Fusion protein and use for bioconversion of molecules

Abstract

The present invention relates to a fusion protein, to a nucleic acid encoding said protein, to a vector comprising said vector, to a host cell comprising said nucleic acid and / or vector, to a method for manufacturing a fusion protein and to a method for bioconversion of a substrate. The fusion protein of the present invention comprises, successively (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 comprising at least 47 amino acids and (iv) at least one polypeptide corresponding to the hydrophilic domain of a plant cytochrome P450 NADPH P450 reductase. Abstract figure: Fig.1

Description

Title of the invention: Fusion protein and use for the bioconversion of molecules Technical field of the invention

[0001] 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.

[0002] 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.

[0003] The present invention also relates to a method of bioconversion of a substrate comprising the use of a fusion protein.

[0004] 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.

[0005] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text. Prior art

[0006] The specialized metabolism of plants is a metabolism of adaptation of plants to changing environmental conditions. Each plant has developed during its evolution an arsenal of molecules allowing it to respond to living conditions specific to it. The diversity of molecules thus produced is almost inexhaustible. These molecules, which can be very complex, have been widely used by humans, particularly for uses in the field of health.

[0007] The synthesis of these molecules is carried out through complex biosynthetic pathways involving numerous steps catalyzed by specific enzymes. Cytochromes P450s (P450s) are part of these enzymes and can be considered as very high-precision tools for producing high-value molecules.

[0008] Cytochromes P450s are therefore enzymes involved in numerous processes linked to the adaptation of plants to their environment. They are at the origin of part of the great diversity of molecules having remarkable physicochemical properties in a physiological context but also for applications in Humans in various fields including medicine, cosmetics, pharmaceuticals and agronomy. Molecular data on P450s have increased through the use of high-throughput sequencing methods.

[0009] 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.

[0010] 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, in order to be active, need to function in tandem with an NADPH P450 reductase which provides electrons necessary for oxidation reactions.

[0011] To carry out a functional characterization of these enzymes, tools / methods have therefore been developed. Since cytochromes P450s function with NADPH-P450 reductases, these two enzymes must be in close interaction in order 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 a 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.

[0012] However, the implementation of this method is not possible in particular when the substrate is hydrophobic. Indeed, when it is hydrophobic, the substrate cannot enter the yeast (pass the membrane) used.

[0013] There is therefore a real need to find a means and / or process allowing production and / or functional characterization of cytochromes P450 and / or allowing bioconversion of a hydrophobic molecule or substrate by cytochromes P450.

[0014] 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 a de gradation of the P450s thus preventing multiple uses of the P450s.

[0015] There is therefore a real need to find a means and / or process allowing production and / or functional characterization of cytochromes P450 while allowing 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. Statement of the invention

[0016] 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.

[0017] The inventors have demonstrated in a surprising and unexpected manner 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 demonstrated in a surprising manner 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.

[0018] 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 binding 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.

[0019] The inventors have also surprisingly demonstrated that the fusion protein according to the invention, advantageously when it is present on the external surface of the cell membrane, can be used in substrate bioconversion processes.

[0020] 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 substrate, advantageously by avoiding steps of extraction of membrane proteins, advantageously making it possible to limit the risks of degradation. gradation of proteins during protein purification steps and to achieve the production of molecules of interest directly in the culture medium thus reducing the purification steps.

[0021] 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 external membrane of a gram-negative bacteria.

[0022] 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 external 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-ll-118

[14] . 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 MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLV-LAVVSTIGNAFAVDHHHHHHLEALFQGPGTQKQRTELENLYFQGEQKLISEED LSRVNNNGSIVINNSIINGNITNDADLSFGTAKLLSATVNGSLVNNKNIILNPTK ESAAAIGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLVVKGNTSGQSDIVYV NEDGSGGQTRDGINIISVEGNSDAEFSLKNRVVAGAYDYTLQKGNESGTDNKG WYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQP ESASVWMKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGI MGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGENATGLFAETW (SEQ ID NO) 1). <h2 style=";text-align:left;direction:ltr">

[0023] Advancing, the polypeptide address and the membrane in the membrane<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> lypeptide of sequence MNKAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLV-LAVVSTIGNAFAVDHHHHHHLEALFQGPGTQKQRTELENLYFQGEQKLISEED LSRVNNNGSIVINNSIINGNITNDADLSFGTAKLLSATVNGSLVNNKNIILNPTK ESAAAIGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLVVKGNTSGQSDIVYV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> NEDGSGGQTRDGINIISVEGNSDAEFSLKNRVVAGAYDYTLQKGNESGTDNKG WYLTSHLPTSDTRQYRPENGSYATNMALANSLFLMDLNERKQFRAMSDNTQP ESASVWMKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFTLGI MGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNGENATGLFAETW MQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHTWTSPEGITGEFW LQPHLQAVWMGVTPDTHQEDNGTVVQGAGKNNIQTKAGIRASWKVKSTLDK DTGRRFRPYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLS VNGGVAYQAGGHGSNAISGALGIKYSF (SEQ ID NO 1).<h2 style=";text-align:left;direction:ltr">

[0024] In the present, by cytochrome P450 is meant proteins with mono-oxygenase activity capable of oxidizing substrates by 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]. These may include, for example, plant cytochromes P450 belonging to the CYP51, CYP71, CYP72, CYP74, CYP85, CYP86, CYP97, CYP710, CYP711 and CYP727 families.Examples include plant cytochrome P450 of 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.

[0025] In the present, by hydrophilic domain of a plant cytochrome P450 is meant the polypeptide sequence of cytochrome P450 comprising the enzymatic domain and the biological activity, advantageously the enzymatic activity, of cytochrome P450. A person skilled in the art, using this general knowledge, knows how to identify the enzymatic domain of 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

[0026] HRNLTDLAKRFGEILLLRMGQRNLVVVSSPELAKEVLHTQGVEFGSRTRNV VFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRYGWEAEAAA VVDDVKKNPAAATEGIVIRRRLQLMMYNNMFRIMFDRRFESEDDPLFLKLKA LNGERSRLAQSFEYNYGDFIPILRPFLRNYLKLCKEVKDKRIQLFKDYFVDERK KIGSTKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINVAAIETTLWSIE WGIAELVNHPEIQAKLRHELDTKLGPGVQITEPDVQNLPYLQAVVKETLRLRM AIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPDQWKKPEEFRPERFL EEEAKVEANGNDFRYLPFGVGRRSCPGIILALPILGITIGRLVQNFELLPPPGQSK IDTDEKGGQFSLHILKH (SEQ ID NO 2), IPVPIFGNWLQVGDDLNHRNLTDLA-KRFGEILLLRMGQRNLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGKGQ DMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRYGWEAEAAAVVDDVKKNP AAATEGIVIRRRLQLMMYNNMFRIMFDRRFESEDDPLFLKLKALNGERSRLAQ SFEYNYGDFIPILRPFLRNYLKLCKEVKDKRIQLFKDYFVDERKKIGSTKKMDN NQLKCAIDHILEAKEKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPE IQAKLRHELDTKLGPGVQITEPDVQNLPYLQAVVKETLRLRMAIPLLVPHMNL HDAKLGGFDIPAESKILVNAWWLANNPDQWKKPEEFRPERFLEEEAKVEANG NDFRYLPFGVGRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKIDTDEKGGQF SLHILKHSTIVAKPRSF (SEQ ID NO 3),MDIFTSLLYLILILFFSLQVFRSFAFP-KHKRLPPGPPPRPIIGSLLELGDQPHRSLARLSYGPFMHLKLGQVTTVVISST TMAKEVLQANSQVVSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHL LSSKALDGNMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFF SMDMADDTNSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNT VTFRKLINLFGRIIDQRLKVREASGSLKDDDILDTLINMMVVDQQLDK TIIEHFLLDLFSAGTETTSTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVK ESDVSRLPYLQAIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQ (SEQ ID NO 4), KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLGQVTTVVISSTTMA-KEVLQANSQVVSSRTITDASRAHRHSDFSMVMLPVSPLWRNLRKISNSHLLSS KALDGNMELRNKVQELLNDVHKSVQAGEAVEIASLSFRATLNLLSTTFSM DMADDTNSVTLKELKEAMSHMMEELGKPNLADYFPFLQKIDPQGIRRRNTVT FRKLINLFGRIIDQRLKVREASGSLKDDDILDTLINMMVVDQEKKEDQLDKTIIE HFLLDLFSAGTETTSTLEWAMAELVKAPEIMSKARAELDQVIGKGNQVKESD VSRLPYLQAIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKDAQ (SEQ ID NO 5)

[0027] Advantageously, this can be a polypeptide isolated from a cytochrome, Plant 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 selected from the group comprising IPVPIFGNWLQVGDDLNHRNLTDLAKRFGEILLLRMG- QRNLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHW RKMRRIMTVPFFTNKVVQQYRYGWEAEAAAVVDDVKKNPAAATEGIVIRRR LQLMMYNNMFRIMFDRRFESEDDPLFLKLKALNGERSRLAQSFEYNYGDFIPI LRPFLRNYLKLCKEVKDKRIQLFKDYFVDERKKIGSTKKMDNNQLKCAIDHIL EAKEKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQAKLRHELD TKLGPGVQITEPDVQNLPYLQAVVKETLRLRMAIPLLVPHMNLHDAKLGGFDI PAESKILVNAWWLANNPDQWKKPEEFRPERFLEEEAKVEANGNDFRYLPFGV GRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKIDTDEKGGQFSLHILKHSTIV AKPRSF (SEQ ID NO 3), a KPRPIIGSLLELGDQPHRSLARLSESYGPFMHLKLG-QVTTVVISSTTMAKEVLQANSQVVSSRTITDASRAHRHSDFSMVMLPVSPLWR NLRKISNSHLLSSSKALDGNMELRNKKVQELLNDVHKSVQAGEAVEIASLSFRA TLNLLSTFFSMDMADDTNSVTLKELKEAMSHMMEELGKPNLADYFPFLQKI DPQGIRRRNTVTFRKLINLFGRIIDQRLKVREASGSLKDDDILDTLINMMVVDQ EKKEDQLDKTIIEHFLLDLFSAGTETTSTTLEWAMAELVKAPEIMSKARAELDQ VIGKGNQVKESDVSRLPYLQAIVKETFRMHPTAPLLIPRKADSDIEISDYIIPKD AQ (SEQ ID NO 5).

[0028] 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 PGGSGGGSGGGG-SGGGGSGGGGSGGGGGGGGGGGGGGGGGGGGSGGGSGGSP (SEQ ID NO 6).

[0029] In this document, by plant cytochrome P450 NADPH P450 reductase is meant proteins with oxidoreductase activity which catalyze the reaction: 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],

[0030] 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 of a plant cytochrome P450 NADPH P450 reductase. 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 a polypeptide chosen from the group comprising TRVSIFFGTQTGTAEGFAKALSEEIKA-RYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARF YKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIE VGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVT HDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDI SRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESA VPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDG KDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAP SRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFK LPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMD FIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEG YLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDV W (SEQ ID NO 7),<h2 style=";text-align:left;direction:ltr">YEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVA-TYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVL DEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVA TPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHHPCRVDVAVQKELH THESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVF SIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATE PSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPR LQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKS HECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELG SSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMME KAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSE (SEQ ID NO 8). ,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0031] After use, it may contain polypeptide isolate with NADPH.<h2 style=";text-align:left;direction:ltr"> Cytochrome P450 reductase comprising the hydrophilic domain of said NADPH P450 cytochrome P450 reductase lacking a transmembrane domain. 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 TRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVK-VIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFYKWFTEENE RDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQS IEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKS MESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETG DHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCT LGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIV ASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSAL VYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIV (SEQ ID NO 7).

[0032] 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.

[0033] The unnatural amino acids may be levorotatory (L-), dextrorotatory (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 may be present at any position in the fusion protein. For example, the unnatural amino acid may be at the N-terminus, the C-terminus, or any position between the N-terminus and the C-terminus.

[0034] 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-aminobutyric acid, β-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. Natural amino acid derivatives may, for example, include the addition of one or more chemical groups to the natural 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., . 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).

[0035] The fusion protein according to the invention can be produced and / or synthesized by any suitable method known to those skilled in the art.

[0036] As used herein, the terms "polypeptide", "peptide" and their grammatical equivalents refer to a polymer of amino acid residues.

[0037] As used herein, a "functional protein" is a protein that is biologically active.

[0038] 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.

[0039] 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 of the longer of the two sequences and multiplying the result by 100.

[0040] 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."

[0041] 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 listed elements are required or mandatory, and that no other elements may be present.

[0042] Another subject of the invention relates to a polynucleotide or nucleic acid coding for a fusion protein according to the invention.

[0043] 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.

[0044] 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 selected from the group consisting of the nucleic acid of sequence: accatgggcaataaggcc-tacagtatcatttggagccactccagacaggcctggattgtggcctcagagttagccagaggacatggttttgtccttgcaaaa aatacactgctggtattggcggttgtttccacaatcggaaatgcatttgcagtcgaccaccatcaccatcaccatctggaagcg ctgttccagggtccgggtaccgctacagtgaatggtagtcttgttaataacaaaaatatcattcttaatcctacaaaagaaagtg cggccgctataggtaatactcttaccgtgtcaaattatactgggacaccgggaagtgttatttctcttggtggtgtgcttgaagg agataattcacttacggaccgtctggtggtgaaaggtaatacctctggtcaaagtgacatcgtttatgtcaatgaagatggcagt ggtggtcagacgagagatggtattaatattatttctgtagagggaaattctgatgcagaattctctctgaagaaccgcgtagttg ccggagcttatgattacacactgcagaaaggaaacgagagtgggacagataataagggatggtatttaaccagtcatcttcc cacatctgatacccggcaatacagaccggagaacggaagttatgctaccaatatggcactggctaactcactgttcctcatgg atttgaatgagcgtaagcaattcagggccatgagtgataatacacagcctgagtctgcatccgtgtggatgaagatcactgga ggaataagctctggtaagctgaatgacgggcaaaataaaacaacaaccaatcagtttatcaatcagctcgggggggatatttataaattccatgctgaacaactgggtgattttaccttagggattatgggaggatacgcgaatgcaaaaggtaaaacgataaatta cacgagcaacaaagctgccagaaacacactggatggttattctgtcggggtatacggtacgtggtatcagaatggggaaaat gcaacagggctctttgctgaaacttggatgcaatataactggtttaatgcatcagtgaaaggtgacggactggaagaagaaaa atataatctgaatggtttaaccgcttctgcaggtgggggatataacctgaatgtgcacacatggacatcacctgaaggaataac aggtgaattctggttacagcctcatttgcaggctgtctggatgggggttacaccggatacacatcaggaggataacggaacg gtggtgcagggagcagggaaaaataatattcagacaaaagcaggtattcgtgcatcctggaaggtgaaaagcaccctggat aaggataccgggcggaggttccgtccgtatatagaggcaaactggatccataacactcatgaatttggtgttaaaatgagtga tgacagccagttgttgtcaggtagccgaaatcagggagagataaagacaggtattgaaggggtgattactcaaaacttgtca gtgaatggcggagtcgcatatcaggcaggaggtcacgggagcaatgccatctccggagcactggggataaaatacagctt ctgataatga (SEQ ID NO 9), atgaataaggcctacagtatcatttggagccactccagacaggcctg-gattgtggcctcagagttagccagaggacatggttttgtccttgcaaaaaatacactgctggtattggcggttgtttccacaatc ggaaatgcatttgcagtcgaccaccatcaccatcaccatctggaagcgctgttccagggtccgggtacccagaaacagcgtaccgagctcgaaaacctgtacttccagggtgaacagaaactgattagcgaagaagatctgtctagagtgaataacaatggaa gcattgtcattaataacagcattataaacgggaatattacgaatgatgctgacttaagttttggtacagcaaagctgctctctgct acagtgaatggtagtcttgttaataacaaaaaatatcattcttaatcctacaaaagaaagtgcggccgctataggtaatactcttac cgtgtcaaattatactgggacaccgggaagtgttatttctcttggtggtgtgcttgaaggagataattcacttacggaccgtctg gtggtgaaaggtaatacctctggtcaaagtgacatcgtttatgtcaatgaagatggcagtggtggtcagacgagagatggtatt aatattatttctgtagagggaaattctgatgcagaattctctctgaagaaccgcgtagttgccggagcttatgattacacactgca gaaaggaaacgagagtgggacagataataagggatggtatttaaccagtcatcttcccacatctgatacccggcaatacaga ccggagaacggaagttatgctaccaatatggcactggctaactcactgttcctcatggatttgaatgagcgtaagcaattcagg gccatgagtgataatacacagcctgagtctgcatccgtgtggatgaagatcactggaggaataagctctggtaagctgaatg acgggcaaaataaaacaacaaccaatcagtttatcaatcagctcgggggggatatttataaattccatgctgaacaactgggt gattttaccttagggattatgggaggatacgcgaatgcaaaaggtaaaacgataaattacacgagcaacaaagctgccagaaacacactggatggttattctgtcggggtatacggtacgtggtatcagaatggggaaaatgcaacagggctctttgctgaaactt ggatgcaatataactggtttaatgcatcagtgaaaggtgacggactggaagaagaaaaatataatctgaatggtttaaccgctt ctgcaggtgggggatataacctgaatgtgcacacatggacatcacctgaaggaataacaggtgaattctggttacagcctcat ttgcaggctgtctggatgggggttacaccggatacacatcaggaggataacggaacggtggtgcagggagcagggaaaa ataatattcagacaaaagcaggtattcgtgcatcctggaaggtgaaaagcaccctggataaggataccgggcggaggttcc gtccgtatatagaggcaaactggatccataacactcatgaatttggtgttaaaatgagtgatgacagccagttgttgtcaggtag ccgaaatcagggagagataaagacaggtattgaaggggtgattactcaaaacttgtcagtgaatggcggagtcgcatatcag gcaggaggtcacgggagcaatgccatctccggagcactggggataaaatacagcttctgataatga (SEQ ID NO 10).

[0045] 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: cctggcccaatcccggttccaattttcggcaactggctacaagttggcgatgatttgaaccaccggaact-taaccgatctggctaagaggtttggtgagatcttgctgctacgcatggggcagaggaatctggtagttgtgtcttcgcctgagc ttgctaaagaggtgttgcatacacaaggagtggagtttggttcgagaacaaggaatgttgtgttcgatatttttactgggaaggg tcaggatatggtgtttacggtttatggtgagcattggaggaagatgaggaggatcatgaccgtaccctttttcaccaacaaagtt gttcagcaatacaggtatgggtgggaggctgaggccgcggcggttgtggacgatgtgaagaagaatccggctgcagcaac tgaaggaatcgtgatccgaagacggttacaactcatgatgtataacaacatgttcagaatcatgttcgacagacgattcgaaa gtgaagatgatcccttgtttttgaaactcaaggcgttgaacggtgagaggagtcgattggcgcagagctttgagtacaactatggcgatttcatccctattttgcggccgtttttgagaaattatttgaagttgtgcaaggaagttaaagataaaaggattcagctcttca aggattacttcgttgacgaaaggaagaagattggaagcactaagaaaatggacaacaatcagttgaaatgtgccattgatcac attcttgaagctaaagagaagggtgagatcaatgaagacaatgttctttacattgttgaaaacatcaatgttgcagcaatcgaga caactctatggtcgatcgaatggggaattgcggagctagttaaccatcccgagatccaagccaaactcaggcacgagctcg acaccaagctcgggcccggtgtccagatcaccgagcccgacgtccaaaacctcccttacctccaagccgtggtcaaggaa accctccgtctccgtatggcgatcccgcttctagtcccacacatgaacctccatgacgctaagctcggcgggtttgacatccc ggccgaaagcaagatcttggtcaacgcgtggtggttagcaaacaaccccgaccaatggaagaaacccgaggagtttaggc cagagaggtttttggaagaggaagcgaaggttgaggctaacgggaatgattttaggtacttgccgtttggagtcgggagaag gagttgccccgggattattcttgcattgccgatacttggtattacaatcgggcgtttggtgcagaatttcgagctgttgcctccac cgggacagtctaagatcgataccgatgagaagggtgggcagtttagtttgcatatcttgaagcactctactatcgtagctaaac ctaggtcattt (SEQ ID NO 11),atggacctcctcctcatagaaaaaaccctcgtcgccttattcgccgccat-tatcggcgcaatactaatctccaaactccgcggtaaaaaattcaagctcccacctggcccaatcccggttccaattttcggcaa ctggctacaagttggcgatgatttgaaccaccggaacttaaccgatctggctaagaggtttggtgagatcttgctgctacgcat ggggcagaggaatctggtagttgtgtcttcgcctgagcttgctaaagaggtgttgcatacacaaggagtggagtttggttcga gaacaaggaatgttgtgttcgatatttttactgggaagggtcaggatatggtgtttacggtttatggtgagcattggaggaagat gaggaggatcatgaccgtaccctttttcaccaacaaagttgttcagcaatacaggtatgggtgggaggctgaggccgcggc ggttgtggacgatgtgaagaagaatccggctgcagcaactgaaggaatcgtgatccgaagacggttacaactcatgatgtat aacaacatgttcagaatcatgttcgacagacgattcgaaagtgaagatgatcccttgtttttgaaactcaaggcgttgaacggt gagaggagtcgattggcgcagagctttgagtacaactatggcgatttcatccctattttgcggccgtttttgagaaattatttgaa gttgtgcaaggaagttaaagataaaaggattcagctcttcaaggattacttcgttgacgaaaggaagaagattggaagcacta agaaaatggacaacaatcagttgaaatgtgccattgatcacattcttgaagctaaagagaagggtgagatcaatgaagacaat gttctttacattgttgaaaacatcaatgttgcagcaatcgagacaactctatggtcgatcgaatggggaattgcggagctagtta accatcccgagatccaagccaaactcaggcacgagctcgacaccaagctcgggcccggtgtccagatcaccgagcccga cgtccaaaacctcccttacctccaagccgtggtcaaggaaaccctccgtctccgtatggcgatcccgcttctagtcccacaca tgaacctccatgacgctaagctcggcgggtttgacatcccggccgaaagcaagatcttggtcaacgcgtggtggttagcaaa caaccccgaccaatggaagaaacccgaggagtttaggccagagaggtttttggaagaggaagcgaaggttgaggctaac gggaatgattttaggtacttgccgtttggagtcgggagaaggagttgccccgggattattcttgcattgccgatacttggtatta caatcgggcgtttggtgcagaatttcgagctgttgcctccaccgggacagtctaagatcgataccgatgagaagggtgggca gtttagtttgcatatcttgaagcactctactatcgtagctaaacctaggtcattttaa (SEQ ID NO 12), atggatatttt-cacctccttactgtatcttgctctcattcttttcttttctcttcaagtcttccgttcctttgcgtttcctaaacacaaaaggcttccacct ggtccaaaacctcgtcccatcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttccgagt cttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaaagaagtcctcc aggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacagacacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttggaaataagcaactcacacttgctttcctccaaggctcttgatggcaaca tggagctgagaaaacaaaaaggtgcaagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcggtggagat cgcgagcctttctttcagagctactctgaatctcttgtccaccacattttctctcatggacatggcggatgacacaaattccgtca ctctaaaaagagctcaaggaggctatgtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgtttctac aaaagattgacccccaaggcattaggcggcgcaacacggtactttccggaaactgatcaacttgtttgggcgtatcatcgac caaagattgaaagtgagagaagcgagtggttctttgaaagatgatgatatttagacactcttatcaacatgatggtggtggatc aggagaagaaagaggatcagcttgacaaaaccataattgaacattttactggatttatttcagcggggactgaaacgacttc aaccacgttggagtgggcaatggctgagctagtaaaagcgccagagattatgtcaaaagcccgagcagagctagatcaagt tataggcaaaggaaaccaagtgaggaatcggacgtatctgactcccttacttcaaagccattgttaaagaaaccttccgca tgcaccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaatctccgactatatcatcccgaaggatgctc agggatgtgtcaatgtatgggccattggtagactcaagcacatgggaaaaatcccgacaagtttataccggagaggtttttggacatcgatatagatgtcggaggccgggattttaagctcattccgttcggtgctggtcggagaatatgtcccggattcccattg gcgatgcgaatgttgcacttgatgttggggtctttgcttcactcgtttgattggaagttggaagatggggttagacctgatgctct aaacatggatgaaaagtttggcctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcattag (SEQ ID NO 13), atcatcggaagcctcttggagctcggcgaccaaccccacaggtccttggccaggctttcc-gagtcttacggcccgtttatgcatttgaagctcggccaagtcacgacggttgtcatttcctccaccaccatggctaaagaagtc ctccaggcaaacagccaagtcgtctccagccggacaatcaccgacgcaagccgcgcccacagacacagcgattttagcat ggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaataagcaactcacacttgctttcctccaaggctcttgatggc aacatggagctgagaaacaaaaaggtgcaagagctcctaaatgatgtccacaaaagcgtccaggccggggaggcggtgg agatcgcgagcctttctttcagagctactctgaatctcttgtccaccacatttttctccatggacatggcggatgacacaaattcc gtcactctaaaagagctcaaggaggctatgtcgcacatgatggaagagttggggaagcctaacttggccgattatttcccgttt ctacaaaagattgacccccaaggcattaggcggcgcaacacggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagattgaaagtgagagaagcgagtggttctttgaaagatgatgatattttagacactcttatcaacatgatggtggt ggatcaggagaagaaagaggatcagcttgacaaaccataattgaacatttttactggatttatttcagcggggactgaaac gacttcaaccacgttggagtgggcaatgctgagctagtaaaaagcgccagagattatgtcaaaagcccgagcagagctaga tcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaacctt ccgcatgcaccctacagctccattattgattcctcgcaaagccgacagtgacatcgaaatctccgactatcatcatcccgaagg atgctcaggtgattgtcaatgtatgggccattggtagagactcaagcacatgggaaaatcccgacaagtttataccggagag gttttggacatcgatatagatgtcggaggccgggatttaagctcattccgttcggtgctggtcggagattgtcccggattc ccattggcgatgcgaatgttgcacttgattggaagttggaagatggggttagacctga tgctctaaacatggatgaaaaagttggcctcaccttgcaaatggctcagcctttgcgagctatccccgtgccgacaaagcatta gséque (SEQ ID NO14), préquence ID SEQ ID NO 11.

[0046] 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: ccgggcggttctggtggcgg-tagcggcggtggcggttctggcggtggcggtagcggcggtggcggttctggcggtggcggtagcggcggtggcggttct ggcggtggcggtagcggcggtggcggttctggtggcggtagcggcggttctccg (SEQ ID NO 15).

[0047] 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: atgacttctgctttgtatgcttccgatttgtttaagcagct-caagtcaattatggggacagattcgttatccgacgatgttgtacttgtgattgcaacgacgtctttggcactagtagctggatttg tggtgttgttatggaagaaaacgacggcggatcggagcggggagctgaagcctttgatgatccctaagtctcttatggctaag gacgaggatgatgatttggatttgggatccgggaagactagagtctctatcttcttcggtacgcagactggaacagctgaggg atttgctaaggcattatccgaagaaatcaaagcgagatatgaaaaagcagcagtcaaagtcattgacttggatgactatgctg ccgatgatgaccagtatgaagagaaattgaagaaggaaactttggcattttctctgtgttgctacttatggagatggagagccta ctgacaatgctgccagattttacaaatggtttacggaggaaaatgaacgggatataaagcttcaacaactagcatatggtgtgtttgctcttggtaatcgccaatatgaacattttaataagatcgggatagttcttgatgaagagttatgtaagaaaggtgcaaagcgt cttattgaagtcggtctaggagatgatgatcagagcattgaggatgattttaatgcctggaaagaatcactatggtctgagctag acaagctcctcaaagacgaggatgataaaagtgtggcaactccttatacagctgttattcctgaataccgggtggtgactcatg atcctcggtttacaactcaaaaatcaatggaatcaaatgtggccaatggaaatactactattgacattcatcatccctgcagagtt gatgttgctgtgcagaaggagcttcacacacatgaatctgatcggtcttgcattcatctcgagttcgacatatccaggacgggt attacatatgaaacaggtgaccatgtaggtgtatatgctgaaaatcatgttgaaatagttgaagaagctggaaaattgcttggcc actctttagatttagtattttccatacatgctgacaaggaagatggctccccattggaaagcgcagtgccgcctcctttccctggt ccatgcacacttgggactggtttggcaagatacgcagaccttttgaaccctcctcgaaagtctgcgttagttgccttggcggcc tatgccactgaaccaagtgaagccgagaaacttaagcacctgacatcacctgatggaaaggatgagtactcacaatggattg ttgcaagtcagagaagtcttttagaggtgatggctgcttttccatctgcaaaacccccactaggtgtattttttgctgcaatagctc ctcgtctacaacctcgttactactccatctcatcctcgccaagattggcgccaagtagagttcatgttacatccgcactagtatatggtccaactcctactggtagaatccacaagggtgtgtgttctacgtggatgaagaatgcagttcctgcggagaaaagtcatga atgtagtggagccccaatctttattcgagcatctaatttcaagttaccatccaacccttcaactccaatcgttatggtgggacctg ggactgggctggcaccttttagaggttttctgcaggaaaggatggcactaaaagaagatggagaagaactaggttcatctttg ctcttctttgggtgtagaaatcgacagatggactttatatacgaggatgagctcaataattttgttgatcaaggcgtaatatctgag ctcatcatggcattctcccgtgaaggagctcagaaggagtatgttcaacataagatgatggagaaggcagcacaagtttggg atctaataaaggaagaaggatatctctatgtatgcggtgatgctaagggcatggcgagggacgtccaccgaactctacacac cattgttcaggagcaggaaggtgtgagttcgtcagaggcagaggctatagttaagaaacttcaaaccgaaggaagatacctc agagatgtctggtga (SEQ ID NO 16), actagagtctctatcttcttcggtacgcagactggaacagctgagg-gatttgctaaggcattatccgaagaaatcaaagcgagatatgaaaaagcagcagtcaaagtcattgacttggatgactatgct gccgatgatgaccagtatgaagagaaattgaagaaggaaactttggcatttttctgtgttgctacttatggagatggagagcct actgacaatgctgccagattttacaaatggtttacggaggaaatgaacgggatataaagcttcaacaactagcatatggtgtg tttgctcttggtaatcgccaatatgaacattttaataagatcgggatagttcttgatgaagagttatgtaagaaaggtgcaaagcg tcttattgaagtcggtctaggagatgatgatcagagcattgaggatgattttaatgcctggaaagaatcactatggtctgagcta gacaagctcctcaaagacgaggatgataaaagtgtggcaactccttatacagctgttattcctgaataccgggtggtgactcat gatcctcggtttacaactcaaaaatcaatggaatcaaatgtggccaatggaaatactactattgacattcatcatccctgcagag ttgatgttgctgtgcagaaggagcttcacacacatgaatctgatcggtcttgcattcatctcgagttcgacatatccaggacgggtattacatatgaaacaggtgaccatgtaggtgtatatgctgaaaatcatgttgaaatagttgaagaagctggaaaattgcttggc cactctttagatttagtattttccatacatgctgacaaggaagatggctccccattggaaagcgcagtgccgcctcctttccctg gtccatgcacacttgggactggtttggcaagatacgcagaccttttgaaccctcctcgaaagtctgcgttagttgccttggcgg cctatgccactgaaccaagtgaagccgagaaacttaagcacctgacatcacctgatggaaaggatgagtactcacaatggat tgttgcaagtcagagaagtcttttagaggtgatggctgcttttccatctgcaaaacccccactaggtgtattttttgctgcaatagc tcctcgtctacaacctcgttactactccatctcatcctcgccaagattggcgccaagtagagttcatgttacatccgcactagtat atggtccaactcctactggtagaatccacaagggtgtgtgttctacgtggatgaagaatgcagttcctgcggagaaaagtcat gaatgtagtggagccccaatctttattcgagcatctaatttcaagttaccatccaacccttcaactccaatcgttatggtgggacc tgggactgggctggcaccttttagaggttttctgcaggaaaggatggcactaaaagaagatggagaagaactaggttcatctt tgctcttctttgggtgtagaaatcgacagatggactttatatacgaggatgagctcaataattttgttgatcaaggcgtaatatctg agctcatcatggcattctcccgtgaaggagctcagaaggagtatgttcaacataagatgatggagaaggcagcacaagtttgggatctaataaaggaaggatatctcttgtatgcggtgatgctaagggcatggcgagggacgtccaccgaactctacac accattgttcaggagcaggaaggtggtcgtcgaggcagaggctatagttaagaaacttcaaccgaaggaagatac ctcagagat gtctgg (SEQ 17), ID de préquence de SEQ ID NO 17.

[0048] As used herein, 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.

[0049] Herein, the nucleic acid or polynucleotide may be produced and / or obtained by any suitable method known to those skilled in the art.

[0050] 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.

[0051] 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 Es-cherichia coli: advances and challenges.” Front. Microbiol. 2014, 5:172. doi: 10.3389 / fmicb.2014.00172

[15] , 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=lf42840e-fbd7-4734-b0cd-el7372a9e5a 4&lang=en [5]. This may be, for example, the expression vector described in WO 83 / 004261 [7].

[0052] 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, pColEl, pR-K, pAIDA1, preferably pAIDA1.

[0053] It may be, for example, a modified plasmid, for example the plasmid pAIDA1, 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 pAIDA1 in which the cmlA gene is replaced by the tetracycline resistance gene of sequence ttctcatgtttgacagcttatcatcga-taagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtca tcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtccatt ccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcac tgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccaca cccgtcctgtggatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatc gccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggc cccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctac . tactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaccgatgccttgagagccttcaacccagtcagctc cttccggtgggcgcggcatgactatcgtcgcgcacttatgactgtcttcttatcatgcaactcgtaggacaggtgcgg cagcgctctgggtcatttcggcgaccgctttcgctggagcgcgacgatcggctgtcgcttgcggtattcggaatc ttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtcggcgaagcaggccattatcgcggcatgg cggccgacgcgctcgtcttgctggcgttcgcgcgcgctggatggccttccccattatgattctctcgcttc ggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcggtagatgacgacgcgcgctcgctc gctcgcggctcttaccagcctaacttcgatcattgaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatgga acgggttggcatggattgtaggcgccctataccttgtctgcctcccgcgttcgcgcgcatgcatggagcccgc ctcgacctgaatggaagccggcggcacccgctaacggattcaccactccaagaattggagccaatcaattcttgcggaga actgtgaatgcgcaaaccaacccttggcagaacatatcgcgtccgccatctccagcgcgcacgcggcgcatctcg ggtcg (SEQ1818lcgcgcacgcgcgcatctcg ggtcg (SEQ18lc lequet IDs dances) AIDA tatacgactcactataggg-gaattgtgagcggataacaattcccctgaaatatttgtttaactttaagaaggagatataccatgggcaataaggcctacagtatcatttggagccactccagacaggcctggattgtggcctcagagttagccagaggacatggttttgtccttgcaaaaaat acactgctggtattggcggttgtttccacaatcggaaatgcatttgcagtcgaccaccatcaccatcaccatctggaagcgctg ttccagggtccgggtacccagaaacagcgtaccgagctcgaaaacctgtacttccagggtgaacagaaactgattagcgaa gaagatctgtctagagtgaataacaatggaagcattgtcattaataacagcattataaacgggaatattacgaatgatgctgact taagttttggtacagcaaagctgctctctgctacagtgaatggtagtcttgttaataacaaaaatatcattcttaatcctacaaaag aaagtgcggccgctataggtaatactcttaccgtgtcaaattatactgggacaccgggaagtgttatttctcttggtggtgtgctt gaaggagataattcacttacggaccgtctggtggtgaaaggtaatacctctggtcaaagtgacatcgtttatgtcaatgaagat ggcagtggtggtcagacgagagatggtattaatattatttctgtagagggaaattctgatgcagaattctctctgaagaaccgc gtagttgccggagcttatgattacacactgcagaaaggaaacgagagtgggacagataataagggatggtatttaaccagtc atcttcccacatctgatacccggcaatacagaccggagaacggaagttatgctaccaatatggcactggctaactcactgttc ctcatggatttgaatgagcgtaagcaattcagggccatgagtgataatacacagcctgagtctgcatccgtgtggatgaagatcactggaggaataagctctggtaagctgaatgacgggcaaaataaaaacaaccaatcagtttatcaatcagctcggggg ggatatttaataattccatcgaaactgggtgatttaccttagggattatgggaggatacgcgaatgcaaaaggtaaac gataaattacacgagcaacaaagctgccagaaacacactggatggttattctctcggggtatacggtacgtggtatcagaatg gggaaaatgcaacagggctttgctgaaacttggatgcaatataactggtttaatgcatcagtgaaaggtgacggactggaa gaagaaaaatataatctgaatggtttaaccgcttctgcaggtgggggatataacctgaatgtgcacacatggacatcacctga aggaataacaggtgaattctggttacagcctcatttgcaggctgtctggatgggttacaccggatacacatcaccagata acggaacggtggtgcagggagcagggaaaataatattcagacaaaagcaggtattcgtgcatcctggaaggtgaaaagc acctggataaggataccgggcggaggttccgtccgtatagaggcaaactggatccataacactcatgaatttggtgttaa aatgagtgatgacagccagttgttgtcaggtagccgaaatcagggagagataaagacaggtattgaaggggtgattactcaa aacttgtcagtgaatggcggagtcgcatatcaggcagggtcacgggagcaatgccatctccgagcactggggataaaa atacagcttctgataatgacagatccggctgctaaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaat aactagcataaccccttggggcctaaacgggtcttgaggggttttttg (SEQ ID NO 44) is replaced by para cloning cassette comprising the promoter sequence taatacgactcactata (SEQ ID NO 19) and the terminator sequence tagcataaccccttggggcctctaaacgggtcttgaggggttttttg (SEQ ID NO 20) of the T7 RNA polymerase gene.

[0054] 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 chosen from the group comprising pYAC-RC, pYAC3(+).

[0055] 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 chosen from the group comprising pUvBBAC, pCCIBAC, pBAC 108L.

[0056] The expression vector of a fusion protein according to the invention may comprise the following functionally linked elements: a) a promoter b) a sequence coding for a fusion protein according to the invention c) transcription termination signals.

[0057] As used herein, "promoter" means a cis-acting DNA sequence located 5' of the transcription initiation site of the sequence coding for a polypeptide 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.

[0058] 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, this may be the T7 terminator of sequence tagcataaccccttggggcctctaaacgggtctt-gaggggttttttg (SEQ ID NO 20),

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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).

[0064] 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.

[0065] 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, by virtue of 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.

[0066] 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.

[0067] The culture time can 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 can be from 24 to 72 hours, for example 53 hours.

[0068] 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 surprisingly and unexpectedly demonstrated 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.

[0069] The present invention also relates to a method for bioconversion of a substrate by a fusion protein according to the invention comprising the steps: - introduction into a culture medium comprising a host cell according to the invention, of the substrate, - incubation of said culture medium for a time sufficient for the bioconversion of said substrate by said fusion protein, and - optionally recovery of metabolites resulting from the bioconversion of said substrate.

[0070] The culture medium is as defined above.

[0071] The host cell is as defined above.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In the present, the recovery of the metabolites can be carried out by any method suitable 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.

[0079] In the present invention, the metabolites obtained may be any plant metabolite. They may, for example, be metabolites belonging to the family of polyphenols, alkaloids and / or terpenes. They may advantageously be metabolites belonging to the family of polyphenols.

[0080] The metabolites obtained can provide access to new products which can be food, cosmetic, pharmaceutical and parapharmaceutical active ingredients usable in the agri-food, cosmetic, pharmaceutical and parapharmaceutical fields. These new products can also be active or non-active products but having a neutrality and / or stability which is very interesting for use in each of these fields.

[0081] Other advantages will become apparent in the light of the following examples, given for illustrative and non-limiting purposes, with reference to the appended figures. Brief description of the figures

[0082] [Fig. 1] represents a diagram of the fusion protein successively comprising a polypeptide for addressing and anchoring to the external membrane (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.

[0083] [Fig.2] represents a schematic representation of the plasmid pAIDAl-TetR-laclQ.

[0084] [Fig.3] represents a schematic representation of the plasmid pAIDAl-T7.

[0085] [Fig.4] represents a schematic representation of the plasmid pAIDAl-T7

[0086] [Fig.5] represents a schematic representation of the plasmid p AID A1-T7-CYP73 A1 - ATR 1.

[0087] [Fig.6] represents a diagram of different elements of an example of a protein of fusion according to the invention. In the figure AIDAI-barrel-beta AIDAI- [31 and AIDAI linker respectively represent an outer membrane addressing 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.

[0088] [Fig.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 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 pAIDAl-T7-CYP73Al-ATRl and Figure 7B represents the diagram after culture of the strain E. Coli BL21 (DE3) pLysE transformed with the plasmid pAIDAl-T7 in [Fig.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.

[0089] [Fig.8] represents an alignment of peptide sequences carried out with the basic local alignment search tool (BLAST also designated in English "Basic Local Alignment Search Tool"). The sequences correspond to the

[0090] cytochromes P450 CYP76F112 and P450 CYP73A1

[0091] [Fig.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). EXAMPLES

[0092] Example 1: Process for preparing a fusion protein and biotransformation process using said protein

[0093] 1) Construction of a generic expression plasmid pAIDA-T7

[0094] The basis of the constructed expression plasmid is based on a commercial plasmid pAIDAl (https: / / www.addgene.org / 79180 /

[11] ) which is a low copy plasmid.

[0095] Replacement of the selection gene

[0096] The plasmid p AID Al 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

[11] ). For this, the plasmid p AID Al 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 LAEWXprl7-lacIQ (tggcgacaccatcgaatggtgc (SEQ ID NO: 21) and LAEWXprO2: Reverse: tttagcttcct-tagctcctg (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 method 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 pAIDA1 without the gene 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%20ManuaPIn / In-Fusion%20Snap%20Ass embly%20User%20Manual_071320.pdf

[16] . The resulting recombinant plasmid was introduced into chemocompetent Escherichia coli TOP 10 bacteria marketed by Life Technologies Corporation according to the TOP 10 transformation protocol described in https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / oneshottoplO_man.pdf

[17] ). 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 LAEWXprl7 (SEQ ID NO 21) and LAEWXprO2 (SEQ ID NO 22).

[0097] The recombinant plasmid pAIDAl-TetR-lacIQ, shown in [Fig.2], thus constructed was used for the construction of the other plasmids. To simplify the nomenclature, the plasmid is also designated pAIDAl. 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 / Instruction-NucleoSpin-Plasmid.pdf

[18] .

[0098] a) Promoter and terminator

[0099] The promoter and terminator of the AIDA cassette of the plasmid pAIDA1 were replaced by two cloning cassettes having the promoter and terminator of T7 RNA Polymerase. To carry out this step, different clonings were carried out

[0100] (i) pAIDA1 was amplified by PCR using the primers LAEWXpr30 (tcatcatcatgcctaatgagtgagaattcc (SEQ ID NO 23)) and LAEWXpr31 (ttggtgcgcaaactattaactgg (SEQ ID NO 24)). This amplification step is included by the DNA fragment contained between the pAIDA1 promoter and the pAIDA1 terminator. The amplification product therefore corresponded to the plasmid without promoter and terminator (ii) In parallel, the T7 terminator was amplified by PCR from the plasmid pET28b-2 using primers LAEWXpr32-l-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-l-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 the plasmid pAIDA1-T7 shown in [Fig.3].

[0101] b) Insertion of the AIDAI cassette

[0102] The AIDA sequence of the original plasmid pAIDAl was amplified by PCR using the primers LAEWXpr36-AIDA-fr (aactttaagaaggagatataccatgggcaataaggcctacagtatcatttgg (SEQ ID NO 29)) and LAEWXpr37-AIDA-rv (tttgttagcagccggatctgtcattatcagaagctgtattttatc (SEQ ID NO 30)) according to Protocol 1 as mentioned above.

[0103] In parallel, plasmid pAIDAl-T7 was digested with restriction enzymes NcoI and KpnI according to Protocol 4: Digestion protocol as described in http: / / assets.thermofisher.com / TFS-Assets / BID / Reference-Materials / fastdigest-restricti on-enzymes-labaid.pdf

[19] . Linearized plasmid pAIDAl-T7-AcoLXpnI and AIDAI amplicon were fused by In-Fusion according to Protocol 2 mentioned above. Generic recombinant plasmid pAIDAl-T7 complete with AIDAI was amplified by transformation of bacteria. The obtained generic plasmid pAIDAl-T7 complete with AIDAI is shown in [Fig.4].

[0104] 2) Construction of recombinant plasmid for the expression of a protein of P450-ATR fusion a. Plasmid pAIDA1-T7 was digested with restriction enzymes KpnI and Sac! according to Protocol 4 as mentioned above. b. The coding sequence for the extramembrane portion of Arabidopsis thaliana NADPH P450 reductase 1 (ATR1) as described in Urban et al, (1997) J Biol Chem 272(31):19176-86

[20] ) was amplified by PCR from a plasmid (pCR8_ATRl) using primers LAEWXpr05 Forward (agcgctgttccagggtccgggtaccactagagtctctatcttc (SEQ ID NO 31)) and LAEWXpr06 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.vl906Da.pdf

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

[20] ) was amplified by PCR from a plasmid pYeDP60-CYP73A1 using primers LAEWXprO7 Forward (gcggtagcggcggttctccgcctggcccaatcccggttcc (SEQ ID NO 35)) and LAEWXpr08 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 17, SEQ ID NO 15, SEQ ID NO 11 and the pAIDAl-T7 plasmid were digested by Kpn\ and SricI restriction enzymes according to Protocol 4 as mentioned above. The sequence of the digested plasmid obtained corresponds to the following sequence: gatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgtagttattgct cagcggtggcagcaacctcagctctttcgggctttgttagcagccggatctgtcattatcagaagctgtat tttatccccagtgctccggagatggcattgctcccgtgacctctgcctgatatgcgactccgccattcactgaca agttttgagtaatcaccccttcaatacctctctctctccctgatttcggctacctgacaacaacctggctcatc actcattttaacaccaaattcatgagtgttatggatccagtttgcctctatacggacggaacctcgcccggtat ccttatccagggtgctttcaccttccaggatgcacgaatacctgcttttgtctgaatattattttcctcctgc accaccgttccgttatcctcctgatgtgtatccggtgtaacccccatccagacagcctgcaaatgaggctgtaaccagaattcacctgttattccttcaggtgatgtccatgtgtgcacattcaggttatatcccccacctgcagaagcggt taaaccattcagattatatttttcttcttccagtccgtcacctttcactgatgcattaaaccagttatattgcatccaagt ttcagcaaagagccctgttgcattttccccattctgataccacgtaccgtataccccgacagaataaccatccagt gtgtttctggcagctttgttgctcgtgtaatttatcgttttaccttttgcattcgcgtatcctcccataatccctaaggta aaatcacccagttgttcagcatggaatttataaatatcccccccgagctgattgataaactgattggttgttgttttat tttgcccgtcattcagcttaccagagcttattcctccagtgatcttcatccacacggatgcagactcaggctgtgta ttatcactcatggccctgaattgcttacgctcattcaaatccatgaggaacagtgagttagccagtgccatattggt agcataacttccgttctccggtctgtattgccgggtatcagatgtgggaagatgactggttaaataccatcccttat tatctgtcccactctcgtttcctttctgcagtgtgtaatcataagctccggcaactacgcggttcttcagagagaatt ctgcatcagaatttccctctacagaaataatattaataccatctctcgtctgaccaccactgccatcttcattgacata aacgatgtcactttgaccagaggtattacctttcaccaccagacggtccgtaagtgaattatctccttcaagcaca ccaccaagagaaataacacttcccggtgtcccagtataatttgacacggtaagagtattacctatagcggccgcactttcttttgtaggattaagaatgatatttttgttattaacaagactaccattcactgtagcagagagcagctttgctg taccaaaacttaagtcagcatcattcgtaatattcccgtttataatgctgttattaatgacaatgcttccattgttattca ctctagacagatcttcttcgctaatcagtttctgttcaccctggaagtacaggttttcgagctccggaccctggaac agcgcttccagatggtgatggtgatggtggtcgactgcaaatgcatttccgattgtggaaacaaccgccaatac cagcagtgtattttttgcaaggacaaaaccatgtcctctggctaactctgaggccacaatccaggcctgtctgga gtggctccaaatgatactgtaggccttattgcccatggtatatctccttcttaaagttaaacaaaattatttctagagg ggaattgttatccgctcacaattcccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgc cggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccga tttggtgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcat tggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtg aagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccttaata agatgatcttcttgagatcgttttggtctgcgcgtaatctcttgctctgaaaacgaaaaaaccgccttgcagggcggtttttcgaaggttctctgagctaccaactctttgaaccgaggtaactggcttggaggagcgcagtcaccaaaac ttgtcctttcagtttagccttaaccggcgcatgacttcaagactaactcctctaaatcaattaccagtggctgctgcc agtggtgcttttgcatgtctttccgggttggactcaagacgatagttaccggataaggcgcagcggtcggactga acggggggttcgtgcatacagtccagcttggagcgaactgcctacccggaactgagtgtcaggcgtggaatg agacaaacgcggccataacagcggaatgacaccggtaaaccgaaaggcaggaacaggagagcgcacgag ggagccgccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccaccactgatttgagcgtcagat ttcgtgatgcttgtcaggggggcggagcctatggaaaaacggctttgccgcggccctctcacttccctgttaagt atcttcctggcatcttccaggaaatctccgccccgttcgtaagccatttccgctcgccgcagtcgaacgaccgag cgtagcgagtcagtgagcgaggaagcggaatatatcctgtatcacatattctgctgacgcaccggtgcagcctt ttttctcctgccacatgaagcacttcactgacaccctcatcagtgccaacatagtaagccagtatacactccgcta gcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccag aaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccac ggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccacgttgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgc ttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcgagtatccgctcatga gattatcaaaaaggatcttcacctagatccttttgtaagttctcatgtttgacagcttatcatcgataagctttaatgcg gtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctc ggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgt ccattccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcaccc gttctcggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgac tacgcgatcatggcgaccacacccgtcctgtggatcctctacgccggacgcatcgtggccggcatcaccggc gccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcggg ctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctcctt gcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagt cgcataagggagagcgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggg gcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctg ggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatctt gcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgcc ggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccatta tgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgac catcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcattggaccgctgatcgtcac ggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtct gcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcg ctaacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaaccctt ggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgtggcgaca ccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaa tgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaac caggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgc acgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgat ggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggct gatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttc ttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagc atctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtc tggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgc catgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacga tcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtg ggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctg gggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgccc gtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgatt cattaatgcagctggcacgacaggtttcccgactggaaagcgggcaagtgagtggataaccgtattaccgcctttgagtgagctgataccgggaattctcactcattaggcatgatgatga (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 plasmid constituent elements was verified by PCR using primers LAEWXpr35-upstream-T7 (tccatccagtctattaattgttgc (SEQ ID NO 37)), LAEWXpr22-ATRl-rv (ccagacatctctgaggtatcttcc (SEQ ID NO 38)), LAEWXpr24-2kbATRl-fr (ggagcaggaaggtgtgagttcgtc (SEQ ID NO 39)) and LAEWXpr25-P540-rv (accctggaagtacaggttttcg (SEQ ID NO 40)). All of the steps a to ea allowed the construction of a plasmid pAIDAl-T7-CYP73Al-ATRl represented in [Fig.5]. 3) Fusion protein

[0105] The resulting fusion protein contains from the N-terminus to the C-terminus: - a tag of 6 Histidines, allowing immunodetection of proteins with anti His antibodies - a cleavage sequence to remove the tag, - a c-MYC sequence, allowing immunodetection of proteins with anti-c-MYC antibodies - an AIDAI binding peptide - an external membrane addressing and anchoring polypeptide: the beta Barel anchoring sequence of AIDAI, namely the sequence SEQ ID NO 1 - a polypeptide sequence of cytochrome P450 CYP73A1 (cinnamate hydroxylase from Helianthus tuberosus, NCBI ID: Sequence ID: Q04468.1) in which the membrane anchor has been removed from the coding sequence, namely the sequence SEQ ID NO 3), - a binding peptide of 51 amino acids PGGGSGGGSGGGGSGGGGSGGGG-SGGGGSGGGGSGGGGSGGGGSGGGSGGSP (SEQ ID NO 6), and - a polypeptide sequence of the Arabidopsis NADPH P450 reductase (Ara-bidopsis thaliana P450 reductase 1 (ATR1), mRNA - Sequence ID: NM_118585.4) in which the membrane anchor has been removed, namely the sequence SEQ ID NO 7.

[0106] 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: - the sequence SEQ ID NO 44 coding for an AIDAI linker, - the sequence SEQ ID NO 10 coding for a polypeptide for targeting and anchoring to the external membrane: the Beta Barrel anchor sequence - the sequence SEQ ID NO 11 coding for the cytochrome P450 CYP73A1 cinnamate hydroxylase of Helianthus tuberosus, NCBI ID: Sequence ID: Q04468.1) - the sequence SEQ ID NO 15 coding for a linker peptide of 51 amino acids, and - the sequence SEQ ID NO 17 coding for Arabidopsis NADPH P450 reductase (Arabidopsis thaliana P450 reductase 1 (ATR1), mRNA - Sequence ID: NM_118585.4)

[0107] [Fig.6] is a schematic representation of the resulting fusion protein.

[0108] 4) Expression of recombinant proteins in E. coli bacteria

[0109] a) The expression system used

[0110] The plasmid pAIDAl and the 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 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.

[0111] b) Preparation of an example of fusion protein

[0112] The fusion protein was expressed from the plasmid whose construction was described previously. After transformation of the 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) comprising 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 D0 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 adding isopropyl [3-Dl-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 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).

[0113] The bacteria were then suspended in KPi buffer (88 mM with additives of 1 mM KCl, 4 rnM MgSO4, 5% v / v glycerol, 5% w / v glucose, at 4°C (products ordered from Sigma Aldrich)).

[0114] [Fig. 1] is a schematic representation of the resulting fusion protein, said fusion protein being anchored in the bacterial membrane. 5) Bioconversion

[0115] A study of the bioconversion of cinnamic acid was carried out. [Fig.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. coli 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. 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 powder obtained was suspended in 150 pL of methanol. The extract thus obtained was analyzed by ultra-high performance liquid chromatography coupled with a mass spectrum (UHPLC / MS / MS)

[0116] The results obtained are shown in [Fig.7]. Figure 7A represents a chromatogram obtained at 300 nm and showing 2 peaks. The major peak corresponds to the substrate, i.e. cinnamate. The minor 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 pAIDAl-T7-CYP73Al-ATRl 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 [Fig.4] were introduced. 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.

[0117] This example therefore clearly demonstrates that an example of a fusion protein successively comprising (i) at least one polypeptide for addressing 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.

[0118] 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.

[0119] 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. Example 2: Fusion protein and bioconversion

[0120] In this example, the cytochrome P450 is cytochrome CYP76F112 (marmesin synthase, Ficus carica cytochrome P450 CYP76F112 mRNA, completed cds Sequence ID: MW348922.1).

[0121] In this example the process for obtaining the fusion protein is identical to the method described in Example 1 except for the following: - La séquence codant pour le cytochrome P450 CYP73A1 est remplacée par la séquence codant pour le cytochrome P450 CYP76F112 de séquence caagtcac-gacggttgtcatttcctccaccaccatggctaaagaagtcctccaggcaaacagccaagtcgtctccagccggacaatcacc gacgcaagccgcgcccacagacacagcgattttagcatggttatgttgcccgtatcccctctgtggcgaaaccttcggaaaat aagcaactcacacttgctttcctccaaggctcttgatggcaacatggagctgagaaacaaaaaggtgcaagagctcctaaat gatgtccacaaaagcgtccaggccggggaggcggtggagatcgcgagcctttctttcagagctactctgaatctcttgtcca ccacatttttctccatggacatggcggatgacacaaattccgtcactctaaaagagctcaaggaggctatgtcgcacatgatg gaagagttggggaagcctaacttggccgattatttcccgtttctacaaaagattgacccccaaggcattaggcggcgcaaca cggttactttccggaaactgatcaacttgtttgggcgtatcatcgaccaaagattgaaagtgagagaagcgagtggttctttga aagatgatgatattttagacactcttatcaacatgatggtggtggatcaggagaagaaagaggatcagcttgacaaaaccata attgaacattttttactggatttattttcagcggggactgaaacgacttcaaccacgttggagtgggcaatggctgagctagtaa aagcgccagagattatgtcaaaagcccgagcagagctagatcaagttataggcaaaggaaaccaagtgaaggaatcggacgtatctcgactcccttacttacaagccattgttaaagaaaccttccgcatgcaccctacagctccattattgattcctcgcaaagc cgacagtgacatcgaaatctccgactatatcatcccgaaggatgctcag (SEQ ID NO 45). - The cinnamic acid substrate is replaced by demethylsuberosin. Bioconversion of the substrate generates marmesin as shown in [Fig.9] (CYP76F112).

[0122] 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 [Fig.8]. The result obtained demonstrates a percentage identity of 28.7%.

[0123] In this example, the bioconversion is carried out according to the method described in Example 1 above in which the substrate used is demethylsuberosin,

[0124] 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 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 the substrate bioconversion.

[0125] 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.

[0126] 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 cell bacterial, and can advantageously allow bioconversion of substrates in the culture medium of said cell.

[0127] Bibliographic references 1. Guengerich and Macdonald, 1990, FASEB J. 4, pp 2453-2459 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 3. Kenneth Jensen et al., Plant NADPH-cytochrome P450 oxidoreductases, Phyto-chemistry Volume 71, 2-3, Février 2010, Pages 132-141 4. https: / / blog.addgene.org / plasmid-101-origin-of-replication 5. https: / / www.qiagen.com / cn / resources / faq?id=lf42840e-fbd7-4734-b0cd-el7372a9e5a 4&lang=en 6. https: / / www.takarabio.com / documents / User%20Manual / R045A_e.v2102Da.pdf 7. WO 83 / 004261 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 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 10. https: / / www.addgene.org / 79180 / 11. https : / / w ww.fishersci.fr / shop / products / fermentas-pbr322-dna / 10191220 12. https: / / www.takarabio.com / products / cloning / in-fusion-cloning 13. https : / / w ww.fishersci.fr / shop / products / fermentas-pbr322-dna / 10191220 14. Jarmander, J., Gustavsson, M., Do, TH. et al. A dual tag System for facilitated détection of surface expressed proteins in Escherichia coli. Microb Cell Fact 11, 118 (2012). https: / / doi.org / 10.1186 / 1475-2859-ll-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 16. https: / / www.takarabio.com / documents / User%20ManuaPIn / In-Fusion%20Snap%20Ass embly%20User%20Manual_071320.pdf 17. https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / oneshottoplO_man.pdf 18. https: / / www.mn-net.com / media / pdf / 45 / 51 / 02 / Instruction-NucleoSpin-Plasmid.pdf 19. http: / / assets.thermofisher.com / TFS-Assets / BID / Reference-Materials / fastdigest-restricti on-enzymes-labaid.pdf 20. Urban et al, (1997) J Biol Chem 272(31):19176-86 21. https: / / www.takara.co.kr / file / manual / pdf / R050A_e.vl906Da.pdf.

Claims

1.

2.

3.

4. Claims Fusion protein successively comprising (i) at least one polypeptide for targeting and anchoring to the bacterial membrane having a sequence with a percentage identity of at least 90% with the polypeptide of sequence SEQ ID NO 1, (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. The fusion protein of claim 1, wherein the quaternary structure of said at least one membrane-targeting and anchoring polypeptide forms a beta barrel. The fusion protein of claim 1 or 2, wherein the targeting and anchoring polypeptide is the polypeptide of sequence MN-KAYSIIWSHSRQAWIVASELARGHGFVLAKNTLLVLAVVSTIGN AFAVDHHHHHHLEALFQGPGTQKQRTELENLYFQGEQKLISEED LSRVNNNGSIVINNSIINGNITNDADLSFGTAKLLSATVNGSLVN NKNIILNPTKESAAAIGNTLTVSNYTGTPGSVISLGGVLEGDNSLT DRLVVKGNTSGQSDIVYVNEDGSGGQTRDGINIISVEGNSDAEFS LKNRVVAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYR PENGSYATNMALANSLFLMDLNERKQFRAMSDNTQPESASVW MKITGGISSGKLNDGQNKTTTNQFINQLGGDIYKFHAEQLGDFT LGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNG ENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGG GYNLNVHTWTSPEGITGEFWLQPHLQAVWMGVTPDTHQEDNG TVVQGAGKNNIQTKAGIRASWKVKSTLDKDTGRRFRPYIEANWI HNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSVNGGV AYQAGGHGSNAISGALGIKYSF (SEQ ID NO 1). The fusion protein of any one of claims 1 to 3, wherein said polypeptide of 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 IPVPIFGNWL-QVGDDLNHRNLTDLAKRFGEILLLRMGQRNLVVVSSPELAKEV LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIM TVPFFTNKVVQQYRYGWEAEAAAVVDDVKKNPAAATEGIVIRR RLQLMMYNNMFRIMFDRRFESEDDPLFLKLKALNGERSRLAQSF EYNYGDFIPILRPFLRNYLKLCKEVKDKRIQLFKDYFVDERKKIG STKKMDNNQLKCAIDHILEAKEKGEINEDNVLYIVENINVAAIET TLWSIEWGIAELVNHPEIQAKLRHELDTKLGPGVQITEPDVQNLP YLQAVVKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVN AWWLANNPDQWKKPEEFRPERFLEEEAKVEANGNDFRYLPFGV GRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKIDTDEKGGQFS LHILKHSTIVAKPRSF (SEQ ID NO 3).

5. A fusion protein according to any one of claims 1 to 4, wherein said binding polypeptide is a polypeptide of sequence PGGSGGGSGGGGSGGGGGGGGGGGGGGGGGGGGGGGGGGGGG SGGGSGGSP (SEQ ID NO 6).

6. The fusion protein of any one of claims 1 to 5, wherein said polypeptide of a hydrophilic domain of a NADPH P450 reductase is a polypeptide having a sequence identity of at least 90% with the polypeptide of sequence TRVSIFFGTQTGTAEGFAKAL-SEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVA TYGDGEPTDNAARFYKWFTEENERDIKLQQLAYGVFALGNRQY EHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKES LWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSME SNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRT GITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKED GSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYAT EPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPP LGVFFAAIAPRLQPRYYSISSSPRLAPSRVHVTSALVYGPTPTGRI HKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVM VGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFI YEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMECAAQV WDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAE AIVKKLQTEGRYLRDVW (SEQ ID NO 7).

7. A fusion protein according to any one of claims 1 to 6, wherein said polypeptide comprising the hydrophilic domain of a plant cytochrome P450 or said polypeptide comprising the hydrophilic domain of a NADPH P450 reductase is free of a transmembrane domain.

8. Nucleic acid encoding a fusion protein according to any one of claims 1 to 7.

9. Vector, preferably for expression, comprising a nucleic acid according to claim 8.

10. A host cell comprising a nucleic acid according to claim 8 and / or a vector according to claim 9.

11. A host cell according to claim 10, said host cell being a bacterial cell, preferably Escherichia coli.

12. A method of producing a fusion protein comprising culturing a host cell according to claim 10 or 11 under conditions suitable for expression of the fusion protein

13. A method of bioconversion of a substrate by a fusion protein according to any one of claims 1 to 7 comprising the steps of: - introducing into a culture medium comprising a host cell according to claim 10 or 11, said substrate, - incubating said culture medium for a time sufficient for the bioconversion of said substrate by said fusion protein, and - optionally recovering the metabolites resulting from the bioconversion of said substrate.

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