgcaa gtcagagaagtcttttagaggtgatggctgcttttccatctgcaaaacccccactaggtgtattttttgctgcaa tagctcctcgtctacaacctcgttactactccatctcatcctcgccaagattggcgccaagtagagttcatgtt acatccgcactagtatatggtccaactcctactggtagaatccacaagggtgtgtgttctacgtggatgaag aatgcagttcctgcggagaaaagtcatgaatgtagtggagccccaatctttattcgagcatctaatttcaagt taccatccaacccttcaactccaatcgttatggtgggacctgggactgggctggcaccttttagaggttttctg caggaaaggatggcactaaaagaagatggagaagaactaggttcatctttgctcttctttgggtgtagaaatcgacagatggactttatatacgaggatgagctcaataattttgttgatcaaggcgtaatatctgagctcatc atggcattctcccgtgaaggagctcagaaggagtatgttcaacataagatgatggagaaggcagcaca agtttgggatctaataaaggaagaaggatatctctatgtatgcggtgatgctaagggcatggcgagggac gtccaccgaactctacacaccattgttcaggagcaggaaggtgtgagttcgtcagaggcagaggctata gttaagaaacttcaaaccgaaggaagatacctcagagatgtctgggagctcccgggcggttctggtggc ggtagcggcggtggcggttctggcggtggcggtagcggcggtggcggttctggcggtggcggtagcggc ggtggcggttctggcggtggcggtagcggcggtggcggttctggtggcggtagcggcggttctccgctcg agaaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggc tttccgagtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccacc atggctaaagaagtcctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagcc gcgcccacagacacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaa ataagcaactcacacttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgc aagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctt tcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctatgcgcacatgatggaagagttggggaagcctaacttggccgattatt tcccgtttctacaaaagattgacccccaaggcattaggcggcgcaacggttactttccggaaactgatc aacttgtttgggcgtatcatcgaccaaagattgaaagtgagagattc atttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaacca taattgaacatttttactggatttattcagcggggactgaaacgacttcaaccacgttggagtgggcaatg gctgagctagtaaaagcgccagagattatgtcaaagccc aggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaaaccttcc gcatgcaccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaatctccgactatatca tcccgaaggatgctcaggtgattctcgactatatca cgacaagtttataccggagaggttttggacatcgatatagatgtcggaggccgggattttaagctcattccg ttcggtgctggtcggagaatgtcccggattcccattggcgatgtgcacttgatgttggggtctttg cttcactcgttt tcaccttgcaaatggctcagcctttcgagctatccccgtgccgacaaagcatgagctcgaaa (SEQ ID NO 57) . f) The ATR1 -FL-76F112 DNA fragment (SEQ ID NO57) obtained by PCR fusion and the linearized plasmid pAIDA1-T7 (SEQ ID NO 50) were then fused by Infusion according to protocol 2. The ligation product was introduced into E. co / / HST08 bacteria (Takara). The transformed bacteria were plated on a solid LB culture medium (lysogenic broth) (10g peptone, 5g yeast extract, 5g NaCl, 16g Agar) containing tetracycline (50pg / mL) at 37°C. An isolated colony was then used to amplify, by culture in liquid LB medium (without Agar) containing tetracycline (50 pg / mL) at 37°C, the plasmid pAIDA1-T7-CYP76F112-ATR1. The presence of the constituent elements of the plasmid was verified by PCR using the primers 27F (GCTAGAGTAAGTAGTTCGCCAGT (SEQ ID NO 72)), 51 F (CCTGAATACCGGGTGGTGAC (SEQ ID NO 58)), 52R (GCATATACACCTACATGGTCAC (SEQ ID NO 59)), 53F (GTGAAGGAGCTCAGAAGGAGTA (SEQ ID NO 60)), 54R (TAGAGTTCGGTGACGTCCCT (SEQ ID NO 61)), 62F (TTTGGGCGTATCATCGACCAA (SEQ ID NO 62)), 63R(ATGGTTTTGTCAAGCTGATCCTC (SEQ ID NO 63)), 57F (TTCTCTTGGTGGTGTGCTTGA (SEQ ID NO 64), 58R (catctctcgtctgaccacca (SEQ ID NO 65)), 59F (ATAACGGAACGGTGGTGCAGG (SEQ ID NO 66)), 60R (AACCTCCGCCCGGTATCCTT (SEQ ID NO 67)) and 61 R (ACGATACCGAAGACAGCTCATG (SEQ ID NO 68)) All of the steps allowed the construction of a plasmid pAIDA1-T7-CYP76F112-ATR1.

[0164] 3) Fusion protein

[0165] The resulting fusion protein contains from the N-terminus to the C-terminus:

[0166] - a tag of 6 Histidines, allowing immunodetection of proteins with anti His antibodies

[0167] - a cleavage sequence to remove the tag,

[0168] - a c-MYC sequence, allowing immunodetection of proteins with anti-c-MYC antibodies

[0169] - an AIDAI binding peptide

[0170] - an external membrane addressing and anchoring polypeptide: the beta Barel anchoring sequence of AIDAI, namely the sequence SEQ ID NO 1

[0171] - a polypeptide sequence of cytochrome P450 CYP76F112 (marmesin synthase from Ficus carica, NCBI ID: GenBank Sequence ID: MW348922.1) in which the membrane anchor has been removed from the coding sequence, namely the sequence SEQ ID NO 70,

[0172] - a binding peptide of 51 amino acids PGGSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGSGGSP (SEQ ID NO 6), and

[0173] - a polypeptide sequence of Arabidopsis NADPH P450 reductase (Arabidopsis thaliana P450 reductase 1 (ATR1), mRNA - NCBI Reference Sequence: NM_118585.4) in which the membrane anchor has been removed, namely the sequence SEQ ID NO 7.

[0174] The coding nucleic sequences corresponding to the different elements of the fusion protein described above were used and assembled successively from 5' to 3'. In particular, these were:

[0175] - the sequence SEQ ID NO 44 coding for an AIDAI linker, - the sequence SEQ ID NO 10 coding for a polypeptide for addressing and anchoring to the external membrane: the Beta Barrel anchor sequence

[0176] - the sequence SEQ ID NO 71 coding for the cytochrome P450 CYP76F112 marmesin synthase of Ficus carica, NCBI ID: Sequence ID: MW348922.1)

[0177] - the sequence SEQ ID NO 15 coding for a linker peptide of 51 amino acids, and

[0178] - the sequence SEQ ID NO 17 coding for Arabidopsis NADPH P450 reductase (Arabidopsis thaliana P450 reductase 1 (ATR1), mRNA - Sequence ID: NM_118585.4)

[0179] 4) Expression of recombinant proteins in E. coli bacteria a) The expression system used

[0180] The pAIDAI plasmid and recombinant plasmids containing the genes encoding the fusion protein are introduced into E. coli BL21 (DE3) plysE bacteria (Novagen's® pET Systems). BL21 (DE3) pLysE bacteria are adapted for the production of proteins under the control of the T7 promoter. BL21 (DE3) pLysE bacteria carry the lambda DE3 lysogen and contain the pLysE plasmid, which constitutively expresses T7 lysozyme. T7 lysozyme reduces the basal expression of target genes by inhibiting T7 RNA polymerase. The BL21 (DE3) pLysE strain therefore allows tighter control of T7 RNA polymerase. b) Preparation of an example fusion protein

[0181] The fusion protein is expressed from the plasmid whose construction was described in point 3 above. The introduction of the plasmid into BL21 plysE bacteria is carried out according to the protocol recommended by the supplier (https: / / tools.thermofisher.com / content / sfs / manuals / oneshotbl21_man.pdf

[0023] ) The presence of the constituent elements of the plasmid is verified by PCR using the primers 27F (GCTAGAGTAAGTAGTTCGCCAGT (SEQ ID NO 72), 51 F (CCTGAATACCGGGTGGTGAC (SEQ ID NO 58)), 52R

[0182] (gcatatacacctacatggtcac (SEQ ID NO 59)), 53F

[0183] (GTGAAGGAGCTCAGAAGGAGTA (SEQ ID NO 60)), 54R

[0184] (TAGAGTTCGGTGGACGTCCCT (SEQ ID NO 61)), 62F

[0185] <h2 style=";text-align:left;direction:ltr">(TTTGGGCGTATCATCGACCAA (SEQ ID NO 62)), 63R (atggttttgtcaagctgatcctc (SEQ ID NO 63)), 57F (TTCTCTTGGTGGTGTGCTTGA (SEQ ID NO 64)), 58R (CATCTCTCGTCTGACCACCA (SEQ ID NO 65)), 59F (ATAACGGAACGGTGGTGCAGG (SEQ ID NO 66)), 60R (aacctccgcccggtatcctt (SEQ ID NO 67)) and 61R (ACGATACCGAAGACAGCTCATG (SEQ ID NO 68)) on the plasmid extract obtained from a culture of BL21 plysE transformed with the plasmid pAIDAI-T7-CYP76F112-ATR1.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0186] A glycerolated stock of the BL21 plysE bacterium containing the plasmid pAIDA1 -T7-CYP76F112-ATR1 is then used to inoculate according to the supplier's recommendations (in-vitrogen) a preculture overnight, i.e. for 12-14 hours, of LB medium (lysogenic broth) (10g peptone, 5g yeast extract, 5g NaCl) containing tetracycline (50pg / mL) and chloramphenicol (20pg / mL) at 37°C. This preculture is used to inoculate 50 mL of LB (lysogenic broth) medium (10g peptone, 5g yeast extract, 5g NaCl) containing tetracycline (50pg / mL) and chloramphenicol (20pg / mL) at an OD 550nm = 0.05 in a sterile 250 mL Erlenmeyer flask. After approximately 2 hours of culture at 37°C with shaking at 180 rpm, the culture is stopped when the optical density of the latter reaches an OD ssonm of 0.4. The cultures are then cooled on ice for 10 minutes.The volume of the culture medium is measured and the antibiotics are re-added to a final concentration of 50 pg / ml of Tetracycline and 20 pg / ml of Chloramphenicol. Production of the fusion protein is induced by adding isopropyl 0-D-1 - thiogalactopyranoside (IPTG) at a final concentration of 20 pM for 24 h at 7 ° C, with shaking at 180 rpm. After 24 h, the optical density is adjusted to 0.4 by dilution in LB medium (4 ° C). The bacteria are harvested by two successive low-speed centrifugations (4 ° C, 20 min, 1000 x g and 4 ° C, 10 min, 4000 x g). The bacteria are then suspended in buffer (88 mM with additives of 1 mM KCl, 44 mM MgSO, 5% v / v glycerol, at 4°C (products ordered from Sigma Aldrich)).

[0187] Figure 1 is a schematic representation of the resulting fusion protein, said fusion protein being anchored in the bacterial membrane.

[0188] 5) Bioconversion

[0189] A study of the bioconversion I of demethyl suberosin is carried out. Figure 9 (CYP76F112) represents the corresponding bioconversion reaction. For this, demethyl suberosin (100 pM) as substrate and nicotinamide adenine dinucleotide phosphate (NADPH) (400 pM) are added to the medium comprising the resuspended E. coli bacteria obtained in point 4 above. The bioconversion process is carried out at 20°C for 1 h and with stirring at 180 rpm. The reaction is stopped by extraction with 1 volume of Ethyl Acetate. The media are mixed by vortexing for 5 minutes followed by centrifugation at 4400xg to separate the organic and aqueous phases. The upper organic phase is recovered and evaporated by Vivaspin. The powder obtained is suspended in 100 pL of methanol. The extract thus obtained is analyzed by ultra-high performance liquid chromatography coupled with a mass spectrum (UHPLC / MS / MS).

[0190] This example therefore clearly demonstrates that an example of a fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane, advantageously to the external membrane, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one binding polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase allows substrate bioconversion.

[0191] This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously be used in a process for the bioconversion of substrates.

[0192] This example also clearly demonstrates that an example of a fusion protein according to the invention can be expressed on the surface of a cell, in particular a bacterial cell, and can advantageously allow bioconversion of substrates in the culture medium of said cell.

[0193] Bibliographic references

[0194] 1. Guengerich and Macdonald, 1990, FASEB J. 4, pp 2453-2459

[0195] 2. Xu Jun et al. “The cytochrome P450 superfamily: Key players in plant development and defense” Journal of Integrative Agriculture 2015, 14(9): 1673-1686

[0196] 3. Kenneth Jensen et al., Plant NADPH-cytochrome P450 oxidoreductases, Phytochemistry Volume 71, 2-3, February 2010, Pages 132-141

[0197] 4. https: / / blog.addgene.org / plasmid-101-origin-of-replication

[0198] 5. https: / / www.qiagen.com / cn / resources / faq?id=1f42840e-fbd7-4734-b0cd-e17372a9e5a4&lang=en

[0199] 6. https: / / www.takarabio.eom / documents / User%20Manual / R045A_e.v 2102Da.pdf

[0200] 7. WO 83 / 004261

[0201] 8. Naso et al 2017, "Adeno-Associated Virus (AAV) as a Vector for Gene Therapy", BioDrugs. 2017 Aug;31 (4):317-334. doi : 10.1007 / s40259- 017-0234-5

[0202] 9. Zincarelli 2008 "Analysis of AAV Serotypes 1 -9 Mediated Gene Expression and Tropism in Mice After Systemic Injection" Mol Ther. 2008 Jun;16(6):1073-80. doi: 10.1038 / mt.2008.76. Epub 2008 Apr 15

[0203] 10. https: / / www.addgene.org / 79180 /

[0204] 11 . https: / / www.fishersci.fr / shop / products / fermentas-pbr322- dna / 10191220

[0205] 12. https: / / www.takarabio.com / products / cloning / in-fusion-cloning

[0206] 13. https: / / www.fishersci.fr / shop / products / fermentas-pbr322- dna / 10191220

[0207] 14. Jarmander, J., Gustavsson, M., Do, TH. et al. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli. Microb Cell Fact 11 , 118 (2012). https: / / doi.org / 10.1186 / 1475-2859- 11 -118 15. Rosano GL and Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5:172. doi: 10.3389 / fmicb.2014.00172

[0208] 16. https: / / www.takarabio.com / documents / User%20Manual / ln / ln- Fusion%20Snap%20Assembly%20User%20Manual_071320.pdf

[0209] 17. https: / / assets.thermofisher.com / TFS- Assets / LSG / manuals / oneshottop10_man.pdf

[0210] 18. https: / / www.mn-net.com / media / pdf / 45 / 51 / 02 / lnstruction-NucleoSpin- Plasmid.pdf 19. http: / / assets.thermofisher.com / TFS-Assets / BID / Reference-

[0211] Materials / fastd igest-restriction-enzymes-labaid.pdf

[0212] 20. Urban et al, (1997) J Biol Chem 272(31 ):19176-86

[0213] 21 . https: / / www.takara.co.com / file / manual / pdf / R050A_e.v1906Da.pdf.

[0214] 22. Villard, C., Munakata, R., Kitajima, S., Van Velzen, R., Schranz, ME, Larbat, R., & Hehn, A. (2021). A novel P450 involved in the furanocoumarin pathway underlies a recent case of convergent evolution. New Phytologist, 231(5), 1923-1939.

[0215] 23. https: / / tools.thermofisher.com / content / sfs / manuals / oneshotbl21_ma n.pdf

Claims

DEMANDS 1. Fusion protein comprising successively (i) at least one bacterial membrane targeting and anchoring polypeptide, (ii) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450, (iii) at least one linking polypeptide comprising at least 47 amino acids, preferably comprising 51 amino acids, and (iv) at least one polypeptide comprising the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase.

2. Fusion protein according to claim 1, wherein the quaternary structure of said at least one membrane-targeting and anchoring polypeptide forms a beta barrel.

3. Protein from the fusion for recovery 1 or 2 times, in the dress and annexation of the polypeptide in the sequence MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAVVSTIGNNAFAVD HHHHHHLEALFQGPGTQKQRTELENLYFQGEQKLISEEDLSRVNNNGSI VINNSIINGNITNDADLSFGTAKLLSATVNGSLVNNKNIILNPTKESAAAIGN TLTVSNYTGTPGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDG SGGQTRDGINIISVEGNSDAEFSLKNRVVAGAYDYTLQKGNESGTDNKG WYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSD NTQPESASVWMKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQL GDFTLGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGEN ATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHT WTSPEGITGEFWLQPHLQAVWMGVTPDTHQEDNGTWQGAGKNNIQTK AGIRASWKVKSTLDKDTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSG SRNQGEIKTGIEGVITQNLSVNGGVAYQAGGHGSNAISGALGIKYSF (SEQ ID NO 1 ).

4. Fusion protein according to any one of claims 1 to 3, wherein said polypeptide from the hydrophilic domain of a plant cytochrome P450 is a polypeptide having a sequence identity of at least 28% identity with the polypeptide of sequence IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMGQRNLWVSSPELA KEVLHTQGVEFGSRTRNWFDIFTGKGQDMVFTVYGEHWRKMRRIMTV PFFTNKVVQQYRYGWEAEAAAWDDVKKNPAAATEGIVIRRRLQLMMYN NMFRIMFDRRFESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPILRPFL RNYLKLCKEVKDKRIQLFKDYFVDERKKIGSTKKMDNNQLKCAIDHILEAK EKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQAKLRHELDT KLGPGVQITEPDVQNLPYLQAWKETLRLRMAIPLLVPHMNLHDAKLGGF DIPAESKILVNAWWLANNPDQWKKPEEFRPERFLEEEAKVEANGNDFRY LPFGVGRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKIDTDEKGGQFS LHILKHSTIVAKPRSF (SEQ ID NO 3).

5. Fusion protein according to any one of claims 1 to 3, wherein said polypeptide from the hydrophilic domain of a plant cytochrome P450 is a polypeptide selected from the group comprising the polypeptide sequence IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMGQRNLVWSSPELA KEVLHTQGVEFGSRTRNWFDIFTGKGQDMVFTVYGEHWRKMRRIMTV PFFTNKVVQQYRYGWEAEAAAWDDVKKNPAAATEGIVIRRRLQLMMYN NMFRIMFDRRFESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPILRPFL RNYLKLCKEVKDKRIQLFKDYFVDERKKIGSTKKMDNNQLKCAIDHILEAK EKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQAKLRHELDT KLGPGVQITEPDVQNLPYLQAWKETLRLRMAIPLLVPHMNLHDAKLGGF DIPAESKILVNAWWLANNPDQWKKPEEFRPERFLEEEAKVEANGNDFRY LPFGVGRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKIDTDEKGGQFS LHILKHSTIVAKPRSF (SEQ ID NO 3) et KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTWISSTTMAKE VLQANSQWSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLS SKALDGNMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTF FSMDMADDTNSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIR RRNTVTFRKLINLFGRIIDQRLKVREASGSLKDDDILDTLINMMVVDQEKK EDQLDKTIIEHFLLDLFSAGTETTSTTLEWAMAELVKAPEIMSKARAELDQ VIGKGNQVKESDVSRLPYLQAIVKETFRMHPTAPLLIPRKADSDIEISDYIIP KDAQVIVNVWAIGRDSSTWENPDKFIPERFLDIDIDVGGRDFKLIPFGAGR RICPGFPLAMRMLHLMLGSLLHSFDWKLEDGVRPDALNMDEKFGLTLQM AQPLRAIPVPTKH (SEQ ID NO 70) 6. Fusion protein according to any one of claims 1 to 5, wherein said linking polypeptide is a polypeptide of sequence PGGSGGGSGGGGSGGGGSGGGGGGSGGGGSGGGGGGGSGGGGSG GGSGGSP (SEQ ID NO 6).

7. Fusion protein according to any one of claims 1 to 6, wherein said polypeptide of a hydrophilic domain of an NADPH P450 reductase is a polypeptide having at least 90% sequence identity with the polypeptide of sequence TRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEE KLKKETLAFFCVATYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFA LGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKES LWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSMESNVAN GNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVG VYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTL GTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQ WIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSR VHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNF KLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCR (SEQ ID NO 7).

8. Fusion protein according to any one of claims 1 to 7, wherein said polypeptide comprising the hydrophilic domain of a plant cytochrome P450 or said polypeptide comprising the hydrophilic domain of an NADPH P450 reductase is free of a transmembrane domain.

9. Nucleic acid encoding a fusion protein according to any one of claims 1 to 8.

10. Vector, preferably an expression vector, comprising a nucleic acid according to claim 9.

11. Host cell comprising a nucleic acid according to claim 8 and / or a vector according to claim 10.

12. Host cell according to claim 11, said host cell being a bacterial cell, preferably Escherichia coli.

13. A method for producing a fusion protein comprising culturing a host cell according to claim 11 or 12 under conditions suitable for the expression of the fusion protein.

14. A method for bioconverting a substrate by a fusion protein according to any one of claims 1 to 8 comprising the steps of: - introduction into a culture medium comprising a host cell according to claim 11 or 12, of said substrate, - incubation of said culture medium for a sufficient time for the bioconversion of said substrate by said fusion protein, and - optionally recovery of metabolites resulting from the bioconversion of said substrate.

Images