Use of type iii polyketide synthases of cyanobacteria as phloroglucinol synthases

Abstract

The present invention relates to the use of a polypeptide selected from type III polyketide synthases of cyanobacteria, a nucleic acid molecule coding for same, a vector comprising a nucleic acid molecule coding for same or a host cell expressing same, to produce phloroglucinol. The invention also provides particular nucleic acid molecules coding for a type III polyketide synthase of cyanobacteria, vectors and host cells, as well as methods of producing phloroglucinol.

Description

[0001] DESCRIPTION

[0002] TITLE: Use of cyanobacterial type III polyketide synthases as phloroglucinol synthases

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the fields of microbial biochemistry and more particularly to the field of synthesis of phloroglucinol by microbial enzymes. It relates to the use of cyanobacterial type III polyketide synthases as phloroglucinol synthases, associated methods for producing phloroglucinol, as well as nucleic acids, vectors and host cells for producing phloroglucinol.

[0005] STATE OF THE ART

[0006] Phloroglucinol is an aromatic organic compound used in particular in the manufacture of pharmaceutical products and explosives.

[0007] Phloroglucinol synthesis is catalyzed by certain type III polyketide synthases called phloroglucinol synthases. Phloroglucinol synthases carry out the condensation of three malonyl-CoA molecules to form one phloroglucinol molecule according to the following reaction scheme (Reaction I): Reaction I

[0008] Many oligomers can then be synthesized from phloroglucinol, such as phlorotannins. Phlorotannins include fucols, phloretols, and fucophloretols, which are derivatives of phloroglucinol, a component of the cell wall of brown algae. In addition, various protective activities of brown algae have also been attributed to phlorotannins.

[0009] Natural phloroglucinol synthesis was initially described in the Gram-negative bacteria Pseudomonas fluorescens (Àchkar et al., 2005; Zha et al., 2006) and in the brown alga Ectocarpus siliculosus (Meslet-Cladière et al., 2013). The enzyme phloroglucinol synthase involved in phloroglucinol synthesis was identified in both species.

[0010] In Pseudomonas fluorescens, phloroglucinol synthase is encoded by the PHLD gene (Àchkar et al., 2005; Zha et al., 2006). The phloroglucinol synthase activity of PHLD has been demonstrated in Escherichia coli expressing a heterologous PHLD gene (Àchkar et al., 2005). This activity was confirmed in vitro by small-scale enzymatic assays performed with recombinant PHLD expressed and purified from Escherichia coli cultures (Zha et al., 2006).

[0011] In Ectocarpus siliculosus, phloroglucinol synthase is encoded by the PKS1 gene (Meslet-Cladière et al., 2013). The phloroglucinol synthase activity of PKS1 has been demonstrated in vitro, from recombinant PKS1 expressed and purified in Escherichia coli and from cell extracts of E. siliculosus (Meslet-Cladière et al., 2013, WO 2013 / 045510).

[0012] However, PHLD and PKS1 enzymes exhibit low enzymatic activities. Furthermore, it has been shown that PHLD enzymes from Pseudomonas fluorescens and PKS1 from Ectocarpus siliculosus produce little or no phloroglucinol when these sequences are expressed in yeast instead of in Escherichia coli (WO2019 / 002799; WO2019 / 002798). However, eukaryotic systems can be advantageous, particularly for large-scale production. They allow the production of enzymes that can be modified at the post-translational level.

[0013] WO2019 / 002799 and WO2019 / 002798 describe phloroglucinol synthases capable of synthesizing phloroglucinol at a much higher level than PKS1 from Ectocarpus siliculosus when sequences coding for these enzymes are transfected into yeast. These phloroglucinol synthases are derived from actinomycete bacteria (WO2019 / 002799) or eukaryotic algae (WO2019 / 002798).

[0014] However, there is a need for other phloroglucinol synthases capable of synthesizing phloroglucinol at high levels, especially when sequences coding for these enzymes are transfected into a host cell, particularly in yeast.

[0015] However, type III polyketide synthases are a large class of enzymes comprising proteins with sequence similarities but exhibiting very dissimilar enzymatic activities (Meslet-Cladière et al., 2013). Thus, phloroglucinol synthase activity is only one of the multiple activities likely to be present in a type III polyketide synthase and only a small proportion of type III polyketide synthases therefore possess phloroglucinol synthase activity. Furthermore, no sequence motifs associated with phloroglucinol synthase activity have been described, making the identification of type III polyketide synthases possessing such activity particularly difficult.

[0016] STATEMENT OF THE INVENTION

[0017] Surprisingly, because genes coding for phloroglucinol synthases had until now only been identified in Pseudomonas fluorescens and in Actinomycetes bacteria and algae, the Inventors were nevertheless able to identify new phloroglucinol synthases from cyanobacteria (phylum Cyanobacteriota, different from the phylum Actinomycetota, to which Actinomycetes bacteria belong), capable of synthesizing phloroglucinol at an interesting level when sequences coding for these enzymes are transfected into a host cell, in particular in a yeast or in a bacterium.

[0018] Thus, according to a first aspect, the invention relates to the use of a polypeptide chosen from cyanobacterium type III polyketide synthases, of a nucleic acid molecule encoding it, of a vector comprising a nucleic acid molecule encoding it or of a host cell expressing it, for producing phloroglucinol.

[0019] According to a second aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide as defined in the use according to the invention, characterized in that: a) the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence; or b) the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence; or c) the nucleic acid sequence is further optimized for expression in a host cell, in particular in yeast or bacteria; or d) any combination of a) to c).

[0020] According to a third aspect, the invention relates to a vector comprising a nucleic acid molecule according to the invention, said vector preferably being a plasmid.

[0021] According to a fourth aspect, the invention relates to a host cell comprising a nucleic acid molecule according to the invention, or a vector according to the invention.

[0022] According to a fifth aspect, the invention relates to a method for producing phloroglucinol, comprising the steps of:

[0023] (i) bringing into contact a host cell expressing the polypeptide chosen from the type III polyketide synthases of cyanobacteria as defined in the use according to the invention, for example a host cell according to the invention, with a suitable substrate;

[0024] (ii) in vitro culture of the host cell of step (i) under conditions allowing the growth of said host cell and / or the expression of the nucleic acid molecule contained in said host cell, so as to produce phloroglucinol; (iii) optionally recovering the culture medium comprising phloroglucinol, obtained after step (ii); and

[0025] (iv) optionally, the purification of phloroglucinol from the culture medium of step (iii).

[0026] According to a sixth aspect, the invention relates to a method for producing phloroglucinol, comprising the steps of:

[0027] (i) bringing into contact a polypeptide chosen from type III polyketide synthases of cyanobacteria as defined in the use according to the invention with malonyl-CoA;

[0028] (ii) incubating the mixture from step (i) under conditions suitable for producing phloroglucinol;

[0029] (iii) optionally the recovery of the reaction medium comprising phloroglucinol, obtained after step (ii); and

[0030] (iv) optionally, the purification of phloroglucinol from the reaction medium of step (iii).

[0031] DESCRIPTION OF FIGURES

[0032] [Fig. 1] Figure 1 represents the alignment of the amino acid sequences SEQ ID NO: 1 (PhlD of Rivularia sp. PCC 7116 PlhD-Rs), SEQ ID NO: 2 (PhlD of Chamaesiphon minutus denoted PlhD-Cm), SEQ ID NO: 3 (PhlD of Filamentous cyanobacterium CCP2 denoted PlhD- Fc), SEQ ID NO: 4 (PhlD of Calothrix sp. HK-06 denoted PlhD-Cs), and SEQ ID NO: 5 (PhlD of Gloeocapsa sp. PCC 7428 denoted PlhD-Gs). Preserved fragments (a) to (g) are indicated by bold boxes.

[0033] [Fig. 2] Figure 2 represents the map of the PBIM5 plasmid used in the examples.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Definitions

[0036] By "a" or "an" is meant one or more. In other words, when "a" or "an" is used with respect to a feature, it covers both embodiments with the feature of interest occurring only once and those with multiple occurrences of the feature of interest. In other words, unless otherwise indicated (such as "a single" or "a single" or "only one"), "a" is used as a synonym for "one or more" or "at least one". In this document, when used to define products, compositions and methods, the terms "comprising" (and any form of "comprising", such as "comprises"), "having" (and any form of "having", such as "has"), "including" (and any form of inclusion, such as "includes") or "containing" (and any form of "containing", such as "contains") are open and do not exclude additional, unmentioned elements or method steps.Thus, a polypeptide "comprises" an amino acid sequence when the amino acid sequence is part of the final amino acid sequence of the polypeptide. Such a polypeptide may have up to several hundred additional amino acid residues. By "consisting essentially of" or "consisting essentially of" is meant the exclusion of other components or steps of any essential importance. Thus, a polypeptide "consists essentially of" an amino acid sequence when such an amino acid sequence is present with possibly only a few additional amino acid residues (e.g., a peptide of at most 20 amino acids, such as a 6-histidine Hisxô tag, may additionally be present). "Consisting of" or "consisting of" means excluding more than trace amounts of other components or steps.For example, a polypeptide "consists of" an amino acid sequence when the polypeptide does not contain any amino acids other than the stated amino acid sequence.

[0037] Type III polyketide synthase means a multifunctional enzyme or enzyme complex that produces polyketides and does not utilize an acyl carrier protein (ACP) domain.

[0038] Polyketide refers to a large family of secondary metabolites in bacteria, fungi, plants, and some animal strains that arise from the iterative condensation of acetyl or malonyl subunits by polyketide synthase enzymes. Polyketides also serve as raw materials for the manufacture of a wide range of natural and semi-synthetic products.

[0039] The term "phloroglucinol" means an aromatic organic compound benzene-1,3,5-triol having the following chemical formula (Formula I: Formula I Phloroglucinol synthase is a multifunctional enzyme or enzyme complex belonging to the type III polyketide synthase family and catalyzing the synthesis of phloroglucinol. A phloroglucinol synthase catalyzes the condensation of three malonyl-CoA molecules to form one phloroglucinol molecule.

[0040] By "enzymatic activity" or "catalytic activity" or even "activity" of an enzyme, we mean the efficiency of an enzyme in converting a substrate into a product in a given environment. The efficiency of the enzyme takes into account here the rate of conversion of the substrate into a product by the enzyme and the rate of conversion of the substrate into a product by the enzyme. By "rate of conversion of the substrate into a product by the enzyme" we mean here the ratio between the quantity of final product obtained compared to the initial quantity of substrate for a defined quantity of enzyme. For example, an enzymatic activity within the meaning of the invention can be expressed as the quantity of phloroglucinol produced in a given volume (in g / L).

[0041] By "bacteria" we mean a microscopic and prokaryotic organism (without an intracellular nucleus) present in all environments.

[0042] By "cyanobacterium" is meant a unicellular organism of the kingdom of bacteria, and of the phylum of cyanobacteria or "Cyanobacteriota", a phylum comprising photosynthetic bacteria, i.e. bacteria which use light energy as a source of energy. This phylum mainly comprises the class Cyanophyceae, which itself includes in particular the orders Nostocales, Synechococcales, and Chroococcales. The order Nostocales includes in particular the family Rivulariaceae, which itself includes in particular the genera Rivularia (to which many species belong, including Rivularia sp. PCC7116 from which PHID-Rs of sequence SEQ ID NO: 1 is derived) and Calothrix (to which many species belong, including Calothrix sp. HK-06 from which PHID-Cs of sequence SEQ ID NO: 4 is derived).The order Synechococcales includes in particular the family Chamaesiphonaceae, which itself includes the genus Chamaesiphon (to which many species belong, including Chamaesiphon minutus from which PHID-Rs with sequence SEQ ID NO: 2 is derived). The order Chroococcales includes in particular the family Chroococcaceae, which itself includes the genus Gloeocapsa (to which many species belong, including Gloeocapsa sp. PCC 7428 from which PHID-Rs with sequence SEQ ID NO: 5 is derived). The species Filamentous cyanobacterium CCP2 (from which PHID-Fc with sequence SEQ ID NO: 3 is derived) has not yet been assigned to a particular genus, family or order of cyanobacteria. By "PHLD.Pf" we mean indifferently the gene coding for the phloroglucinol synthase PH LD of Pseudomonas fluorescens, or the polypeptide encoded by this gene.

[0043] By "PKS1.Es" or "PHLD.Es" is meant indifferently the gene coding for the phloroglucinol synthase PKS1 of Ectocarpus siliculosus, or the polypeptide encoded by this gene.

[0044] “PhlD” or “PHLD” herein refers to a candidate gene encoding a candidate phloroglucinol synthase enzyme, or the polypeptide encoded by this gene. According to the nomenclature chosen by the Inventors, “PhlD.ii” or “PHLD.ii” herein refers to the candidate gene or the candidate polypeptide from a given organism. The letters “ii” represent the initials of the genus and species to which said organism belongs. For example, PhlD.Hm corresponds to a candidate phloroglucinol synthase enzyme from a species Hirsutella minnesotensis, and PhlD.At corresponds to a candidate phloroglucinol synthase enzyme from a species Aspergillus tanneri. A number may follow the initials when several candidate phloroglucinol synthase enzymes have been identified in the same species.

[0045] The term "nonpolar amino acids" or "nonpolar amino acids" refers to a family of amino acids whose average positions of positive and negative partial charges are the same. Among the 20 conventional amino acids, the nonpolar amino acids are: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M), and cysteine ​​(C).Those other than glycine (G) can be subdivided into three subfamilies: 1) "aliphatic nonpolar amino acids," which have an aliphatic-type side chain and include alanine (A), valine (V), leucine (L), isoleucine (I), and proline (P), 2) "aromatic nonpolar amino acids," which have an aromatic-type side chain and include tryptophan (W), phenylalanine (F), and tyrosine (Y), and 3) "sulfur-containing nonpolar amino acids" whose side chain includes a sulfur atom and which include methionine (M) and cysteine ​​(C).

[0046] Polar amino acids are defined as a family of amino acids whose average positions of positive and negative partial charges are not confused. Among the 20 conventional amino acids, polar amino acids are: serine (S), threonine (T), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), histidine (H). They can be subdivided into three subfamilies: 1) "neutral (or uncharged) polar amino acids," which have an uncharged side chain and include serine (S), threonine (T), asparagine (N), and glutamine (Q), 2) "negatively charged polar amino acids," which have a negatively charged side chain and include glutamic acid (E) and aspartic acid (D), and 3) "positively charged polar amino acids," which have a positively charged side chain and include lysine (K), arginine (R), and histidine (H).

[0047] “Nucleic acid molecule” means a polymer of any length of deoxyribonucleic acid (DNA), or polydeoxyribonucleotides, including, but not limited to, complementary DNA or cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers, and any mixture thereof; or a polymer of any length of ribonucleic acid (RNA), or polyribonucleotides, including, but not limited to, messenger RNA or mRNA, antisense RNA; or mixed polyribo-polydeoxyribonucleotides. They include single- or double-stranded, linear or circular, natural or synthetic polynucleotides. In addition, a polynucleotide may include non-naturally occurring nucleotides and may be interrupted by non-nucleotide components.

[0048] As used herein, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" and "nucleotide sequence" are used interchangeably.

[0049] An "isolated molecule" means a molecule, including a protein, polypeptide, peptide, nucleic acid molecule, plasmid vector, viral vector, or host cell, that is removed from its natural environment (i.e., separated from at least one other component with which it is naturally associated).

[0050] By “polypeptide”, “protein” and “peptide” are meant polymers of amino acid residues that comprise at least nine amino acids linked by peptide bonds. The polymer may be linear, branched or cyclic. The polymer may comprise naturally occurring amino acids and / or amino acid analogues and may be interrupted by non-amino acid residues. As a general indication and without being bound thereto in the present application, if the polymer of amino acids contains more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein, whereas if the polymer consists of 50 amino acids or less, it is preferably referred to as a “peptide”.

[0051] By "identity" is meant an exact sequence match between two polypeptides or two amino acid molecules. The "identity percentages" referred to in the disclosure of the present invention are determined on the basis of a global alignment of the sequences (nucleic or protein) to be compared, that is to say on an alignment of the sequences taken in their entirety over their entire length using any algorithm well known to those skilled in the art such as the Needleman and Wunsch-1970 algorithm. This sequence comparison can be carried out using any software well known to those skilled in the art, for example the needle software using the "Gap open" parameter equal to 10.0, the "Gap extend" parameter equal to 0.5 and a "Blosum 62" matrix. The needle software is for example available on the website ebi.ac.uk worldwide under the name "Align".

[0052] By "vector" is meant a vehicle, preferably a nucleic acid molecule or a viral particle, which contains the elements necessary to enable the delivery, propagation and / or expression of one or more nucleic acid molecules in a host cell or organism.

[0053] From a functional point of view, this term encompasses vectors for maintenance (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids), or integrating vectors (e.g., designed to integrate into the genome of a host cell and produce additional copies of the nucleic acid molecule it contains when the host cell replicates). This term also encompasses shuttle vectors (e.g., functioning in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., for the transfer of nucleic acid molecule(s) into the genome of a host cell).

[0054] From a structural point of view, the vectors according to the invention may be natural, synthetic or artificial genetic sources, or a combination of natural and artificial genetic elements.

[0055] Thus, in the context of the invention, the term "vector" should be understood broadly to include plasmid (or plasmid) and viral vectors.

[0056] A "plasmid" as used herein means a replicable DNA construct. Typically, plasmid vectors contain selection marker genes that allow host cells carrying the plasmid to be identified and / or selected positively or negatively in the presence of the compound corresponding to the selection marker. A variety of positive and negative selection marker genes are known in the art. For illustration, an antibiotic resistance gene can be used as a positive selection marker gene to select a host cell in the presence of the corresponding antibiotic.

[0057] The term "viral vector" as used herein refers to a nucleic acid vector that comprises at least one element of a virus genome and may be packaged into a viral particle or virus-like particle. Viral vectors may be replication competent or selective (e.g., designed to replicate better or selectively in specific host cells), or may be genetically disabled so as to be defective or deficient in replication.

[0058] A "host cell" means a cell containing a heterologous nucleic acid molecule. "Heterologous" or "exogenous" means that the nucleic acid molecule originates from a species different from the host cell's species. Thus, a host cell is not a naturally occurring cell but is a molecular biology tool obtained through genetic manipulation techniques.

[0059] The host cell may consist of a single type of cell or a group of different types of cells (in which case we have a mixture of host cells of distinct types). The host cell may also be a hybrid cell, i.e. resulting from the fusion of at least two cells of different types. The host cell may belong to cultured cell lines, primary cells, stem cells or proliferative cells. In the context of the invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. "Prokaryote" means a unicellular microorganism whose cellular structure does not include a nucleus. Prokaryotes include the kingdoms of bacteria and archaea. "Eukaryote", as opposed to prokaryotes, means any unicellular or multicellular organism whose cells have a structured nucleus.Eukaryotic cells include yeast, insect, plant, and animal cells (including non-human mammalian cells). The host cell may, for example, be isolated or organized into a tissue, an organ, or within a complete organism. If the host cell is within a complete organism, the organism is not human.

[0060] In the following detailed description, the embodiments may be taken alone or combined as appropriate by those skilled in the art.

[0061] Use of isolated polypeptides, nucleic acid molecules, vectors, host cells for the production of phloroglucinol

[0062] Here we are interested in the use of isolated polypeptides selected from type III polyketide synthases of cyanobacteria.

[0063] Indeed, quite surprisingly, the Inventors identified genes encoding novel type III polyketide synthases in the genome of cyanobacteria. Moreover, type III polyketide synthases are a broad class of enzymes that bring together proteins with sequence similarities while exhibiting very dissimilar enzymatic activities (Meslet-Cladière et al., 2013). Thus, phloroglucinol synthase activity is only one of the multiple activities that may be present in a type III polyketide synthase. Furthermore, no sequence motif associated with phloroglucinol synthase activity has been described, making the identification of type III polyketide synthases possessing such activity particularly difficult. However, the inventors have further demonstrated that the new type III polyketide synthases identified in cyanobacteria have phloroglucinol synthase activity.

[0064] In a first aspect, the invention therefore relates to the use of a polypeptide selected from cyanobacteria type III polyketide synthases, an isolated nucleic acid molecule encoding it, a vector comprising a nucleic acid molecule encoding it or a non-human host cell expressing it, for producing phloroglucinol.

[0065] Polypeptide used

[0066] The polypeptide used in the invention is chosen from type III polyketide synthases of cyanobacteria, and advantageously has phloroglucinol synthase activity.

[0067] It may in particular be chosen from type III polyketide synthases of cyanobacteria of the families Rivulariaceae, Chamaesiphonaceae and Chroococcaceae, advantageously of the genera Rivularia, Caltothrix, Chamaesiphon and Gloeocapsa, more advantageously of the species Rivularia sp. PCC 7116, Calothrix sp. HK-06, Chamaesiphon minutus, Gloeocapsa sp. PCC 7428, and Gloeocapsa sp. PCC 73106. Alternatively, it may be chosen from type III polyketide synthases of cyanobacteria of the species Filamentous cyanobacterium CCP2.

[0068] Advantageously, the polypeptide used in the invention corresponds to those identified here as having phloroglucinol synthase activity or to derivatives thereof. In particular, the polypeptide used in the invention comprises, is essentially composed of or is composed of an amino acid sequence having at least 70% identity, at least 75% identity, advantageously at least 80% identity, at least 85% identity, more advantageously at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, even more advantageously at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, or even 100% identity with a sequence chosen from SEQ ID NO: 1 (PhlD of Rivularia sp.PCC 7116 PlhD-Rs), SEQ ID NO:2 (PhlD of Chamaesiphon minutus denoted PlhD-Cm), SEQ ID NO:3 (PhlD of Filamentous cyanobacterium CCP2 denoted PlhD-Fc), SEQ ID NO:4 (PhlD of Calothrix sp. HK-06 denoted PlhD-Cs), and SEQ ID NO: 5 (PhlD of Gloeocapsa sp. PCC 7428 denoted PlhD-Gs).

[0069] The sequences SEQ ID NO: 1 to 5 are presented in Table 1 below:

[0070] [Table 1]

[0071] Furthermore, the inventors have identified conserved motifs between the five sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 (see the alignment of Figure 1), which are therefore advantageously present in the sequence of the polypeptide used in the invention.

[0072] Thus, in a general advantageous embodiment, the polypeptide used in the invention further comprises (in addition to a minimum percentage of identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 as described above) at least one fragment chosen from:

[0073] (a) ATGTP (SEQ ID NO:6);

[0074] (b) STGFXiAPG, wherein Xi is selected from apolar amino acids (i.e. glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M) and cysteine ​​(C), SEQ ID NO:7);

[0075] (c) VX2FMGCAAA, wherein X2 is selected from polar amino acids (i.e. serine (S), threonine (T), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H), SEQ ID NO:8);

[0076] (d) VCLELSS (SEQ ID NO:9);

[0077] (e) IHSIFX3DGCAA, wherein X3 is any amino acid (i.e. X3 is selected from glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M), cysteine ​​(C), serine (S), threonine (T), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H), SEQ ID NO: 10); and (f) IDLWX HPGGTRI, wherein X4 is selected from apolar amino acids (i.e. glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M) and cysteine ​​(C), SEQ ID NO:11).

[0078] Advantageously, in an intermediate embodiment, the polypeptide used in the invention further comprises (in addition to a minimum percentage of identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as described above) at least one fragment chosen from:

[0079] (a) ATGTP (SEQ ID NO:6);

[0080] (b) STGFX1APG, wherein X1 is selected from aliphatic apolar amino acids (i.e. alanine (A), valine (V), leucine (L), isoleucine (I), and proline (P), SEQ ID NO: 12);

[0081] (c) VX2FMGCAAA, wherein X2 is selected from neutral polar or positively charged polar amino acids (i.e. serine (S), threonine (T), asparagine (N), glutamine (Q), lysine (K), arginine (R), and histidine (H), SEQ ID NO: 13);

[0082] (d) VCLELSS (SEQ ID NO:9);

[0083] (e) IHSIFX3DGCAA, wherein X3 is selected from neutral apolar or polar amino acids (i.e. glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M), cysteine ​​(C), serine (S), threonine (T), asparagine (N), and glutamine (Q), SEQ ID NO: 14); and

[0084] (f) IDLWX4VHPGGTRI, wherein X4 is selected from aliphatic apolar amino acids (i.e. alanine (A), valine (V), leucine (L), isoleucine (I), and proline (P), SEQ ID NO:15).

[0085] Even more advantageously, in a limited embodiment, the polypeptide used in the invention further comprises (in addition to a minimum percentage of identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as described above) at least one fragment chosen from:

[0086] (a) ATGTP (SEQ ID NO:6);

[0087] (b) STGFX1APG, wherein X1 is selected from leucine (L), valine (V), and isoleucine (I) (SEQ ID NO: 16), (c) VX2FMGCAAÀ, wherein X2 is selected from asparagine (N) and histidine (H) (SEQ ID NO: 17);

[0088] (d) VCLELSS (SEQ ID NO:9);

[0089] (e) IHSIFX3DGCAÀ, wherein X3 is selected from glycine (G) and serine (S) (SEQ ID NO: 18); and

[0090] (f) IDLWX4VHPGGTRI, wherein X4 is selected from alanine (A) and valine (V) (SEQ ID NO:19).

[0091] Advantageously, the polypeptide used in the invention further comprises (in addition to a minimum percentage of identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as described above) several of the fragments (a) to (g) as described above (general, intermediate or limited embodiment). In particular, the polypeptide used in the invention may further comprise (in addition to a minimum percentage of identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as described above (general, intermediate or limited embodiment)):

[0092] - fragments (b), (c), and (e),

[0093] - fragments (d) and (f),

[0094] - fragments (b) to (f, or

[0095] - all fragments (a) to (f).

[0096] Particularly advantageously, the polypeptide used in the invention comprises, is essentially constituted, or is constituted of an amino acid sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0097] Isolated nucleic acid molecule used

[0098] The isolated nucleic acid molecule used in the invention may be any nucleic acid molecule comprising a nucleic acid sequence encoding any polypeptide selected from the type III polyketide synthases of cyanobacteria as defined above. According to one embodiment, the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence encoding the polypeptide selected from the type III polyketide synthases of cyanobacteria as defined above.Advantageously, the promoter is an exogenous promoter, in particular a yeast promoter, preferably a promoter chosen from ÀDH2 (pÀDH2), this promoter allows expression in particular when the culture medium contains ethanol as a carbon source), CCW12 (pCCW12, this promoter allows expression in particular when the culture medium contains glucose as a carbon source), and TEF1 (pTEF1, this promoter allows expression in particular when the culture medium contains glucose or sucrose as a carbon source), more preferably a promoter chosen from ÀDH2 (pÀDH2) from Saccharomyces cerevisiae and CCW12 (pCCW12) from S. cerevisiae, and TEF1 (pTEF1) from S. cerevisiae, more preferably a promoter chosen from ÀDH2 of sequence SEQ ID NO: 20, CCW12 sequence of SEQ ID NO: 21 and TEF1 of sequence SEQ ID NO: 22.

[0099] According to an alternative embodiment or one that can be combined with the previous one, the isolated nucleic acid molecule further comprises a transcription terminator of the nucleic acid sequence coding for the polypeptide chosen from the type III polykeetide synthases of cyanobacteria as defined above. Advantageously, the terminator is an exogenous terminator, in particular a yeast terminator, preferably the RPL3 terminator (tRPL3) or the ΔDH1 terminator, more preferably the RPL3 terminator of S. cerevisiae or the ΔDH1 terminator of S. cerevisiae, more preferably the RPL3 terminator of sequence SEQ ID NO: 23 or the ΔDH1 terminator of sequence SEQ ID NO: 24.

[0100] According to a preferred embodiment, the isolated nucleic acid molecule further comprises both a promoter and a terminator which are as defined above.

[0101] According to an alternative embodiment or one that can be combined with each of the embodiments relating to the isolated nucleic acid molecule used in the invention, the nucleic acid molecule further comprises an export sequence. Advantageously, this export sequence allows the secretion or excretion of the polypeptide(s) encoded by the nucleic acid molecule into the cellular environment.

[0102] According to a preferred embodiment, the isolated nucleic acid molecule further comprises both a promoter, a terminator and an export sequence as defined above. The nucleic acid molecule may be isolated from homologous strains in culture, preferably selected from the families Rivulariaceae, Chamaesiphonaceae and Chroococcaceae, advantageously from the genera Rivularia, Caltothrix, Chamaesiphon and Gloeocapsa, more advantageously from the species Rivularia sp. PCC 7116, Calothrix sp. HK-06, Chamaesiphon minutus, Gloeocapsa sp. PCC 7428, and Gloeocapsa sp. PCC 73106, or from the species Filamentous cyanobacterium CCP2. Alternatively, the nucleic acid molecule may be isolated from a heterologous vector or host cell comprising said molecule, said vector or host cell being as defined above and described below in the sections "Host Cells" or "Vectors".Alternatively, the isolated nucleic acid molecule may be synthesized in vitro by nucleic synthesis techniques known to those skilled in the art.

[0103] According to an alternative embodiment or one that can be combined with each of the embodiments relating to the isolated nucleic acid molecule used in the invention, the nucleic acid sequence included in the isolated nucleic acid molecule used in the invention is further optimized for expression in a host cell, in particular in a yeast or a bacterium.

[0104] To optimize expression in a host cell, in particular in yeast or bacteria, the person skilled in the art knows how to use the usage bias of the genetic code, which designates the preferential use for a given organism of one of the possible triplets of nucleotides or codons to code for the same amino acid. Indeed, there are generally several combinations of three nucleotides (called "codons") coding for the same amino acid (except for methionine and tryptophan), called synonymous codons, but some of these combinations are generally used preferentially by a given organism. For the production of an amino acid sequence of interest, optimal expression can thus be obtained when the codons chosen to code for the amino acid sequence are those used preferentially by the organism of origin of the host cell.Depending on the production organism chosen (in particular yeast or bacteria), different optimal nucleic acid sequences will therefore be used when the nucleic acid sequence included in the isolated nucleic acid molecule used in the invention is further optimized for expression in the production organism chosen.

[0105] Various software programs are available to those skilled in the art for optimizing codons for expression in a host cell, particularly in yeast or bacteria. Examples of such software programs include the Twist Codon Optimization tool software (provided by Twist Biosciences), the GenSmart™ Codon Optimization software (provided by GenScript and the functionalities of which are described in application WO2020024917A1), the IDT Codon Optimization Tool software (provided by Integrated DNA technologies), and Azenta's codon optimization tool software (provided by Azenta).

[0106] Examples of optimized nucleic acid sequences respectively encoding the polypeptides of amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID

[0107] NO:4 and SEQ ID NO:5 are the nucleic acid sequences SEQ ID NO:25 to SEQ ID NO:29, respectively, shown in Table 2 below and which are optimized (at least partially) for expression in yeast, in particular Saccharomyces cerevisiae:

[0108] [Table 2] However, the person skilled in the art would be able to generate other nucleic sequences optimized for expression in yeast, in particular Saccharomyces cerevisiae, or in another type of host cell (in particular another species of yeast or a bacterium).

[0109] Once the optimized nucleic acid sequence has been defined, the person skilled in the art can obtain this sequence by in vitro synthesis directly with the optimized codons. When the number of nucleotides to be modified compared to the original sequence is not too high, the optimized sequence can also be obtained by in vitro site-directed mutagenesis from a sample of the nucleic acid molecule whose codons are to be adapted, using amplification by polymerase chain reaction (PCR).

[0110] According to a preferred embodiment, the isolated nucleic acid molecule further comprises both a promoter and a terminator as defined above, and the nucleic acid sequence comprised in the isolated nucleic acid molecule used in the invention is further optimized for expression in a host cell, advantageously in a yeast or a bacterium, preferably in a yeast, as described above.

[0111] According to another preferred embodiment, the isolated nucleic acid molecule further comprises both a promoter, a terminator and an export sequence as defined above, and the nucleic acid sequence comprised in the isolated nucleic acid molecule used in the invention is further optimized for expression in a host cell, advantageously in a yeast or a bacterium, preferably a yeast, as described above.

[0112] Vector used

[0113] The vector used in the invention may be any vector comprising any nucleic acid molecule as defined above.

[0114] Vectors that are suitable for use in the present invention include, but are not limited to, bacteriophage, plasmid, or cosmid vectors for expression in prokaryotic host cells such as bacteria (e.g., E. coli, or bacteria of the genus Pseudomonas); vectors for expression in yeast (e.g., Saccharomyces cerevisiae, Schyzosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g., Sf 9 cells); viral and plasmid vectors for expression in plant cell systems (e.g., Ti plasmid, Cauliflower mosaic virus, CaMV, Tobacco mosaic virus TMV); and viral and plasmid vectors for expression in vertebrate, including mammalian, cells or organisms.

[0115] Depending on the host of interest, the skilled person knows suitable expression vectors. These vectors are generally commercially available (e.g., from suppliers such as Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.), available from depository institutions such as the American Type Culture Collection (ATCC, Rockville, Md.), or have been the subject of numerous publications describing their sequence, structures and production methods, so that the skilled person can apply them without difficulty.

[0116] The invention is particularly concerned with the production of phloroglucinol in yeasts, and the vector used in the invention is therefore advantageously suitable and even optimized for the transfection of yeasts. The vector used in the invention may in particular advantageously be a plasmid vector suitable for the transfection of yeasts. Representative examples of plasmid vectors suitable for the transfection of yeasts include, without limitation, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen) and pgWiz (Gene Therapy System Inc) and YCplac22 (ATCC 87585), as well as any derivative of these vectors (in particular a derivative of YCplac22 in which the pair (PGK promoter and CYC terminators) and the pair (TEF1 promoter and ADH1 terminator) have been integrated), but any other vector suitable for yeast transfection may be used.

[0117] Host cell used

[0118] The host cell used in the invention may be any host cell expressing any polypeptide chosen from the type III polyketide synthases of cyanobacteria as defined above. It may in particular be chosen from host cells comprising an isolated nucleic acid molecule as defined above or a vector as defined above.

[0119] As defined above, a host cell is a cell containing a heterologous nucleic acid molecule. In the context of the invention, the heterologous nucleic acid molecule corresponds to the isolated nucleic acid molecule as defined above or to the vector as defined above.

[0120] The host cell used can be a prokaryotic cell or a eukaryotic cell. Among prokaryotic cells, it can be chosen from bacteria. Among eukaryotic cells, it can be chosen from yeast cells, fungal cells, algal cells, insect cells, plants or non-human mammalian cells.

[0121] The host cell used is preferably a yeast, said yeast being in particular selected from the genera Saccharomyces, Candida, Eremothecium, Dekkera, Pichia (Hansenula), Debaryomyces, Lodderomyces, Yarrowia, Zigosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, Brettanomycces, Cryptococcus and Malassezia.

[0122] Even more specifically, the yeast is selected from the species Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, Zi osaccharomyces bailii , Schizosaccharomyces pombe, Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens, Torulaspora globosa and Torulaspora glabrata.

[0123] Even more particularly, the yeast is of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae.

[0124] The host cell used may also be a bacterium, said bacterium being in particular selected from among the proteobacteria, the actinomycete bacteria and the Firmicutes bacteria. Among the proteobacteria, the host cell may advantageously be chosen from among the genera Escherichia (in particular among the strains of the species Escherichia coli, the species most used for the production of recombinant proteins) and Pseudomonas. Among the actinomycete bacteria, the host cell may advantageously be chosen from among the genera Streptomyces and Corynebacterium. Among the Firmicutes bacteria, the host cell may advantageously be chosen from among the genera Bacillus and Lactobacillus.

[0125] The host cell used comprises at least one copy of the isolated nucleic acid molecule as defined above integrated into its genome. It may in particular comprise a single copy of the isolated nucleic acid molecule as defined above integrated into its genome.

[0126] When the host cell used is a yeast cell (in particular of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae), the copy(ies) of the nucleic acid molecule may be integrated at different loci, preferably at the URÀ3 locus, at the JLP1 locus, at the LEU2 locus, or at the TRP1 locus of the genome of said yeast cell. When the host cell is a yeast cell and several copies of the nucleic acid molecule are integrated, the different copies may be integrated at the same locus, or at different loci, preferably at any of the combinations of the URÀ3, JLP1, LEU2, and / or TRP1 loci. Advantageously, the codons used in the nucleic acid molecule encoding the polypeptide selected from the type III polyketide synthases of cyanobacteria included in the host cell or in the vector included in the host cell have been adapted for optimal expression in the selected host cell.As explained previously, in producing an amino acid sequence of interest, optimal expression can be achieved when the codons chosen to encode the amino acid sequence are those preferentially used by the organism of origin of the host cell. Regardless of the type of host cell, the person skilled in the art will be able to find which codons are to be favored in the literature or by using codon optimization software. The examples of codon optimization software mentioned above for yeast can also be used for other types of host cells.Yeasts, and in particular those of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae, being particularly preferred as host cells, the codons used in the nucleic acid molecule coding for the polypeptide chosen from the type III polyketide synthases of cyanobacteria included in the host cell or in the vector included in the host cell have advantageously been adapted for optimal expression in yeasts, and in particular in yeasts of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae.

[0127] For the use according to the invention, the host cells can be cultured in aerobic or anaerobic bioreactors, on a small and large scale, in flasks or Petri dishes. The culture can be carried out at a temperature, pH, culture medium and oxygen content appropriate for a given host cell. For yeasts, and more particularly for Saccharomyces cerevisiae, the culture is advantageously carried out by any suitable method. Methods for culturing a Saccharomyces cerevisiae strain are known in the art, and the person skilled in the art knows how to optimize the culture conditions for each strain according to its nature. Conventional methods are described in particular in the reference work "Yeast Technology", 2nd Edition, 1991, Reed and Nagodawithana, published by Van Nostrand Reinhold (ISBN 0-442-31892-8).In particular, the cultivation of a yeast strain, in particular of the species Saccharomyces cerevisiae, can generally be carried out at a temperature between 20 and 37°C, in a rich liquid medium (for example YPD medium available from VWR) or synthetic (defined to precisely meet the needs of the strain), in aerobic or anaerobic culture. Use.

[0128] The polypeptide selected from cyanobacteria type III polyketide synthases, the nucleic acid molecule encoding it, the vector comprising a nucleic acid molecule encoding it or the host cell expressing it, each as defined above, is used to produce phloroglucinol, regardless of the production method used.

[0129] According to the method, the person skilled in the art will be able to choose the most appropriate between the polypeptide chosen from the type III polyketide synthases of cyanobacteria, the nucleic acid molecule encoding it, the vector comprising a nucleic acid molecule encoding it or the host cell expressing it, each being as defined above.

[0130] In particular, the polypeptide selected from the type III polyketide synthases of cyanobacteria as described above can be used directly to produce phloroglucinol in vitro, provided that it is brought into contact with malonyl-CoA. Indeed, phloroglucinol synthases carry out the condensation of three molecules of malonyl-CoA to form one molecule of phloroglucinol according to the reaction scheme Reaction I previously described.

[0131] The nucleic acid molecule encoding it and the vector comprising a nucleic acid molecule encoding it can be used to transfect a host cell, which can then be used either to produce the polypeptide (which can then produce phloroglucinol in vitro) or to directly produce phloroglucinol when cultured in the presence of a suitable substrate (such as a carbon source like glucose or ethanol).

[0132] Examples of more specific methods in which the polypeptide selected from cyanobacterial type III polyketide synthases, the nucleic acid molecule encoding it, the vector comprising a nucleic acid molecule encoding it or the host cell expressing it as described above are described in the section below concerning methods according to the invention for producing phloroglucinol.

[0133] Isolated nucleic acid molecule according to the invention

[0134] The invention also relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide selected from cyanobacteria type III polyketide synthases as defined above, characterized in that: a) the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence; or b) the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence; or c) the nucleic acid sequence is further optimized for expression in a host cell, in particular in a yeast or a bacterium; or d) any combination of a) to c).

[0135] Such isolated nucleic acid molecules are useful either for the production of a polypeptide selected from the type III polyketide synthases of cyanobacteria as defined above, or for directly producing phloroglucinol when transfected (directly or within a vector) into a host cell, cultured in the presence of an appropriate substrate.

[0136] In embodiment a), the isolated nucleic acid molecule further comprises a promoter controlling expression of the nucleic acid sequence.

[0137] In embodiment b), the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence.

[0138] In embodiment c), the nucleic acid sequence is further optimized for expression in a host cell, in particular in a yeast or in a bacterium, advantageously in a yeast.

[0139] In other embodiments, the isolated nucleic acid molecule may combine several of the features a) to c). In particular, the isolated nucleic acid molecule may be characterized in that: d) the isolated nucleic acid molecule further comprises a promoter and a transcription terminator controlling the expression of the nucleic acid sequence (combination of a) and b)); e) the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence and the nucleic acid sequence is further optimized for expression in a host cell, in particular in a yeast or in a bacterium, advantageously in a yeast (combination of a) and c));f) the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence and the nucleic acid sequence is further optimized for expression in a host cell, in particular in a yeast or in a bacterium, advantageously in a yeast (combination of b) and c)); or g) the isolated nucleic acid molecule further comprises a promoter and a transcription terminator controlling the expression of the nucleic acid sequence and the nucleic acid sequence is further optimized for expression in a host cell, in particular in a yeast or in a bacterium, advantageously in a yeast (combination of a), b) and c)).;

[0140] When the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence (embodiments a), d), e), and g) above), the promoter is advantageously an exogenous promoter, in particular a yeast promoter, and preferably a promoter selected from ÀDH2 (pÀDH2) and CCW12 (pCCW12) and TEF1 (pTEF1), more preferably a promoter selected from ÀDH2 (pÀDH2) of Saccharomyces cerevisiae and CCW12 of S. cerevisiae, CCW12 (pCCW12) of S. cerevisiae and TEF1 (pTEF1) of S. cerevisiae, more preferably a promoter selected from ÀDH2 of sequence SEQ ID NO: 20, CCW12 of sequence SEQ ID NO: 21 and TEF1 of sequence SEQ ID NO: 22.

[0141] When the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence (embodiments b), d), f), and g) above), the transcription terminator is advantageously an exogenous terminator, such as a yeast terminator, and preferably the RPL3 terminator (tRPL3) or the ΔDH1 terminator (tΔDH1), more preferably the S. cerevisiae RPL3 terminator or the S. cerevisiae ΔDH1 terminator, more preferably the RPL3 terminator of sequence SEQ ID NO: 23 or the ΔDH1 terminator of sequence SEQ ID NO: 24.

[0142] When the nucleic acid sequence is further optimized for expression in yeast (embodiments c), e), f), and g) above), it is advantageously optimized for expression in yeast of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae. In particular, it is advantageously selected from SEQ ID NO: 25 to SEQ ID NO: 29.

[0143] When the nucleic acid sequence is further optimized for expression in a bacterium (embodiments c), e), f), and g) above), it is advantageously optimized for expression in a bacterium of the genera Escherichia (in particular strains of the species Escherichia coli, the species most used for the production of recombinant proteins) and Pseudomonas. Vector according to the invention

[0144] The invention also relates to a vector comprising a nucleic acid molecule according to the invention.

[0145] The vector according to the invention may comprise any nucleic acid molecule according to the invention described above. It may be chosen from any type of vector described previously in the section relating to the vectors capable of being used in the use according to the invention.

[0146] Host cell according to the invention

[0147] The invention also relates to a host cell comprising a nucleic acid molecule according to the invention, or a vector according to the invention.

[0148] The host cell according to the invention may comprise any nucleic acid molecule according to the invention described above or any vector according to the invention as described above. It may further comprise any characteristic or combination of characteristics described previously in the section relating to the host cells capable of being used in the use according to the invention.

[0149] Methods for producing a polypeptide selected from cyanobacterial type III polyketide synthases

[0150] The present invention further relates to a method for producing a polypeptide selected from cyanobacterial type III polyketide synthases as defined above.

[0151] According to one embodiment, the method for producing such a polypeptide comprises, consists essentially of, or consists of the following steps:

[0152] (i) introducing a nucleic acid molecule or a vector comprising a nucleic acid molecule as described above (nucleic acid molecule or vector used in the invention or according to the invention) into a suitable host cell in accordance with the foregoing description; and

[0153] (ii) in vitro culture of said host cell obtained in step (i) under conditions allowing the expression of said nucleic acid molecule, so as to produce said polypeptide. According to another embodiment, the method for producing such a polypeptide comprises, essentially consists of, or consists at least of the step consisting of:

[0154] (ii) in vitro culture of a host cell expressing said polypeptide, for example a host cell as described above (host cell used in the invention or according to the invention), under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce said polypeptide.

[0155] Step (i) of introducing the nucleic acid molecule or vector into a suitable host cell may be carried out by any suitable method known to those skilled in the art, such as transfection by calcium phosphate, transfection by liposomes comprising the nucleic acid molecule or vector to be transfected, transfection by polycationic agents, electroporation, and transfection by heat shock. In particular, step (i) may be carried out by transfection (by any method described above) into the host cell of a transfer plasmid comprising the sequence coding for the polypeptide chosen from the type III polyketide synthases of Ascomycetes fungi as defined above, flanked in 5' and 3' by sequences homologous to genomic sequences of the host cell, thus allowing homologous recombination between the transfer plasmid and the genome of the host cell.This method is particularly applicable when the host cell is a yeast, in particular Saccharomyces, preferably of the species Saccharomyces cerevisiae, or a bacterium.

[0156] Step (ii) of in vitro culture of the host cell is carried out under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce said polypeptide. These conditions vary depending on the host cell used and the person skilled in the art will be able to determine them on the basis of his general knowledge. In particular, when the host cell is a yeast, in particular Saccharomyces, preferably of the species Saccharomyces cerevisiae, step (ii) can be carried out by any appropriate method, in particular as described in the section above concerning the host cells used in the use according to the invention.

[0157] The method for producing the polypeptide may further comprise at least one additional step selected from the following steps:

[0158] (a) recovering the host cells expressing said polypeptide and / or the supernatant comprising the polypeptide, obtained after the culturing step; and (B) purifying the polypeptide from the host cells and / or the supernatant recovered in step (a).

[0159] The optional step (a) of recovering the host cells expressing said polypeptide and / or the supernatant comprising the polypeptide may be carried out by any suitable technique known to those skilled in the art. The cells and the supernatant may in particular be separated by decantation or centrifugation.

[0160] The optional step (B) of purifying the polypeptide from the host cells and / or the supernatant recovered in step (a) may be carried out by any suitable technique known to the person skilled in the art. When the polypeptide is purified from the supernatant, any liquid-phase protein purification technique may be used, such as, for example, the various types of chromatography (gel filtration, ion exchange, hydrophobic interaction, affinity when the protein comprises an affinity tag, high-performance liquid chromatography “HPLC”). When the polypeptide is purified from the cells or from the cells and the supernatant, the purification further comprises one or more preliminary steps of lysis of the cells and optionally removal of cell debris, before using a liquid-phase purification technique.

[0161] Methods for producing phloroglucinol

[0162] As explained in the subsection "Use" of the section relating to the use according to the invention, different methods for producing phloroglucinol can be implemented depending on whether they are based on the polypeptide chosen from the type III polyketide synthases of cyanobacteria, the nucleic acid molecule encoding it, the vector comprising a nucleic acid molecule encoding it or the host cell expressing it, each being as defined above.

[0163] In particular, the nucleic acid molecule encoding the polypeptide selected from the cyanobacterial type III polyketide synthases as described above and the vector comprising a nucleic acid molecule encoding this polypeptide can be used to transfect a host cell, which can then be used either to produce the polypeptide (which can then produce phloroglucinol in vitro) or to directly produce phloroglucinol when cultured in the presence of an appropriate substrate.

[0164] Thus, according to an embodiment M1, the method for producing phloroglucinol uses a host cell expressing the polypeptide chosen from the type III polyketide synthases of cyanobacteria as defined above (for example a host cell according to the invention), which is cultured in the presence of a substrate suitable for producing phloroglucinol. This embodiment can be subdivided into two sub-embodiments, depending on whether the method comprises (embodiment M1A) or not (embodiment M1B) a prior step of obtaining the host cells.

[0165] In embodiment M1A, the invention relates to a method for producing phloroglucinol which comprises, consists essentially of, or consists of the following steps:

[0166] (ii) introducing a nucleic acid molecule or a vector comprising a nucleic acid molecule as described above (nucleic acid molecule or vector used in the invention or according to the invention) into a suitable host cell in accordance with the preceding description;

[0167] (ii1) bringing the host cells obtained in step (ii) into contact with a suitable substrate;

[0168] (iii1) in vitro culture of the host cell of step (ii1) under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce phloroglucinol;

[0169] (iv1) optionally recovering the culture medium comprising phloroglucinol, obtained after step (iii1); and

[0170] (v1) optionally, the purification of phloroglucinol from the culture medium of step (iv1).

[0171] In embodiment M1 B, the invention relates to a method for producing phloroglucinol, which comprises, consists essentially of, or consists of the following steps:

[0172] (ii1) bringing into contact a non-human host cell expressing the polypeptide chosen from the type III polyketide synthases of cyanobacteria as defined above (for example a host cell according to the invention), with an appropriate substrate;

[0173] (iii1) in vitro culture of the host cell of step (ii1) under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce phloroglucinol;

[0174] (iv1) optionally recovering the culture medium comprising phloroglucinol, obtained after step (iii1); and

[0175] (v1) optionally, the purification of phloroglucinol from the culture medium of step (iv1). For the purposes of methods M1 A and M1 B, and in particular when the host cell is a yeast (in particular of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae):

[0176] - step (il) of introducing the nucleic acid molecule or vector into a suitable host cell may be carried out by any method described herein in the context of step (i) of the methods of producing the polypeptide selected from the type III polyketide synthases of cyanobacteria as described above.

[0177] - the substrate brought into contact with the host cell in step (ii1) is advantageously a carbon source. Advantageously, the carbon source is a pure carbon source or an industrial co-product (such as molasses or lean sewage, for example from the sugar industry). Preferably, the substrate in the pure carbon source or industrial co-product is a simple sugar, such as glucose (or dextrose), fructose, galactose, mannose, sucrose, lactose, or maltose; a complex sugar, such as a monosaccharide, a disaccharide or trisaccharides, or a polysaccharide such as starch; an alcohol, such as ethanol; an acid; a fatty acid and an ester derivative thereof; or a mixture of sugars, alcohols, acids and / or fatty acids or their ester derivatives. Preferably, the substrate is glucose or ethanol.

[0178] - step (iii1) of in vitro culture of the host cell under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce phloroglucinol can be carried out by any method described here in the context of step (ii) of the methods of production of the polypeptide chosen from the type III polyketide synthases of cyanobacteria as described above.

[0179] - when present, step (iv1) of recovering the culture medium comprising phloroglucinol may be carried out by any technique described here for the separation of cells and supernatant in the context of optional step (a) of the methods for producing the polypeptide selected from the type III polyketide synthases of cyanobacteria as described above.

[0180] - when present, step (v1) of purification of phloroglucinol from the culture medium can be carried out by any suitable technique, such as liquid-liquid extraction.

[0181] Alternatively, the polypeptide selected from the type III polyketide synthases of cyanobacteria as described above can be used directly to produce phloroglucinol in vitro, provided that it is brought into contact with malonyl-CoA. Indeed, phloroglucinol synthases carry out the condensation of three molecules of malonyl-CoA to form one molecule of phloroglucinol according to the reaction scheme Reaction I previously described.

[0182] Thus, according to an embodiment M2, the method for producing phloroglucinol uses the polypeptide selected from the type III polyketide synthases of cyanobacteria as described above to produce phloroglucinol in vitro in the presence of malonyl-CoA. This embodiment can be subdivided into two sub-embodiments, depending on whether the method comprises (embodiment M2A) or not (embodiment M2B) a prior step of producing the polypeptide selected from the type III polyketide synthases of cyanobacteria as defined above.

[0183] In embodiment M2A, the invention relates to a method for producing phloroglucinol, which comprises, consists essentially of, or consists of the following steps:

[0184] (i2) producing a polypeptide selected from cyanobacteria type III polyketide synthases as defined above by one of the polypeptide production methods described above;

[0185] (ii2) bringing the polypeptide obtained in step (i2) of the method as described above into contact with malonyl-CoA;

[0186] (iii2) incubating the mixture from step (ii2) under conditions suitable for producing phloroglucinol;

[0187] (iv2) optionally the recovery of the reaction medium comprising phloroglucinol, obtained after step (iii2); and

[0188] (v2) optionally, the purification of phloroglucinol from the reaction medium of step (iv2).

[0189] In embodiment M2B, the invention relates to a method for producing phloroglucinol, which comprises, consists essentially of, or consists of the following steps:

[0190] (ii2) contacting a polypeptide selected from type III polyketide synthases of cyanobacteria as defined above with malonyl-CoA;

[0191] (iii2) incubating the mixture from step (ii2) under conditions suitable for producing phloroglucinol;

[0192] (iv2) optionally the recovery of the reaction medium comprising the phloroglucinol, obtained after step (iii2); and (v2) optionally, the purification of the phloroglucinol from the reaction medium of step (iv2).

[0193] For the purposes of these methods M2A and M2B, and in particular when the host cell is a yeast (in particular of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae):

[0194] - step (i2) can be carried out by any method described herein to produce a polypeptide chosen from type III polyketide synthases of cyanobacteria.

[0195] - step (ii2) consists of bringing the polypeptide into contact with its substrate malonyl-CoA to allow the production of phloroglucinol. This contact is carried out in a medium and at a temperature which do not alter the enzymatic activity of the polypeptide. A suitable medium may in particular be chosen from a sodium phosphate buffer at pH 7 (in particular at a concentration of approximately 25-75 mM) comprising NaCl (in particular at a concentration of approximately 5-15 mM) and bovine serum albumin (BSA, in particular at a concentration of approximately 150-250 mg / L). A suitable temperature is in the range 20 to 37°C.

[0196] - step (iii2) consists of incubating the polypeptide and its substrate under conditions allowing the production of phloroglucinol. These conditions involve a medium and an incubation temperature which do not alter the enzymatic activity of the polypeptide, as described above for step (ii2). The incubation time is chosen according to the quantity of substrate present in the medium. The person skilled in the art will know how to adapt the initial concentration of substrate to the incubation time. It may also be envisaged to add substrate during the incubation time to continue the production of phloroglucinol.

[0197] - when present, step (iv2) of recovery of the reaction medium does not require any particular technique since there are no cells to exclude or lyse before purification.

[0198] - when present, step (v2) of purification of phloroglucinol from the culture medium can be carried out by any suitable technique, such as liquid-liquid extraction.

[0199] The following examples are intended to illustrate the present invention.

[0200] EXAMPLES Example 1. Identification of type III polyketide synthases from cyanobacteria

[0201] PhlDs are type III polyketide synthases (PKSs) involved in the biocatalytic synthesis of phloroglucinol from three malonyl-CoA molecules (Zha et al., 2006). To identify novel PhlDs by a sequence-based approach, we used the sequence of PhlD from Tsukamurella pulmonis (PhlD_Tpu). PhlD was indeed found to be the best phloroglucinol-producing enzyme among the 12 enzymes previously tested in S. cerevisiae, the host organism used for cloning and protein production (W02019 / 002799).

[0202] A blast (Johnson et al., 2008) [NCBI] of the PhlD_Tpu protein sequence was performed against standard protein databases (non-redundant protein sequences, protein databank, Swissprot, metagenome-derived proteins, and proprietary protein sequences, versions of 22.11.2019). The obtained sequences were filtered by collecting all sequences with a percentage identity greater than 20% to the input sequence. Redundant sequences were then removed (CD-HIT, sequence identity >95%), reducing the dataset to approximately 3,320 sequences. In addition, sequences of 45 characterized PKSs from the literature were included if they were not present in the previously generated dataset. The sequences were finally clustered using a sequence similarity network (SSN, Gerlt, et al., 2015), each group corresponding to a different phylum or class. SSNs are useful for analyzing sequence datasets and studying sequence-function relationships. SSN is a convenient way to visualize the relationships between protein sequences by constructing a graph where each member of a protein family is represented by a node connected by an edge to the nodes of all other members that share sequence similarity greater than a user-specified value. These graph networks are easy to visualize and are more readily interpretable than traditional dendrogram and phylogenetic tree approaches. This representation makes it easier to assign a putative function to a protein in a given cluster if a member has already been characterized and to track evolutionary relationships between clusters.

[0203] These graphs can be visualized using visualization software such as Cytoscape (Shannon et al., 2003), where subregions of the graphs (edges and nodes) can be zoomed in or manipulated iteratively, allowing better identification of sequence similarity at the microscopic scale of the network.

[0204] After careful cluster analysis, we identified PKS sequences previously characterized as producing phloroglucinol or other related compounds known to be produced by PKS family enzymes. The majority of these enzymes are found in the plant, brown algae, and actinobacteria groups. However, other enzymes were also characterized in other isolated clusters. The P. fluorescens PhlD and PhlD_Tpu sequences from P. fluorescens known to produce phloroglucinol were also identified, validating the method used.

[0205] To further the classification of sequences and their interconnections, clusters of sequences from different classes were generated with an E value <1 O' 130 . This allowed the recovery of two type III PKS from brown macroalgae (Ectocarpus siliculosus-PKS .Es and Sar assium binderi-PHLD.Sbi) already reported to produce phloroglucinol in Saccharomyces cerevisiae (W02019 / 002799). The use of a stricter threshold score (E value <10 170 ) allowed the separation of a cluster containing type III PKSs of the genus Tsukamurella, also known to have high phloroglucinol production activity in S. cerevisiae (WO2019 / 002799). These results further validate the method used.

[0206] Finally, we pooled the information from the two clustering analyses with an E value <10' 130 and a value E <1 O' 170and selected a total of 23 sequences from seven clusters and different classes to evaluate their ability to produce phloroglucinol.

[0207] Example 2. Sequences of five cyanobacterial type III polyketide synthases identified in Example 1

[0208] The polypeptides of amino acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are among the 23 sequences selected in Example 1 and all five are derived from cyanobacteria.

[0209] More specifically:

[0210] - SEQ ID NO: 1 corresponds to the amino acid sequence of a PhlD of Rivularia sp. PCC 7116 (denoted PlhD-Rs);

[0211] - SEQ ID NO: 2 corresponds to the amino acid sequence of a PhlD of Chamaesiphon minutus (denoted PlhD-Cm);

[0212] - SEQ ID NO: 3 corresponds to the amino acid sequence of a PhlD of Filamentous cyanobacterium CCP2 (denoted PlhD-Fc);

[0213] - SEQ ID NO: 4 corresponds to the amino acid sequence of a PhlD of Calothrix sp. HK-06 (denoted PlhD-Cs); and - SEQ ID NO: 5 corresponds to the amino acid sequence of a PhlD of Cloeocapsa sp. PCC 7428 (denoted PlhD-Gs).

[0214] A multiple alignment of their amino acid sequences is shown in Figure 1. Their pairwise identity percentages, as well as to other prior art PhlDs, are further shown in Table 3 below.

[0215] [Table 3]

[0216] Table 3 above shows that the 5 sequences newly identified as possible phloroglucinol synthases have only low sequence identity with the sequences of previously identified phloroglucinol synthases, and belong to a different kingdom (cyanobacteria versus actinomycete bacteria or eukaryotic algae).

[0217] Example 3. Ability of type III polyketide synthases of cyanobacteria to produce phloroglucinol in yeast

[0218] The 5 previously identified putative phloroglucinol synthases (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5) were tested for their ability to produce phloroglucinol when expressed by a yeast of the species Saccharomyces cerevisiae. Their activity was further compared to that of prior art PHLDs. Materials and methods

[0219] Yeast cells Cells of the yeast S. cerevisiae CEN.PK2-1 D (see Entian KD and Kôtter P, 2007, commercially available in particular from Euroscarf) were used.

[0220] Preparation of S. cerevisiae CEN.PK2-1D cell clones expressing the 5 putative phloroglucinol synthases or prior art phloroglucinol synthases

[0221] The nucleic sequences SEQ ID NO: 25 to 29 coding respectively for the 5 putative phloroglucinol synthases previously identified (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5) were modified by adding to their 5' and 3' ends respectively sequences homologous to the desired site of integration in the genome of S. cerevisiae CEN.PK2-1 D yeast cells, leading to the sequences SEQ ID NO: 30 to 34. For the phloroglucinol synthases PHLD.Pf, PHLD.Tsp and PHLD.Tt, the sequences SEQ ID NO: 35 to 37, which include sequences homologous to the desired site of integration in the genome, were used.

[0222] The sequences SEQ ID NO: 30 to 37 were then cloned into a plasmid called “PBIM5” or “YCplac22_PGK_CYC_TEF_ÀDH” (derived from the commercial plasmid YCplac22 by addition of the pair (PGK promoter and CYC terminator, see Gietz RD et al., 1988) as well as the pair (TEF1 promoter and ÀDH1 terminator), see Figure 2), under the control of the TEF1 promoter and the ÀDH1 terminator. The sequences SEQ ID NO: 30 to 37 were then integrated into the genome of S. cerevisiae CEN.PK2-1 D yeast cells by in vivo homologous recombination, and the cloned sequences verified by Sanger sequencing.

[0223] 96-well plate culture for phloroglucinol production

[0224] Each well was filled with 500 µl of complete synthetic Trp_drop out medium marketed by Formedium.

[0225] A single cell clone obtained as described above was collected and inoculated into a well.

[0226] The plate was incubated at 30°C for approximately 20 hours at 800 rpm in a shaking incubator. The following day, a new 96-well plate filled with 500 μl of complete synthetic medium was inoculated with 10 μl of culture. Three individual plates were inoculated for 24-, 48-, and 72-hour samplings.

[0227] Quantification of phloroglucinol production by calorimetric test

[0228] Every 24 hours, each plate was removed from the incubator and measurements were performed as described below: -First, absorbance was measured in the microtiter plate at 600 nm in the Tecan plate reader. For this, 20 μl of culture was diluted with 180 μl of milliQ water.

[0229] -Then, the 96-well plate was centrifuged at 3700 rpm for 10 minutes and the supernatant was used for colorimetric assay. -150 μl of 500mg / L 4-hydroxy-3methyoxycinnamaldehyde in HCl / EtOH v:v (1:3) was added to the sample and Phloroglucinol standard. Incubation was carried out at room temperature for 30min.

[0230] Results

[0231] The amounts of phloroglucinol produced in the test used by yeasts expressing the different PhlDs are presented in Table 4 below.

[0232] [Table 4]

[0233] Table 4 shows that the 5 previously identified putative phloroglucinol synthases (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5), although derived from cyanobacteria, are capable of producing phloroglucinol when expressed in yeast.

[0234] BIBLIOGRAPHICAL REFERENCES

[0235] Àchkar J et al., (2005) « Biosynthesis of phloroglucinol » J Àm Chem Soc. 127:5332-5333. Entian K-D,e et al., (2007) « Yeast genetic strain and plasmid collections », Methods in microbiology, 36, 0580-9517.

[0236] Gietz R.D et al., (1988) « New yeast-escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites », Gene, 74, 527-534.

[0237] Gerlt, J. À. et al. Enzyme Function Initiative- Enzyme Similarity Tool (EFI-EST): À web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta BBA - Proteins Proteomics 1854, 1019-1037 (2015).

[0238] Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5-W9 (2008).

[0239] Meslet-Cladière L, Delage L, Leroux CJ, Goulitquer S, Leblanc C, Creis E, Gall EA, Stiger- Pouvreau V, Czjzek M, and Potin P. (2013) “Structure / function analysis of a type III polyketide synthase in the brown alga Ectocarpus siliculosus reveals a biochemical pathway in phlorotannin monomer biosynthesis.” Plant Cell. 25:3089-3103.

[0240] Needleman et Wunsch. J. Mol. Biol. 48,443-453, 1970.

[0241] Shannon, P. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 13, 2498-2504 (2003).

[0242] WO2013 / 045510

[0243] WO2019 / 002798

[0244] WO2019 / 002799

[0245] W02020 / 024917A1

[0246] Zha W, Rubin-Pitel SB and Zhao H. (2006) “Characterization of the substrate specificity of PHLD, a type III polyketide synthase from Pseudomonas fluorescens.” J Biol Chem. 281 :32036-32047.

Claims

CLAIMS 1. Use of a polypeptide selected from cyanobacteria type III polyketide synthases, a nucleic acid molecule encoding it, a vector comprising a nucleic acid molecule encoding it or a non-human host cell expressing it, for producing phloroglucinol; the use being characterized in that said polypeptide comprises an amino acid sequence having at least 70% identity with a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

2. Use according to claim 1, characterized in that said polypeptide is chosen from type III polyketide synthases: - of the families Rivulariaceae, Chamaesiphonaceae and Chroococcaceae, advantageously of the genera Rivularia, Caltothrix, Chamaesiphon and Gloeocapsa, more advantageously of the species Rivularia sp. PCC 7116, Calothrix sp. HK-06, Chamaesiphon minutus, Gloeocapsa sp. PCC 7428, and Gloeocapsa sp. PCC 73106 or - of the species Filamentous cyanobacterium CCP2.

3. Use according to claim 1 or claim 2, characterized in that said polypeptide comprises an amino acid sequence having at least 80% identity with a sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

4. Use according to any one of claims 1 to 3, characterized in that said polypeptide further comprises at least one fragment chosen from: (a) ATGTP (SEQ ID NO:6); (b) STGFXiAPG, wherein Xi is selected from apolar amino acids (i.e. glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M) and cysteine ​​(C), SEQ ID NO:7); (c) VX2FMGCAAA, wherein X2 is selected from polar amino acids (i.e. serine (S), threonine (T), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H), SEQ ID NO:8); (d) VCLELSS (SEQ ID NO:9); (e) IHSIFX3DGCAA, wherein X3 is any amino acid (i.e. X3 is selected from glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M), cysteine ​​(C), serine (S), threonine (T), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), and histidine (H), SEQ ID NO:10); (f) IDLWX HPGGTRI, wherein X4 is selected from apolar amino acids (i.e. glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M) and cysteine ​​(C), SEQ ID NO: 11).

5. Use according to claim A, characterized in that said polypeptide further comprises at least one fragment chosen from: (a) ÀTGTP (SEQ ID NO:6); (b) STGFX1ÀPG, wherein X1 is selected from aliphatic amino acids (i.e. alanine (À), valine (V), leucine (L), isoleucine (I), and proline (P), SEQ ID NO: 12); (c) VX2FMGCAAÀ, wherein X2 is selected from neutral polar or positively charged polar amino acids (i.e. serine (S), threonine (T), asparagine (N), glutamine (Q), lysine (K), arginine (R), and histidine (H), SEQ ID NO: 13); (d) VCLELSS (SEQ ID NO:9); (e) IHSIFX3DGCAÀ, wherein X3 is selected from neutral apolar or polar amino acids (i.e. glycine (G), alanine (À), valine (V), leucine (L), isoleucine (I), proline (P), tryptophan (W), phenylalanine (F), tyrosine (Y), methionine (M), cysteine ​​(C), serine (S), threonine (T), asparagine (N), and glutamine (Q), SEQ ID NO: 14); (g) IDLWX4VHPGGTRI, wherein X4 is selected from aliphatic nonpolar amino acids (i.e. alanine (A), valine (V), leucine (L), isoleucine (I), and proline (P), SEQ ID NO:15).

6. Use according to claim 5, characterized in that said polypeptide further comprises at least one fragment chosen from: (a) ÀTGTP (SEQ ID NO:6); (b) STGFX1ÀPG, wherein X1 is selected from leucine (L), valine (V), and isoleucine (I) (SEQ ID NO: 16), (c) VX2FMGCÀÀÀ, wherein X2 is selected from asparagine (N) and histidine (H) (SEQ ID NO: 17); (d) VCLELSS (SEQ ID NO:9); (e) IHSIFX3DGCÀÀ, wherein X3 is selected from glycine (G) and serine (S) (SEQ ID NO: 18); (g) IDLWX HPGGTRI, wherein X4 is selected from alanine (A) and valine (V) (SEQ ID NO:19).

7. Use according to any one of claims 1 to 6, characterized in that said polypeptide comprises an amino acid sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide as defined in any one of claims 1 to 7, characterized in that: a) the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence; or b) the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence; or c) the nucleic acid sequence is further optimized for expression in a host cell, in particular a yeast or a bacterium; or d) any combination of a) to c).

9. Isolated nucleic acid molecule according to claim 8, characterized in that when the isolated nucleic acid molecule further comprises a promoter controlling the expression of the nucleic acid sequence, the promoter is an exogenous promoter, in particular a yeast promoter.

10. Isolated nucleic acid molecule according to claim 8, characterized in that when the isolated nucleic acid molecule further comprises a transcription terminator controlling the expression of the nucleic acid sequence, the transcription terminator is an exogenous terminator, such as a yeast terminator.

11. Isolated nucleic acid molecule according to claim 8, characterized in that the nucleic acid sequence is further optimized for expression in yeast, advantageously it is chosen from SEQ ID NO: 25 to SEQ ID NO:

29.

12. Vector comprising a nucleic acid molecule according to any one of claims 8 to 11, said vector preferably being a plasmid.

13. A non-human host cell comprising a nucleic acid molecule according to any one of claims 8 to 11, or a vector according to claim 12.

14. Non-human host cell according to claim 13, characterized in that said host cell is a yeast, advantageously selected from the genera Saccharomyces, Candida, Ashbya, Dekkera, Pichia (Hansenula), Debaryomyces, Clavispora, Lodderomyces, Yarrowia, Zigosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, Brettanomycces, Cryptococcus and Malassezia, more particularly among the species Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, Zigosaccharomyces bailii, Schizosaccharomyces pombe, Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens, Torulaspora globosa or Torulaspora glabrata; even more particularly, the yeast is of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae.

15. A method for producing phloroglucinol, which comprises the following steps: (111) contacting a non-human host cell expressing the polypeptide selected from cyanobacterial type III polyketide synthases as defined in any one of claims 1 to 7, with a suitable substrate; (iii1) in vitro culture of the host cell of step (ii1) under conditions allowing the expression of the nucleic acid molecule contained in said host cell, so as to produce phloroglucinol; (iv1) optionally recovering the culture medium comprising phloroglucinol, obtained after step (iii1); and (v1) optionally, the purification of phloroglucinol from the culture medium of step (iv1).

16. A method for producing phloroglucinol, which comprises the following steps: (112) contacting a polypeptide selected from cyanobacteria type III polyketide synthases as defined in any one of claims 1 to 7 with malonyl-CoA; (iii2) incubating the mixture from step (ii2) under conditions suitable for producing phloroglucinol; (iv2) optionally the recovery of the reaction medium comprising phloroglucinol, obtained after step (iii2); and (v2) optionally, the purification of phloroglucinol from the reaction medium of step (iv2).

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