Optimized phloroglucinol synthase

Abstract

The present invention relates to a mutant polypeptide comprising an amino acid sequence of phloroglucinol synthase having at least 80% identity with the amino acid sequence of tsukamurella pneumoniae phloroglucinol synthase as shown in SEQ ID NO: 1, with one or more substitutions of one or more amino acid residues. The invention also relates to a nucleic acid molecule encoding the mutant polypeptide, as well as a vector and a host cell comprising the nucleic acid molecule. The invention also relates to a method for producing phloroglucinol.

Description

Technical Field

[0001] This invention relates to the field of microbial biochemistry, and more particularly to the field of synthesizing phloroglucinol from microbial enzymes. The invention especially relates to optimized, mutant phloroglucinol synthases, particularly optimized, mutant bacterial phloroglucinol synthases, especially those from actinomycete bacteria, particularly those from the genus *Tsukamura*. Tsukamurella Optimized and mutant phloroglucinol synthases of bacteria, and their methods and uses for the production of phloroglucinol. Background Technology

[0002] Phloroglucinol (chemically named 1,3,5-phenylpyroglucinol) is an aromatic organic compound, particularly used in the production of pharmaceutical products and explosives.

[0003] The synthesis of phloroglucinol is catalyzed by a type III polyketide synthase called phloroglucinol synthase. Phloroglucinol synthase undergoes the condensation of three malonyl-CoA molecules to form one phloroglucinol molecule, following the reaction scheme (reaction I): Reaction I Subsequently, many oligomers, such as phlorotannins, can be synthesized from phloroglucinol. Phlorotannins specifically include fucols, phloretols, and fucophloretols, which are phloroglucinol derivatives that constitute the cell walls of brown algae. Furthermore, various protective activities of brown algae are also attributed to phlorotannins.

[0004] In Gram-negative fluorescent pseudomonas ( Pseudomonas fluorescens (Achkar et al., 2005; Zha et al., 2006) and the brown algae *Fragaria fringeii* ( Ectocarpus siliculosus The synthesis of phloroglucinol has been described in (Meslet-Cladière et al., 2013). In *Pseudomonas fluorescens*, the phloroglucinol synthase is encoded by the PHLD gene (Achkar et al., 2005; Zha et al., 2006). In *Bryophyta fringeii*, the phloroglucinol synthase is encoded by the PKS1 gene (Meslet-Cladière et al., 2013). In *Escherichia coli* expressing the heterologous PHLD gene (…),… Escherichia coliThe activity of PHLD phloroglucinol synthase has been confirmed (Achkar et al., 2005). This activity was also confirmed in vitro using a small-scale enzymatic assay with recombinant PHLD expressed and purified from Escherichia coli cultures (Zha et al., 2006). Recombinant PKS1 expressed and purified from Escherichia coli and from *Tricholoma matsutake* (*P. spp.*) has been shown to have activity in vitro. E. siliculosus The activity of PKS1 phloroglucinol synthase was demonstrated in vitro in the cell extract of PKS1 (Meslet-Cladière et al., 2013, WO 2013 / 045510).

[0005] However, PHLD and PKS1 enzymes exhibited low enzymatic activity. Furthermore, the feasibility of using these enzymes for large-scale in vitro synthesis of resorcinol has not yet been confirmed. Finally, the resorcinol synthase used in these studies was derived from *Escherichia coli* (…). E. coli ) or fluorescent pseudomonas ( P. fluorescens These enzymes are produced by bacteria. Therefore, when these enzymes are produced by eukaryotes (such as yeast, insects, or mammalian cells), their activity is unknown. However, eukaryotic systems may have advantages, especially for large-scale production. Other advantages of production in eukaryotes are their ability to modify proteins (including enzymes) at the post-translational level.

[0006] Phloroglucinol synthase was also identified in actinomycetes (WO2019 / 002799) and ochrophyte algae (WO2019 / 002798). Among these, the actinomycete *Pneumocystis pulmonaria* (… Tsukamurella pulmonis Phld_Tpu (WT) was previously identified in Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae It is a good candidate for the production of phloroglucinol (WO2019 / 002799).

[0007] The PhlD_Tpu amino acid sequence (as shown in SEQ ID NO: 1) is 390 amino acids long (41 kDa) and is encoded by the gene SAMN04489765_2627 (protein accession number: WP_068567598 in the NCBI database). It is assumed that this enzyme is biologically active in a homodimer form, similar to other enzymes in the type III polyketide synthase (PKS) family to which PhlD_Tpu belongs. Figure 1 This enzyme is directly involved in the synthesis of phloroglucinol, which is achieved through the condensation of three malonyl-CoA (MLC) molecules.

[0008] Although Phld_Tpu is in brewer's yeast ( S. cerevisiaePhlo showed satisfactory production levels, but there is still a need to identify new phloroglucinol synthases with optimized activity suitable for large-scale industrial production, which can be produced by eukaryotic systems and have high phloroglucinol synthase activity in vitro or in vivo.

[0009] However, the catalytic sites associated with phloroglucinol synthase activity have not been described, and no conserved domains or motifs among phloroglucinol synthases have been identified, making the design of optimized phloroglucinol synthases particularly difficult. Summary of the Invention

[0010] In the context of this invention, the inventors have developed a novel phloroglucinol synthase with optimized activity, which is particularly suitable for large-scale industrial production.

[0011] The inventors devised an original and comprehensive mutagenesis strategy to identify mutant peptides with improved properties, including increased phloroglucinol synthase activity, from the phloroglucinol synthase (Phld_Tpu, wild type (WT)) of the actinomycete Pneumotsukumura.

[0012] Specifically, computational studies using state-of-the-art techniques were employed to predict the three-dimensional structure of its functional homodimeric form, Phld_Tpu, and to reveal the molecular determinants involved in substrate and product recognition. Using an innovative combination of computer-aided methods, multiple amino acid positions were predicted as mutagenic targets for improving the enzyme's catalytic activity and stability. Subsequently, multiple mutant libraries were constructed and screened to improve Phlo production.

[0013] This complementary approach strategy led to the identification of several simple mutants, as well as mutants with double or even multiple mutations, exhibiting significantly higher Phld activity (i.e., phloroglucinol production activity) compared to WT Phld_Tpu. In fact, the new phloroglucinol synthase mutants allowed for up to 90% higher phloroglucinol concentrations obtained under the same conditions than those obtained using wild-type Phld (i.e., using the same experimental setup, the inventors were able to obtain up to 190% of the phloroglucinol concentrations obtained using wild-type). The inventors unexpectedly identified several key amino acid residues, allowing for optimization of enzyme activity.

[0014] The inventors hereby demonstrate that these phloroglucinol synthase mutants are functional when produced in both prokaryotic and eukaryotic systems and are suitable for large-scale industrial production.

[0015] Therefore, the present invention relates to a mutant polypeptide comprising an amino acid sequence having at least 80% identity, preferably at least 85% identity, and more preferably at least 90% identity with the amino acid sequence of the *Pneumocystis tectorum* resorcinol synthase shown in SEQ ID NO: 1, having one or more substitutions of one or more amino acid residues selected from the following: a) Any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, any amino acid residue or combination of amino acid residues located at positions from position 268 to position 271 of SEQ ID NO: 1; or b) The amino acid residue located at position 49 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 49 of SEQ ID NO: 1; or c) The amino acid residue located at position 339 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 339 of SEQ ID NO: 1; or d) Amino acid residues located at positions 93 and 262 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 93 and 262 of SEQ ID NO: 1; or e) Amino acid residues located at positions 129 and 176 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 129 and 176 of SEQ ID NO: 1; and f) Any combination thereof.

[0016] The present invention also relates to nucleic acid molecules encoding these mutant polypeptides, particularly nucleic acid molecules encoding mutant phloroglucinol synthases obtained from wild-type phloroglucinol synthase (Phld_Tpu, SEQ ID NO: 1) of *Pneumocystis jirovecii*. More specifically, the present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding the mutant polypeptide of the present invention, preferably wherein: a) The nucleic acid molecule is isolated; or b) The nucleic acid molecule further contains a promoter that controls the expression of the nucleic acid sequence; or c) The isolated nucleic acid molecule further contains a transcription terminator that controls the expression of the nucleic acid sequence; or d) The nucleic acid sequence was further optimized for expression in host cells (particularly yeast or bacteria); or e) Any combination of a) to d).

[0017] The present invention also relates to a vector comprising at least one nucleic acid molecule encoding such a mutant polypeptide.

[0018] The present invention also relates to host cells that contain at least one nucleic acid molecule or at least one vector according to the present invention.

[0019] The present invention also relates to a method for producing functional phloroglucinol synthase.

[0020] The present invention also relates to methods and uses for producing phloroglucinol.

[0021] Detailed Description of the Invention In the context of this invention, the inventors have developed a novel phloroglucinol synthase (Phld) with optimized activity, which is particularly suitable for large-scale industrial production.

[0022] The inventors devised an original and comprehensive mutagenesis strategy to identify mutant peptides with improved properties, including increased phloroglucinol synthase activity, from the phloroglucinol synthase (Phld_Tpu, wild type (WT)) of the actinomycete Pneumotsukumura.

[0023] Specifically, state-of-the-art computational studies were conducted to predict the three-dimensional structure of its functional homodimeric form, Phld_Tpu, and to reveal the molecular determinants involved in substrate and product recognition. Using these original computer-aided strategies, multiple amino acid positions were predicted as mutagenic targets for improving the enzyme's catalytic activity and stability. Multiple mutant libraries were then constructed and screened to improve Phlo production.

[0024] This combination of complementary strategies led to the identification of several simple mutants, as well as mutants with double or even multiple mutations, exhibiting significantly higher Phlo yields compared to WT Phld_Tpu. In fact, the new phloroglucinol synthase mutants allowed for up to 90% higher phloroglucinol concentrations obtained under the same conditions than with wild-type Phld (i.e., using the same experimental setup, the inventors were able to obtain up to 190% of the phloroglucinol concentrations obtained with wild-type). The inventors unexpectedly identified several key amino acid residues, allowing for optimization of enzyme activity. The inventors here demonstrate that these mutant phloroglucinol synthases are functional when produced in both prokaryotic and eukaryotic systems and are suitable for large-scale industrial production.

[0025] definition Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry, biochemistry, microbiology, cell biology, molecular biology, and medical sciences.

[0026] As used throughout this text, the terms “a” and “an” are used in the sense that they mean “at least one,” “at least the first,” “one or more,” or “one or more” of the compound or step mentioned, unless the context otherwise indicates.

[0027] The term “and / or” as used anywhere in this document includes the meaning of “and”, “or”, and “all or any other combination of the elements connected by the term”.

[0028] As used herein, the terms “about” or “approximately” mean within 10% of a given value or range, preferably within 8%, more preferably within 5%, more preferably within 3%, and even more preferably within 1%.

[0029] As used herein, when used to define products, compositions, cells, uses, and methods, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are open-ended and do not exclude additional, unlisted elements or method steps. Thus, a polypeptide “comprising” is said amino acid sequence when the amino acid sequence may be part of the final (and / or complete) amino acid sequence of the polypeptide. Such polypeptides may have up to hundreds of additional amino acid residues (e.g., corresponding to linkers, tags, and any other parts as described herein). “Comprising” means excluding any other components or steps. "Mainly composed of..." means excluding other components or steps that have any substantial significance (however, it does not exclude other minor / non-substantial components or steps). In this disclosure, the terms "comprising," "composed of," and "mainly composed of..." may be used interchangeably if necessary.

[0030] The term “type III polyketide synthase” in this article refers to a multifunctional enzyme or enzymatic complex that produces polyketide compounds without using an acyl carrier protein (or ACP) domain.

[0031] The term "polyketide" refers to a large family of secondary metabolites found in many eukaryotes and prokaryotes (including bacteria, fungi, algae, plants, and animals) that originate from the iterative condensation of acetyl or malonyl subunits under the action of polyketide synthases. Polyketides are also used as starting materials for the production of a variety of natural and semi-synthetic products.

[0032] The term "phloroglucinol" (abbreviated as "Phlo") in this article refers to the aromatic organic compound benzene-1,3,5-triol, which has the following chemical formula (Formula I) and CAS number 108-73-6: Formula I The term "phloroglucinol synthase" (abbreviated as PHLD or PhlD) refers to a multifunctional enzyme or enzymatic complex belonging to the type III polyketide synthase family that catalyzes the synthesis of phloroglucinol. Phloroglucinol synthase catalyzes the condensation of three malonyl-CoA molecules to form one phloroglucinol molecule.

[0033] As used herein, the term "enzyme" refers to a polypeptide that possesses catalytic properties. Enzymes function by lowering the activation energy of a chemical reaction, thereby increasing the rate of reaction. Enzymes are not altered during the reaction. The initial molecule is the enzyme's "substrate," and the molecules formed from these substrates are the "reaction products." Enzymes are highly specific. Furthermore, enzymes are reusable. Enzymes are typically globular proteins that function individually or in complexes composed of multiple enzymes or subunits. Like all proteins, enzymes consist of one or more polypeptide chains that fold to form a three-dimensional structure corresponding to their native state. Enzyme sizes can range from approximately 50 residues to over 2000 residues. Only a very small fraction of an enzyme (typically between two and four residues, sometimes more) is directly involved in catalysis; these constitute the so-called catalytic site. The catalytic site is typically located near one or more binding sites, where the substrate binds and is directed to catalyze the chemical reaction. The catalytic site and the binding site form the enzyme's active site.

[0034] The term "enzyme activity," "catalytic activity," or simply "activity" refers to the amount of product formed or substrate consumed per unit time (second, minute, or day) in a given environment (in moles, micromoles, or grams per liter). The expression "degree of conversion of an enzyme into a product" is intended herein to refer to the ratio between the amount of final product obtained and the initial amount of substrate for a given amount of enzyme. For example, for the purposes of this invention, enzyme activity can be expressed as the amount of phloroglucinol produced per unit time in a given volume (in g / L / time).

[0035] In this article, the term "bacteria" refers to tiny prokaryotes that exist in all media.

[0036] The term “Gram-positive bacterium” or “Gram-positive bacterium” in this article refers to bacteria that are Gram-positive (that is, bacteria that retain crystal violet (also known as crystal violet) and remain pale purple-purple or blue-dark purple).

[0037] The terms "actinomycetes" or "Gram-positive actinomycete bacterium" used in this article refer to bacteria belonging to the order Actinomycetes in the classification of actinomycetes. Actinomycetales Actinomycetes are bacteria that are Gram-positive. They grow to produce colonies composed of hyphae, that is, filamentous structures that radiate outwards from the microorganisms that produce them through centrifugal growth.

[0038] The term "species of the genus *Tsukamura*" in this article refers to... Tsukamurella "sp." refers to the genus *Tsukamura*. Tsukamurella Tsukamura is a rod-shaped, obligate aerobic, non-spore-forming actinomycetes. The genus *Tsukamura* specifically includes the species *Oligometabolous Tsukamura*. Tsukamurella paurometabola (hereinafter also referred to as Tp), Tyrosine Tsukamura ( Tsukamurella tyrosinosolvens (hereinafter also referred to as Tt), *Pseudomonas tsukumura* ( Tsukamurella pseudospumae (hereinafter also referred to as Tps), Pneumothorax ( Tsukamurella pulmonis (hereinafter also referred to as Tpu) and species 1534 of the genus Tsukamura ( Tsukamurella sp. 1534 (also referred to below as Tsp).

[0039] The term "Nocardia species" in this article Nocardia "sp." refers to the genus Nocardia (sp.) Nocardia Filamentous actinomycetes. The genus *Nocardia* specifically includes the species *Nocardia dermatophyte* (…). Nocardia farcinica (hereinafter also referred to as Nf).

[0040] The term "mycobacterial species" in this article Mycobacterium "sp." refers to the genus Mycobacterium ( Mycobacterium Mycobacteria are bacillus-shaped, non-spore-forming, aerobic actinomycetes. The genus *Mycobacterium* specifically includes the species *Mycobacterium marinei* (…). Mycobacterium marinum ), Mycobacterium Kansas ( Mycobacterium kansasii) and Mycobacterium tuberculosis ( Mycobacterium tuberculosis (Hereinafter referred to as Mma, Mk and Mt respectively).

[0041] The term "Gordonella species" in this article Gordonia "sp." refers to the genus *Goldenella* (*Goldenella*). Gordonia Actinobacteria. The genus *Gordonella* specifically includes the species *Gordonella hydrophobicum* (…). Gordonia hydrophobica (hereinafter also referred to as Gh).

[0042] The term "pseudomonas species" in this article Pseudomonas "sp." refers to the genus *Pseudomonas* (sp.). Pseudomonas Gram-negative bacteria that do not form spores (or are non-sporogenic), are bacilli in appearance, and are obligate aerobes. The genus *Pseudomonas* specifically includes the species *Pseudomonas fluorescens* (hereinafter also referred to as *Pf*).

[0043] The term "species of the genus *Fragaria*" in this article ( Ectocarpus "sp." refers to algae belonging to the family Phaeophyceae ( Ectocarpaceae The genus *Fragaria*, a type of brown algae ( ) Ectocarpus Algae. The genus *Flammulina* specifically includes the species *Flammulina floribunda* (…). Ectocarpus siliculosus ).

[0044] In this article, the term "PHLD.Pf" refers indiscriminately to the gene encoding PHLD phloroglucinol synthase in Pseudomonas fluorescens, or the polypeptide encoded by that gene.

[0045] The terms “PKS1.Es” or “PHLD.Es” are intended to refer indiscriminately to the gene encoding PKS1 phloroglucinol synthase in brown algae, or the polypeptide encoded by that gene.

[0046] The term “PhlD_Tpu” or “PHLD_Tpu” in this document refers to the wild-type, unmutated phloroglucinol synthase of *Pneumocystis jirovecii*, having the amino acid sequence shown in SEQ ID NO: 1.

[0047] The terms “obtained from,” “derived from,” “originating from,” or “having a source of XXX” (where XXX is any source) are used to identify the original source of a component (e.g., a polypeptide, a nucleic acid molecule) but are not intended to limit the method of manufacturing the component, which may be, for example, by chemical synthesis, mutation, and / or recombination (including replacing codons encoding amino acid residues).

[0048] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to any covalently linked amino acid polymer, regardless of its length or post-translational modifications. There is no limitation on the maximum number of amino acids contained in a polypeptide. As a general indication, these terms refer to both short polymers (commonly referred to in the art as peptides or protein fragments) and longer polymers (commonly referred to in the art as polypeptides or proteins). As a general indication and not limited thereto, if an amino acid polymer contains more than 50 amino acid residues, it is preferably referred to as a polypeptide or protein, while if the polymer consists of 50 or fewer amino acids, it is preferably referred to as a “peptide.”

[0049] A polypeptide can be the translation product of any polynucleotide, regardless of its size. Alternatively, a polypeptide can be any product of a chemical synthesis reaction.

[0050] The polymer can be linear, branched, or cyclic, with linear being preferred. The polymer can contain naturally occurring amino acids and / or amino acid analogs, and it can be interrupted by non-amino acid compounds.

[0051] Amino acids in a polypeptide are typically covalently linked by peptide bonds. Preferably, all chemical bonds in a polypeptide are peptide bonds. In some cases, a polypeptide may contain one or more chemical bonds that are not peptide bonds. The term "polypeptide" encompasses both natural and non-natural polypeptides, including derivatives, mutant polypeptides, engineered polypeptides, fusion polypeptides, etc. The term "polypeptide" also encompasses polypeptide fragments and polypeptide multimers (e.g., dimers), including homopolymers and heteropolymers. Polypeptides available herein may be further modified by chemical or enzymatic modifications. Such chemically and / or enzymatically modified polypeptides contain chemical groups in addition to those of the 20 naturally occurring amino acids. Examples of such chemical or enzymatic modifications include post-translational modifications, the addition of markers / tags, etc. Chemical or enzymatic modifications of a polypeptide can alter one or more properties of the polypeptide. For example, some modifications may alter stability, biological half-life, solubility (e.g., water solubility), activity, etc.

[0052] The polypeptide amino acid sequences used in this article are read and written in the conventional direction. The convention for reading and writing polypeptide amino acid sequences is to place the amino terminus on the left and then write and read the sequence from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) from left to right.

[0053] The terms “amino acid,” “residue,” and “amino acid residue” are used interchangeably to cover natural amino acids obtained by removing one molecule of water, as well as amino acid analogs (e.g., non-natural, synthetic, and modified amino acids, including D or L optical isomers). The term “amino acid” is used interchangeably to cover whole natural amino acids, as well as amino acid analogs (e.g., non-natural, synthetic, and modified amino acids, including D or L optical isomers).

[0054] The terms “peptide tag” or “tag peptide” or “peptide label” or “label peptide” refer to a short amino acid sequence (particularly 2 to 25 amino acids) fused to another polypeptide and used to detect the presence of that polypeptide, or to facilitate its purification or solubilization. Examples of such labeled peptides are described in Kimple et al. [Kimple ME et al. 2013] and are presented in Table 1 below.

[0055] Table 1: Examples of peptide tags As used herein, “post-translational modification” refers to a chemical or enzymatic modification, occurring naturally or unnaturally, on a protein or protein fragment after or accompanying translation (e.g., biological or biochemical protein synthesis, such as using cellular machinery) or after or accompanying protein synthesis (e.g., artificial and / or chemical synthesis). This means modifying at least one naturally occurring amino acid of a protein or protein fragment by adding at least one chemical group and / or modifying (including, but not limited to, removing) at least one chemical group of a naturally occurring amino acid. Examples of such chemical or enzymatic modifications include, but are not limited to, glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, esterification, amidation, ubiquitination, SUMOylation, deamination, etc. “Post-translational modified protein” as used herein refers to a protein having at least one (i.e., one or more) post-translational modifications. “Post-translational modified protein fragment” as used herein refers to a protein fragment having at least one (i.e., one or more) post-translational modifications.

[0056] The terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably herein and are understood to refer to polymers or oligomeric macromolecules composed of nucleotide monomers (preferably at least five nucleotide monomers, also referred to as nucleotide residues). A nucleotide monomer consists of a nucleobase, a pentose sugar (e.g., but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Nucleotide monomers may be chemically and / or enzymatically modified. Typically, polynucleotides are formed via phosphodiester bonds or thiophosphate bonds between individual nucleotide monomers. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids (mixed polyribose-polydeoxyribonucleotides). These terms encompass their single-stranded or double-stranded, straight-chain or circular, natural or synthetic, unmodified or modified versions (e.g., genetically modified polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, and chimeric mixtures (e.g., RNA-DNA hybrids). Furthermore, polynucleotides may contain nucleotides that are not naturally occurring and may be interrupted by non-nucleotide components. Exemplary DNA nucleic acids include, but are not limited to, complementary DNA (cDNA), genomic DNA, plasmid DNA, DNA vectors, viral DNA (e.g., viral genomes, viral vectors), oligonucleotides, probes, primers, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof. Exemplary RNA nucleic acids include, but are not limited to, messenger RNA (mRNA), pre-mRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), RNA vectors, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc. The polynucleotides described herein can be synthesized by standard methods known in the art, such as by using automated DNA synthesizers (e.g., those available from Biosearch, Applied Biosystems, etc.), or obtained from naturally occurring sources (e.g., genomes, cDNA, etc.) or artificial sources (e.g., commercially available libraries, plasmids, etc.) using molecular biology techniques well known in the art (e.g., cloning, PCR, etc.). Nucleic acids can be synthesized, for example, by chemical methods, such as the phosphate triester method (see, for example, Uhlmann, E. and Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0057] The term “vector” is intended to refer to a carrier, preferably a nucleic acid molecule or viral particle, which contains elements necessary to enable one or more nucleic acid molecules to be applied to, reproduce in and / or expressed in a host cell or organism.

[0058] From a functional perspective, the term encompasses maintenance vectors (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids), or integration vectors (e.g., designed to integrate into the genome of a host cell and produce additional copies of the nucleic acid molecules they contain during host cell replication). The term also encompasses shuttle vectors (e.g., vectors that function in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., vectors for transferring nucleic acid molecules into the genome of a host cell).

[0059] From a structural perspective, the vector according to the present invention can be a natural, synthetic or artificial genetic source, or a combination of natural and artificial genetic elements.

[0060] Therefore, in the context of this invention, the term "vector" should be interpreted broadly to include both plasmid vectors (or plasmids) and viral vectors.

[0061] As used herein, "plasmid" refers to a reproducible DNA construct. Typically, plasmid vectors contain selection marker genes that allow for recognition and / or positive or negative selection of the host cell carrying the plasmid in the presence of a compound corresponding to the selection marker. Various positive and negative selection marker genes are known in the art. For illustrative purposes, antibiotic resistance genes can be used as positive selection marker genes for selecting host cells in the presence of the corresponding antibiotic.

[0062] As used in this article, the term "viral vector" refers to a nucleic acid vector containing at least one element of a viral genome that can be packaged in a viral particle, or to a viral particle itself. Viral vectors can be replication-capable or selective (e.g., designed to replicate better or selectively in a particular host cell), or they can be genetically deactivated to make them defective or inadequate in terms of replication.

[0063] As used herein, the term "host cell" should be understood broadly and without any limitation regarding a specific tissue, organ, or isolated cell. The term "host cell" is intended to refer to a cell containing a polypeptide according to the invention, a nucleic acid molecule according to the invention, a vector according to the invention, or any mixture thereof. Advantageously, the host cell is capable of expressing a polypeptide having phloroglucinol synthase activity and / or capable of producing the vector according to the invention. Again, advantageously, the host cell is capable of synthesizing phloroglucinol.

[0064] The host cell can be a productive cell, i.e., a cell capable of expressing one or more nucleic acid molecules (including one or more genes) encoded by the vector according to the invention and / or capable of producing the vector according to the invention (i.e., a cell expressing one or more nucleic acid molecules (including one or more genes) encoded by the vector according to the invention and / or producing the vector according to the invention). The term also includes cells expressing polypeptides of the invention, as well as cells that can serve or have served as receptors for nucleic acid molecules encoding polypeptides of the invention (or vectors containing said nucleic acid molecules), and progeny of such cells. The term "host cell" more broadly includes cells containing or formerly containing nucleic acid molecules according to the invention, and progeny of such cells.

[0065] A host cell can consist of a single type of cell or a group of different types of cells. A host cell can also be a hybrid cell, i.e., formed by the fusion of at least two different types of cells. A host cell can be, for example, isolated, or organized within a tissue, organ, or even a whole organism. In the case where the host cell is located within a whole organism, the organism is not a human being.

[0066] Host cells can belong to cultured cell lines, primary cells, stem cells, proliferating cells, or dividing cells. The term "host cell" includes prokaryotic cells, lower eukaryotic cells (e.g., yeast cells), and other eukaryotic cells (e.g., archaea, fungi, insects, plants, algae, microalgae, parasites, animals, and mammalian cells (e.g., human or non-human, preferably non-human)). Host cells can be differentiated cells, pluripotent cells, totipotent cells, stem cells, induced pluripotent stem cells (iPSCs), induced totipotent stem cells, or even embryonic cells or embryonic stem cells. In the case of embryonic cells or embryonic stem cells, the cell is a non-human cell.

[0067] Therefore, it is clear that the "host cell" according to the present invention is a recombinant host cell, that is, a cell that contains exogenous genetic material. Thus, the host cell is not a naturally occurring cell, but a molecular biology tool obtained through gene manipulation techniques.

[0068] The term "isolated molecule" is intended to refer to molecules, in particular proteins, polypeptides, peptides, nucleic acid molecules, plasmid vectors, viral vectors, or host cells, which are extracted from their natural environment (that is, isolated from at least one other component naturally associated with them).

[0069] As used herein, “identity” or “sequence identity” refers to an exact sequence match between two polypeptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The “identity percentage” mentioned in the context of this invention is determined after optimal alignment of the sequences to be compared, and thus optimal global alignment may include one or more insertions, deletions, truncations, and / or substitutions. An alignment is “global”, meaning it includes sequences compared as a whole over their entire length. An alignment is “optimal”, meaning the number of insertions, deletions, truncations, and / or substitutions is as small as possible. Optimal global alignment and identity percentage calculation can be performed using any sequence analysis method well known to those skilled in the art. In addition to manual comparison, global alignment can be determined using the Needleman and Wunsch algorithm (A general method applicable to the search for similarities in the amino acid sequence of two proteins, J. Mol. Biol., 1970, Mar;48(3):443-453). Optimal global alignment and identity percentage calculation can be performed using any software well known to those skilled in the art (e.g., Emboss Needle software). For example, the Emboss Needle software is available on the website ebi.ac.uk under the name "Align". This software reads two input sequences and writes their best global sequence alignment to a file. It uses the Needleman-Wunsch alignment algorithm to find the best alignment (including gaps) of the two sequences along their entire length. This algorithm uses a dynamic programming approach to ensure the alignment is optimal by exploring all possible alignments and selecting the best. A scoring matrix is ​​read, containing the value for each possible residue or nucleotide match. The Emboss Needle software finds the alignment with the highest possible score, where the score of the alignment is equal to the sum of matches obtained from the scoring matrix, minus the penalty points for open and extended gaps in the aligned sequence. The substitution matrix and the penalty points for open and extended gaps are specified by the user. In the case of this invention, the Emboss Needle software with default parameters can be used to obtain the best global alignment. For nucleotide sequences, the parameters used can be, in particular, the following: “Gap open” equals 10.0, “Gap extend” equals 0.5, and the EDNAFULL matrix (NCBI EMBOSS version NUC4.4).For the amino acid sequence, the parameters used can be, in particular, the following: "Gap open" equals 10.0, "Gap extend" equals 0.5, and the BLOSUM62 matrix; or the parameters used can be: "Gap open" equals 10.0, "Gap extend" equals 0.5, "End gap penalty" is "false", "End gap open" is 10.0, and the "Blosum 62" matrix.

[0070] When two (or more) amino acid sequences or two (or more) nucleotide sequences are input, the EmbossNeedle software returns the best global alignment, along with several values ​​characterizing the alignment: • “Identity” is the identity percentage, that is, the percentage of identical matches between two sequences in the reported alignment region (including any gaps in the length). • “Similarity” is a similarity percentage, which takes into account the percentage of identical matches between two sequences in the reported alignment region (including any gaps in the length) and the percentage of conservative substitutions between the two.

[0071] • “Score” is the total score of the alignment, corresponding to the best score obtained by the software for the global alignment of all tests (i.e., over the entire length of the two sequences).

[0072] This score can be called a "similarity score" because the higher the score between two (or more) sequences being compared, the higher the similarity between the two (or more) sequences. This takes into account not only the same amino acids being compared, but also conserved substitutions.

[0073] The score can be calculated using any software well-known to those skilled in the art (such as Emboss Needle software), or it can be calculated manually.

[0074] Preferably, the identity percentage defined as in the case of this invention is determined by a global alignment of sequences compared over their entire length. For illustrative purposes, “at least 80% identity” herein means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0075] In this paper, "identical match" between two (or more) residues (e.g., amino acid or nucleic acid residues) means that the two residues are identical.

[0076] A peptide or nucleic acid molecule “mutant” is defined herein as a peptide or nucleic acid molecule containing one or more mutations relative to a reference amino acid or nucleic acid sequence. Mutations may include substitution (replacing one amino acid or nucleic acid with another), deletion (omitting one or more amino acids or nucleic acids at one end or inside the reference sequence), and / or insertion (adding one or more amino acids or nucleic acids at one end or inside the reference sequence).

[0077] A mutant of the reference peptide preferably retains the activity of the reference peptide. The reference peptide can be, for example, a wild-type peptide. In the case of an enzyme, the mutant preferably retains the enzymatic activity of the reference enzyme.

[0078] In this article, "silent mutation" or "silent substitution" refers to the replacement of a nucleotide residue (also called a "original nucleotide") with a different nucleotide residue in the protein-coding region of a nucleic acid molecule. This does not affect the amino acid sequence encoded by the nucleic acid molecule. A silent mutation results in a change of one letter in the triplet code representing a codon, but despite the single-base change, the encoded amino acid remains unchanged. This is allowed by the degeneracy of the genetic code. Table 2 below shows an example of the standard genetic code, illustrating the codons encoding each standard amino acid residue.

[0079] Table 2: Standard genetic code In this article, "conservative substitution" or "conservative mutation" refers to replacing amino acid residues in a polypeptide (e.g., a protein) with different amino acid residues that have similar physicochemical properties (molar mass, charge, functional groups, hydrophobicity, etc.) (also known as "original amino acids"). Table 3 below shows examples of equivalent amino acids that can be used for conservative substitution.

[0080] Table 3: Examples of amino acids with equivalent physicochemical properties "Functional mutants / derivatives of peptides" refer to any peptide, peptide, or protein fragment derived from a peptide, peptide, or protein fragment that has at least one original function of the peptide, peptide, or protein from which it originates. Preferably, the functional mutant / derivative performs the function at a power equal to at least 30% of the power of the peptide, peptide, or molecule, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, preferably at least 100%. Examples of functional mutants / derivatives of peptides include, for example, mutated proteins or peptides (including proteins or peptides mutated by substitution, deletion, insertion, fusion, or any combination thereof), protein domains (e.g., protease domains), protein epitopes, etc. Substitution includes replacing at least one amino acid residue with at least one different amino acid residue, preferably via one or more exogenous proteins, one or more exogenous protein fragments, or one or more exogenous polypeptides (i.e., wherein the protein, protein fragment, or polypeptide is derived from another species). Insertion includes inserting (adding) at least one amino acid residue, preferably inserting one or more exogenous proteins, one or more exogenous protein fragments, or one or more exogenous polypeptides (i.e., wherein the protein, protein fragment, or polypeptide is derived from another species). Fusion includes, with or without a linker sequence, covalently binding at least one amino acid residue, preferably covalently binding one or more exogenous proteins, one or more exogenous protein fragments, or one or more exogenous polypeptides (i.e., wherein the protein, protein fragment, or polypeptide is derived from another species).

[0081] Substitution, insertion, deletion, and fusion can be generated in a variety of ways known to those skilled in the art, such as site-directed mutagenesis, PCR mutagenesis, DNA shuffling, chemical synthesis techniques (e.g., resulting in synthetic nucleic acid molecules encoding engineered proteins) and / or the use of restriction sites.

[0082] The functional mutants / derivatives available in this article can be further modified by chemical or enzymatic modifications (e.g., post-translational modifications). Chemically / enzymatically modified polypeptides, peptides, or protein fragments contain chemical groups other than the 20 naturally occurring amino acids (e.g., may contain post-translational modifications and / or non-naturally occurring amino acids).

[0083] In the following detailed description, the implementation schemes may be used individually or in appropriate combinations by those skilled in the art.

[0084] Mutant peptides In the context of this invention, the inventors have developed a novel phloroglucinol synthase (Phld) with optimized activity, which is particularly suitable for large-scale industrial production.

[0085] The inventors devised an original and comprehensive mutagenesis strategy to identify mutant peptides with improved properties, including increased phloroglucinol synthase activity and increased substrate affinity, from the phloroglucinol synthase (Phld_Tpu, wild type (WT)) of the actinomycete Pneumotsukumura.

[0086] Specifically, state-of-the-art computational studies were conducted to predict the three-dimensional structure of its functional homodimeric form, Phld_Tpu, and to reveal the molecular determinants involved in substrate and product recognition. Using these original computer-aided strategies, multiple amino acid positions were predicted as mutagenic targets for improving the enzyme's catalytic activity and stability. Multiple mutant libraries were then constructed and screened to improve Phlo production.

[0087] This combination of complementary strategies led to the identification of several simple mutants, as well as mutants with double or even multiple mutations, exhibiting significantly higher Phlo activities than WT Phld_Tpu. In fact, the novel phloroglucinol synthase mutants allowed for phloroglucinol concentrations up to 90% higher than those obtained using wild-type Phld (i.e., the inventors were able to obtain up to 190% of the phloroglucinol concentrations obtained using wild-type). The inventors unexpectedly identified several key amino acid residues, allowing for optimization of enzyme activity.

[0088] The inventors hereby demonstrate that these mutant phloroglucinol synthases are functional when produced in both prokaryotic and eukaryotic systems and are suitable for large-scale industrial production.

[0089] Therefore, the present invention relates to a mutant polypeptide comprising, primarily composed of, or composed of the amino acid sequence of phloroglucinol synthase, said amino acid sequence having at least 80% identity with the amino acid sequence of *Pneumocystis tectoris* phloroglucinol synthase described in SEQ ID NO: 1, preferably having at least 81% identity with the amino acid sequence of *Pneumocystis tectoris* phloroglucinol synthase described in SEQ ID NO: 1, and with SEQ ID NO: 1. Compared to the amino acid sequence of *Pneumocystis tumefaciens* phloroglucinol synthase described in NO:1, an amino acid sequence of phloroglucinol synthase with at least 82% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 83% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 84% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 85% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 86% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 87% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 88% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 89% identity is preferred, and an amino acid sequence of phloroglucinol synthase with at least 90% identity is preferred. The amino acid sequence of the resorcinol synthase is preferably an amino acid sequence of a resorcinol synthase having at least 91% identity, preferably an amino acid sequence of a resorcinol synthase having at least 92% identity, preferably an amino acid sequence of a resorcinol synthase having at least 93% identity, preferably an amino acid sequence of a resorcinol synthase having at least 94% identity, preferably an amino acid sequence of a resorcinol synthase having at least 95% identity, preferably an amino acid sequence of a resorcinol synthase having at least 96% identity, preferably an amino acid sequence of a resorcinol synthase having at least 97% identity, preferably an amino acid sequence of a resorcinol synthase having at least 98% identity, preferably an amino acid sequence of a resorcinol synthase having at least 99% identity, and even more preferably an amino acid sequence of a resorcinol synthase having the amino acid sequence of the *Pneumocystis tectoris* resorcinol synthase as described in SEQ ID NO: 1. The amino acid sequence of the phloroglucinol synthase has one or more substitutions selected from one or more of the following amino acid residues: a) Any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, any amino acid residue or combination of amino acid residues located at positions from position 268 to position 271 of SEQ ID NO: 1; or b) The amino acid residue located at position 49 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 49 of SEQ ID NO: 1; or c) The amino acid residue located at position 339 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 339 of SEQ ID NO: 1; or d) Amino acid residues located at positions 93 and 262 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 93 and 262 of SEQ ID NO: 1; or e) Amino acid residues located at positions 129 and 176 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 129 and 176 of SEQ ID NO: 1; and f) Any combination thereof.

[0090] Preferably, the present invention may relate to a mutant polypeptide comprising, primarily composed of, or composed of the amino acid sequence of phloroglucinol synthase, said amino acid sequence having at least 80% identity with the amino acid sequence of *Pneumocystis tectoris* phloroglucinol synthase described in SEQ ID NO: 1, preferably having at least 81% identity with the amino acid sequence of *Pneumocystis tectoris* phloroglucinol synthase described in SEQ ID NO: 1, and with SEQ ID NO: 1. Compared to the amino acid sequence of *Pneumocystis tumefaciens* phloroglucinol synthase described in NO:1, an amino acid sequence of phloroglucinol synthase with at least 82% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 83% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 84% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 85% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 86% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 87% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 88% identity is preferred, an amino acid sequence of phloroglucinol synthase with at least 89% identity is preferred, and an amino acid sequence of phloroglucinol synthase with at least 90% identity is preferred. The amino acid sequence of the resorcinol synthase is preferably an amino acid sequence of a resorcinol synthase having at least 91% identity, preferably an amino acid sequence of a resorcinol synthase having at least 92% identity, preferably an amino acid sequence of a resorcinol synthase having at least 93% identity, preferably an amino acid sequence of a resorcinol synthase having at least 94% identity, preferably an amino acid sequence of a resorcinol synthase having at least 95% identity, preferably an amino acid sequence of a resorcinol synthase having at least 96% identity, preferably an amino acid sequence of a resorcinol synthase having at least 97% identity, preferably an amino acid sequence of a resorcinol synthase having at least 98% identity, preferably an amino acid sequence of a resorcinol synthase having at least 99% identity, and even more preferably an amino acid sequence of a resorcinol synthase having the amino acid sequence of the *Pneumocystis tectoris* resorcinol synthase as described in SEQ ID NO: 1. The amino acid sequence of the phloroglucinol synthase has one or more substitutions selected from one or more of the following amino acid residues: - Any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1; or - Any amino acid residue or combination of amino acid residues located at positions from position 268 to position 271 of SEQ ID NO:1, after optimal global alignment with SEQ ID NO:1.

[0091] As detailed in the section titled "Definitions" above, the percentage of identity mentioned in the context of this invention is determined based on the best global alignment of the sequences to be compared (as defined above).

[0092] Once the best global alignment is achieved between SEQ ID NO:1 and another sequence (also referred to as "the other sequence" or "the sequence aligned with SEQ ID NO:1"), the percentage of identity and the position corresponding to a specific position in SEQ ID NO:1 can be defined. Specifically, the position corresponding to the Xth position in SEQ ID NO:1 is the position in the other sequence that is aligned with the Xth position in SEQ ID NO:1.

[0093] The score (which may be referred to as the "similarity score" as explained in the "Definitions" section above) can also be determined after a best global alignment of SEQ ID NO:1 with another sequence (or "the sequence aligned with SEQ ID NO:1"), as explained in the "Definitions" section above. The higher the score between SEQ ID NO:1 and another sequence, the higher the similarity between that other sequence and SEQ ID NO:1, taking into account not only the aligned identical amino acids but also conserved substitutions.

[0094] In a preferred embodiment, the mutant polypeptide comprises one or more substitutions of any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1, or, after optimal global alignment with SEQ ID NO: 1, one or more substitutions of any amino acid residue or combination of amino acid residues located at positions corresponding to positions 268 to 271 of SEQ ID NO: 1. Indeed, data obtained by the inventors indicate that mutants with substitutions contained in this region exhibit particularly high Phlo activity.

[0095] Advantageously, the mutant polypeptide comprises: a) Substitution of the glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or substitution of an amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, preferably wherein the glutamic acid residue at position 271 of SEQ ID NO: 1, or substitution of an amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is located at position 271 of SEQ ID NO: 1. The amino acid residue at position 271 of NO:1 is replaced by an amino acid residue selected from arginine (R), histidine (H), lysine (K), glutamine (Q), valine (V), methionine (M), phenylalanine (F), leucine (L), tyrosine (Y), and proline (P); preferably replaced by an amino acid residue selected from arginine (R), histidine (H), lysine (K), valine (V), methionine (M), and phenylalanine (F); more preferably replaced by an amino acid residue selected from arginine (R), lysine (K), valine (V), and methionine (M); even more preferably replaced by an amino acid residue selected from arginine (R), lysine (K), and valine (V); even more preferably replaced by an amino acid residue selected from arginine (R) and lysine (K); b) Substitution of the glutamic acid (E) residues at positions 268 and 271 of SEQ ID NO: 1, or substitution of the amino acid residues at positions corresponding to positions 268 and 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, preferably wherein: - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; or c) Substitution of amino acid residues at positions 124, 154, 177, 236, 268, 271, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 124, 154, 177, 236, 268, 271, and 298 of SEQ ID NO: 1, preferably wherein: - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by alanine (A); and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or d) Substitution of amino acid residues at positions 124, 154, 177, 236, 268, 270, 271, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 124, 154, 177, 236, 268, 270, 271, and 298 of SEQ ID NO: 1, preferably wherein: - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by alanine (A); and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 270 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or e) Substitution of amino acid residues at positions 35, 60, 91, 123, 124, 154, 177, 236, 270, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 35, 60, 91, 123, 124, 154, 177, 236, 270, and 298 of SEQ ID NO: 1, preferably wherein: - The serine (S) residue at position 35 of SEQ ID NO: 1, or the amino acid residue at position 35 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a glycine (G) residue; and - The alanine (A) residue at position 60 of SEQ ID NO: 1, or the amino acid residue at position 60 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 91 of SEQ ID NO: 1, or the amino acid residue at position 91 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 123 of SEQ ID NO: 1, or the amino acid residue at position 123 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 270 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or f) Substitution of amino acid residues at positions 35, 46, 60, 91, 123, 124, 154, 177, 191, 236, 238, 270, 290, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 35, 46, 60, 91, 123, 124, 154, 177, 191, 236, 238, 270, 290, and 298 of SEQ ID NO: 1, preferably wherein: - The serine (S) residue at position 35 of SEQ ID NO: 1, or the amino acid residue at position 35 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a glycine (G) residue; and - The serine (S) residue at position 46 of SEQ ID NO: 1, or the amino acid residue at position 46 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The alanine (A) residue at position 60 of SEQ ID NO: 1, or the amino acid residue at position 60 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 91 of SEQ ID NO: 1, or the amino acid residue at position 91 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 123 of SEQ ID NO: 1, or the amino acid residue at position 123 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The methionine (M) residue at position 191 of SEQ ID NO: 1, or the amino acid residue at position 191 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a valine (V) residue; and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamine (Q) residue at position 238 of SEQ ID NO: 1, or the amino acid residue at position 238 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a lysine (K) residue; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The aspartic acid (D) residue at position 290 of SEQ ID NO: 1, or the amino acid at position 290 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The alanine (A) residue at position 298 of SEQ ID NO:1, or the amino acid residue at position 298 of SEQ ID NO:1 after optimal global alignment with SEQ ID NO:1, is replaced by an aspartic (D) residue.

[0096] The mutant polypeptide preferably comprises, is mainly composed of, or is composed of the amino acid sequence defined in SEQ ID NO: 2: in: a) Amino acid residue X 271 It is any amino acid residue selected from arginine (R), histidine (H), lysine (K), glutamine (Q), valine (V), methionine (M), phenylalanine (F), leucine (L), tyrosine (Y), and proline (P); preferably amino acid residue X. 271 Selected from arginine (R), histidine (H), lysine (K), valine (V), methionine (M), and phenylalanine (F); more preferably, amino acid residue X. 271 Selected from arginine (R), lysine (K), valine (V), and methionine (M), or even more preferably, amino acid residue X. 271 Selected from arginine (R), lysine (K), and valine (V); and b) Amino acid residue X 35 X 46 X 49 X 60 X 91 X 93 X 123 X 124 X 129 X 154 X 176 X 177 X 191 X 236 X 238 X 262 X 268 X 270 X 290 X 298 and X 339 Independently selected from any amino acid; Among them, the following are preferred: i. Amino acid residue X35 It is an amino acid residue S or G, preferably G; ii. Amino acid residue X 46 It is an amino acid residue S or A, preferably A; iii. Amino acid residue X 49 It is any amino acid residue selected from R, A, and S, preferably X. 49 It can be any amino acid residue A or S; iv. Amino acid residue X 60 It is amino acid residue A or R, preferably R; v. Amino acid residue X 91 It is amino acid residue A or R, preferably R; vi. Amino acid residue X 93 It is an amino acid residue P or C, preferably C; vii. Amino acid residue X 123 It is amino acid residue A or S, preferably S; viii. Amino acid residue X 124 It is amino acid residue A or P, preferably P; ix. Amino acid residue X 129 It is amino acid residue Q or M, preferably M; x. Amino acid residue X 154 It is amino acid residue A or P, preferably P; xi. Amino acid residue X 176 It is any amino acid residue selected from V, I, and L; preferably X. 176 It can be L or I, preferably I; xii. Amino acid residue X 177 It is an amino acid residue S or A, preferably A; xiii. Amino acid residue X 191 It is an amino acid residue M or V, preferably V; xiv. Amino acid residue X 236 It is an amino acid residue S or P, preferably P; xv. amino acid residue X 238 It is amino acid residue Q or K, preferably K; xvi. Amino acid residue X 262 It is amino acid residue A or C, preferably C; xvii. Amino acid residue X 268 It is any amino acid residue selected from E, Q, T, S, C, G, K, M, and R; preferably X. 268 It is any amino acid residue selected from Q, T, S, C, G, K, M, and R; preferably X. 268 It is any amino acid residue selected from Q, T, and S; xviii. Amino acid residue X 270 It is amino acid residue A or S, preferably S; xix. Amino acid residue X 290 It is amino acid residue D or A, preferably A; xx. Amino acid residue X 298 It is amino acid residue A or D, preferably D; xxi. Amino acid residue X 339 It is any amino acid residue selected from M, I, Q, and T; preferably X. 339 It is any amino acid residue selected from I, Q, and T, preferably X. 339 For I; or xxii. Any combination thereof.

[0097] In a preferred embodiment, the mutant polypeptide comprises, is mainly composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 1, having one or more substitutions selected from the following (having one substitution or a group of substitutions): 1a. A124P, A154P, S177A, S236P, E268Q, E271R and A298D (mutant peptide M27); 2a. A124P, A154P, S177A, S236P, E268T, E271K and A298D (mutant peptide M28); 3a. A124P, A154P, S177A, S236P, E268S, A270S, E271K and A298D; 4a. A124P, A154P, S177A, S236P, E268Q, E271K and A298D; 5a. A124P, A154P, S177A, S236P, E268S, E271R and A298D; 6a. E268Q and E271K; 7a. E268T and E271K; 8a. E268T and E271R; 9a. A124P, A154P, S177A, S236P, E268Q, A270S, E271K and A298D; 10a. A124P, A154P, S177A, S236P, E268T, A270S, E271R and A298D; 11a. A124P, A154P, S177A, S236P, E268T, A270S, E271V and A298D; 12a. A124P, A154P, S177A, S236P, E268S, E271K and A298D; 13a. Q129M and V176I; 14a. E268Q and E271R (mutant peptide M29); 15a. A124P, A154P, S177A, S236P, E268Q, A270S, E271R and A298D; 16a. A124P, A154P, S177A, S236P, E268S, A270S, E271R and A298D; 17a. E271K; 18a. S35G, A60R, A91R, A123S, A124P, A154P, S177A, S236P, A270S and A298D (mutant peptide M22); 19a. E268S and E271K; 20a. P93C and A262C; 21a. E268T and E271V; 22a. A124P, A154P, S177A, S236P, E268T, A270S, E271K and A298D; 23a. E271R; 24a. S35G, S46A, A60R, A91R, A123S, A124P, A154P, S177A, M191V, S236P, Q238K, A270S, D290A and A298D (mutant peptide M23); 25a. R49A; and 26a. M339I.

[0098] In a preferred embodiment, the mutant polypeptide comprises, is mainly composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 1, having one or more substitutions selected from the following (having one substitution or a group of substitutions): 1b. A124P, A154P, S177A, S236P, E268Q, E271R and A298D (mutant peptide M27); 2b. A124P, A154P, S177A, S236P, E268T, E271K and A298D (mutant peptide M28); 3b. A124P, A154P, S177A, S236P, E268S, A270S, E271K and A298D; 4b. A124P, A154P, S177A, S236P, E268Q, E271K and A298D; 5b. A124P, A154P, S177A, S236P, E268S, E271R and A298D; 6b. E268Q and E271K; 7b. E268T and E271K; 8b. E268T and E271R; 9b. A124P, A154P, S177A, S236P, E268Q, A270S, E271K and A298D; 10b. A124P, A154P, S177A, S236P, E268T, A270S, E271R and A298D; 11b. A124P, A154P, S177A, S236P, E268T, A270S, E271V and A298D; 12b. A124P, A154P, S177A, S236P, E268S, E271K and A298D; 13b. E268Q and E271R (mutant peptide M29); 14b. A124P, A154P, S177A, S236P, E268Q, A270S, E271R and A298D; 15b. A124P, A154P, S177A, S236P, E268S, A270S, E271R and A298D; 16b. E271K; 17b. S35G, A60R, A91R, A123S, A124P, A154P, S177A, S236P, A270S and A298D (mutant peptide M22); 18b. E268S and E271K; 19b. E268T and E271V; 20b. A124P, A154P, S177A, S236P, E268T, A270S, E271K and A298D; 21b. E271R; 22b. S35G, S46A, A60R, A91R, A123S, A124P, A154P, S177A, M191V, S236P, Q238K, A270S, D290A and A298D (mutant peptide M23).

[0099] In fact, the inventors have shown that these mutants have been specially optimized to have improved properties, including increased phloroglucinol synthase activity.

[0100] Advantageously, the mutant polypeptide comprises, is mainly composed of, or is composed of amino acid sequences selected from SEQ ID NO: 3-28.

[0101] According to one embodiment, the mutant polypeptide comprises, is mainly composed of, or is composed of at least one amino acid sequence, said amino acid sequence preferably having at least 60% identity with an amino acid sequence selected from SEQ ID NO: 3-28, more preferably at least 65% identity, more preferably at least 70% identity, even more preferably at least 75% identity, even more preferably at least 80% identity, even more preferably at least 85% identity, even more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity.

[0102] The mutant peptide preferably has higher Phld activity than the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1). In an advantageous embodiment, the mutant peptide allows for the acquisition of a phloroglucinol concentration at least 10% higher than that obtained using the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) under the same conditions. In a more advantageous embodiment, the mutant peptide allows for the acquisition of a phloroglucinol concentration that is at least 15% higher (preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and even more preferably at least 100%) higher than that obtained using the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1).

[0103] In other words, the mutant peptide advantageously allows for the acquisition of a phloroglucinol concentration of at least 110% of that obtained with the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) using the same experimental setup. Preferably, the mutant peptide allows for the acquisition of a phloroglucinol concentration of at least 115% (preferably at least 120%, preferably at least 125%, preferably at least 130%, preferably at least 135%, preferably at least 140%, preferably at least 145%, preferably at least 150%, preferably at least 155%, preferably at least 160%, preferably at least 165%, at least 170%, preferably at least 175%, preferably at least 180%, preferably at least 185%, preferably at least 190%, preferably at least 195%, and even more preferably at least 200%) of that obtained with the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) using the same experimental setup.

[0104] Therefore, in the same experimental setup, the mutant peptide is preferably synthesized at an efficiency of at least 110% (preferably at least 115%, preferably at least 120%, preferably at least 125%, preferably at least 130%, preferably at least 135%, preferably at least 140%, preferably at least 145%, preferably at least 150%, preferably at least 155%, preferably at least 160%, preferably at least 165%, at least 170%, preferably at least 175%, preferably at least 180%, preferably at least 185%, preferably at least 190%, preferably at least 195%, even more preferably at least 200%) of that of the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1).

[0105] In some embodiments, the mutant polypeptide further comprises one or more additional mutations, such as mutations selected from substitution, deletion, and insertion; preferably, the one or more additional mutations are conserved mutations, particularly when the one or more additional mutations are substitutions. The nature of the introduced mutation does indeed affect the activity of the resulting mutant, and it is well known that conserved substitutions are less likely to alter the enzyme activity of the mutant compared to non-conserved substitutions.

[0106] Preferably, when the mutant peptide further contains one or more additional mutations, the mutant peptide at least partially retains Phlo activity. Therefore, in the same experimental setup, the mutant peptide further containing one or more additional mutations is preferably used for Phlo synthesis at an efficiency of at least 100% (preferably at least 101%, preferably at least 102%, preferably at least 103%, preferably at least 104%, more preferably at least 105%, more preferably at least 110%, more preferably at least 115%, more preferably at least 120%) that of the wild-type peptide (i.e., the unmutated peptide). In other words, the mutant peptide further containing one or more additional mutations allows for obtaining a phloroglucinol concentration at least equal to that obtained using the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) under the same conditions. In a more advantageous embodiment, the mutant peptide further comprising one or more additional mutations allows for obtaining a phloroglucinol concentration at least 1% (preferably at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 10%, preferably at least 15%, more preferably at least 20%) higher than that obtained using the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) under the same conditions (e.g., using the same experimental setup). Therefore, the mutant peptide further comprising one or more additional mutations advantageously allows for obtaining a phloroglucinol concentration at least 100% (preferably at least 101%, preferably at least 102%, preferably at least 103%, preferably at least 104%, more preferably at least 105%, more preferably at least 110%, more preferably at least 115%, more preferably at least 120%) higher than that obtained using the wild-type peptide (i.e., the unmutated peptide, preferably the peptide of SEQ ID NO: 1) under the same experimental setup.

[0107] Mutant polypeptides may advantageously further include the insertion of a single amino acid residue, a peptide, or a polypeptide, and any combination thereof.

[0108] Therefore, the present invention also relates to a fusion polypeptide comprising, primarily composed of, or composed of the mutant polypeptide of the present invention, and additional peptides, polypeptides, or proteins. The additional peptides, polypeptides, or proteins may be inserted into: a) At the N-terminus of the mutant polypeptide (i.e., at the N-terminal position), and / or b) At the C-terminus of the mutant polypeptide (i.e., at the C-terminal position), and / or c) Between any two consecutive amino acid residues of the mutant polypeptide.

[0109] The additional peptide, polypeptide, or protein can have various functions. One useful function is to allow the identification and / or purification of the fusion. Indeed, it is well known that adding a tagged peptide or polypeptide that makes the mutant easier to detect or purify generally does not alter enzyme activity, especially when the tagged peptide or polypeptide is inserted at the N-terminus or C-terminus (rather than inside the sequence).

[0110] When an additional peptide, polypeptide, or protein is inserted into the N-terminus and / or C-terminus of a mutant polypeptide, it can be fused directly to the N-terminal and / or C-terminal endpoints of the mutant polypeptide, or separated from the N-terminal and / or C-terminal endpoints of the mutant polypeptide by a linker peptide.

[0111] Typically, the linker is a peptide of 1 to 30 amino acids in length, composed of amino acid residues such as glycine (G), serine (S), threonine (T), asparagine (N), glutamine (Q), alanine (A), proline (P), and / or phenylalanine (F). In the case of this invention, the preferred linker contains 2 to 15 amino acids, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids.

[0112] Nucleic acid molecules The present invention also relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a mutant polypeptide according to the present invention, preferably wherein: a) Nucleic acid molecules are isolated; or b) The nucleic acid molecule further contains a promoter that controls the expression of the nucleic acid sequence; or c) The isolated nucleic acid molecule further contains a transcription terminator that controls the expression of the nucleic acid sequence; or d) The nucleic acid sequence is further optimized for expression in host cells (particularly in yeast or bacteria); or e) Any combination of a) to d).

[0113] Advantageously, a mutant polypeptide is any mutant polypeptide defined in the above-mentioned "Mutant Polypeptides" section.

[0114] When introduced or transfected (directly or in a vector) into host cells cultured in the presence of a suitable substrate, such nucleic acid molecules can be used to produce mutant peptides as defined above, or to directly produce phloroglucinol.

[0115] Advantageously, the nucleic acid molecule comprises, is mainly composed of, or is composed of nucleic acid sequences selected from SEQ ID NO: 46-72.

[0116] According to one embodiment, the nucleic acid molecule comprises, is mainly composed of, or is composed of at least one nucleic acid sequence, said nucleic acid sequence preferably having at least 60% identity with nucleic acid sequences selected from SEQ ID NO: 46-72, more preferably at least 65% identity, more preferably at least 70% identity, even more preferably at least 75% identity, even more preferably at least 80% identity, even more preferably at least 85% identity, even more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity.

[0117] In some embodiments, the nucleic acid molecule contains one or more additional mutations, such as mutations selected from substitution, deletion, and insertion; preferably, the one or more additional mutations are silent mutations, particularly when the one or more additional mutations are substitutions. The nature of the introduced mutation does indeed affect the activity of the resulting mutant, and it is well known that silent substitutions do not alter the enzyme activity of the mutant.

[0118] Advantageously, in the nucleic acid molecule according to the invention: a) When the nucleic acid molecule further includes a promoter that controls the expression of the nucleic acid sequence, this promoter is an exogenous promoter, especially the yeast promoter; or b) When the isolated nucleic acid molecule further contains a transcription terminator that controls the expression of the nucleic acid sequence, the transcription terminator is an exogenous terminator, such as the yeast terminator; or c) The nucleic acid sequence was further optimized for expression in yeast.

[0119] According to one embodiment, the nucleic acid molecule contains a promoter that controls the expression of at least one nucleic acid sequence encoding a mutant polypeptide as defined above.

[0120] Advantageously, the promoter is an exogenous promoter, particularly a yeast promoter, preferably selected from the ADH2 promoter (pADH2), CCW12 promoter (pCCW12), TEF1 promoter (pTEF1), PGK1 promoter (pPGK1), TDH3 promoter (pTDH3), and TPI1 promoter (pTPI1), and more preferably selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiaePromoters selected from the ADH2 promoter (pADH2), CCW12 promoter, TEF1 promoter, PGK1 promoter, TDH3 promoter, and TPI1 promoter of Saccharomyces cerevisiae; more preferably promoters selected from the ADH2 promoter of SEQ ID NO: 29, CCW12 promoter of SEQ ID NO: 30, TEF1 promoter of SEQ ID NO: 31, PGK1 promoter of SEQ ID NO: 73, TDH3 promoter of SEQ ID NO: 74, and TPI1 promoter of SEQ ID NO: 75, especially promoters selected from the ADH2 promoter of SEQ ID NO: 29, CCW12 promoter of SEQ ID NO: 30, and TEF1 promoter of SEQ ID NO: 31.

[0121] According to one implementation, the nucleic acid molecule contains a transcription terminator for at least one nucleic acid sequence encoding a mutant polypeptide as defined above.

[0122] Advantageously, the terminator is an exogenous terminator, particularly a yeast terminator, preferably an RPL3 terminator (tRPL3), an ADH1 terminator (tADH1), or a CYC1 terminator (tCYC1); more preferably an RPL3 terminator or an ADH1 terminator of Saccharomyces cerevisiae; even more preferably the RPL3 terminator of SEQ ID NO: 32, the ADH1 terminator of SEQ ID NO: 33, or the CYC1 terminator of SEQ ID NO: 76, particularly the RPL3 terminator of SEQ ID NO: 32 or the ADH1 terminator of SEQ ID NO: 33.

[0123] According to a preferred embodiment, the nucleic acid molecule contains both a promoter and a terminator as defined above.

[0124] According to one embodiment, the nucleic acid molecule further comprises an exported sequence. Advantageously, this exported sequence allows the polypeptide encoded by the nucleic acid molecule to be secreted or excreted into the cellular medium.

[0125] According to a preferred embodiment, the nucleic acid sequence is further optimized for expression in host cells (particularly yeast or bacteria). To optimize expression in host cells (particularly yeast or bacteria), those skilled in the art know how to utilize genetic code usage bias, which refers to the preferential use by a particular organism of one of the possible nucleotide triplets or codons to encode the same amino acid. In fact, there are often combinations of multiple nucleotide triplets (called "codons") to encode the same amino acid (except for methionine and tryptophan), called degenerate codons, but some of these combinations are generally preferred by a particular organism. For the production of the target amino acid sequence, optimal expression can be achieved when the codons selected to encode the amino acid sequence are those preferred by the host cell-derived organism. Depending on the selected production organism (particularly yeast or bacteria), different optimal nucleic acid sequences will therefore be used when the nucleic acid sequence contained in the isolated nucleic acid molecule used in this invention is further optimized for expression in the selected production organism. Those skilled in the art can use various software packages to optimize the codons for expression in host cells (including yeast, bacteria, or filamentous fungi). Examples of such software include Twist Codon Optimization Tool (provided by Twist Biosciences), GenSmart™ Codon Optimization Tool (provided by GenScript and whose functionality is described in application WO2020024917A1), IDT Codon Optimization Tool (provided by IntegratedDNA technologies), and Azenta's codon optimization tool (provided by Azenta).

[0126] When the nucleic acid sequence is further optimized for expression in yeast, it is advantageously optimized for expression in the genus *Saccharomyces* (Yeast). Saccharomyces It is expressed in yeast, preferably in the species Saccharomyces cerevisiae.

[0127] When the nucleic acid sequence is further optimized for expression in bacteria, it is advantageously optimized for expression in Escherichia (especially strains of the species Escherichia coli, which is the most widely used species in recombinant protein production) and Pseudomonas bacteria.

[0128] According to one embodiment, the nucleic acid molecule is isolated from a heterologous vector or host cell containing the molecule, said vector or said host cell being any vector or any host cell as defined above and below in the “Host Cell” or “Vector” section.

[0129] According to one embodiment, the nucleic acid molecule is synthesized in vitro using nucleic acid synthesis techniques that are fully known to those skilled in the art as defined. According to another embodiment, the nucleic acid molecule is recombinant.

[0130] carrier This invention relates to a vector comprising at least one nucleic acid molecule according to the invention, or expressing a mutant polypeptide according to the invention.

[0131] Advantageously, a nucleic acid molecule is any nucleic acid molecule as defined in the “Nucleic Acid Molecules” section above.

[0132] Advantageously, a mutant polypeptide is any mutant polypeptide defined in the “Mutant Polypeptides” section above.

[0133] Suitable vectors in the context of this invention include, but are not limited to, bacteriophage, plasmid, or cosmid vectors for expression in prokaryotic host cells (e.g., bacteria, such as Escherichia coli or Pseudomonas spp.); vectors for expression in yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g., Sf9 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 higher eukaryotic cells or organisms.

[0134] These vectors are typically commercially available (e.g., from suppliers such as Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.), obtainable from depository institutions such as the American Center for Type Culture Collection (ATCC, Rockville, Md.), or have their sequences, structures, and production methods described in numerous publications, allowing those skilled in the art to use them without difficulty.

[0135] Advantageously, the vector is a plasmid.

[0136] Representative examples of suitable plasmid vectors (especially for expression in yeast species, particularly Saccharomyces cerevisiae) include, but are not limited to, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen), and pgWiz (Gene Therapy System Inc.).

[0137] Host cell-combination In another aspect, the present invention relates to a host cell that contains or expresses a mutant polypeptide according to the invention, or contains a nucleic acid molecule according to the invention, or contains a vector according to the invention, or any combination thereof.

[0138] Advantageously, a mutant polypeptide is any mutant polypeptide defined in the “Mutant Polypeptides” section above.

[0139] Advantageously, a nucleic acid molecule is any nucleic acid molecule as defined in the “Nucleic Acid Molecules” section above.

[0140] Advantageously, a carrier is any carrier as defined in the “Carrier” section above.

[0141] According to various embodiments, the host cell (particularly the aforementioned heterologous host cell) can be a prokaryotic cell, a lower eukaryotic cell (such as a yeast cell), or other eukaryotic cells, such as insect cells, plant cells, and mammalian cells (e.g., human or non-human cells, preferably non-human cells). When the host cell is an embryonic cell or embryonic stem cell, the cell is a non-human cell.

[0142] Advantageously, the host cell is a microorganism selected from bacteria, yeast, fungi, algae and cyanobacteria, more preferably a microorganism selected from yeast, fungi, algae and bacteria, and even more preferably a microorganism selected from yeast and bacteria.

[0143] The host cell is preferably yeast, specifically selected from the genus *Saccharomyces* (Yeast). Saccharomyces ), Candida ( Candida ), Ashu pseudocystis ( Ashbya ), Decker yeast ( Dekkera Pichia pastoris ( ) Pichia (Hansenula polymorpha) Hansenula ), Debali yeast ( Debaryomyces ), *Corydalis* genus ( Clavispora ), Lode yeast ( Lodderomyces ), Yeastra genus ( Yarrowia ), Zygosaccharomyces ( Zigosaccharomyces ), genus *Fissionyomyces* ( Schizosaccharomyces ), Rhodotorula spp. ( Torulaspora Kluyveromyces ( ) Kluyveromyces ), Brett's yeast ( Brettanomycces Cryptococcus ( Cryptococcus ) and Malassezia ( Malassezia ).

[0144] Even more specifically, the yeast is selected from the species *Saccharomyces cerevisiae* and *Saccharomyces boulardii*. Saccharomyces boulardii ), Douglas yeast ( Saccharomyces douglasii Pasteur yeast ( Saccharomycesbayanus ), Bydion conjugated yeast ( Zigosaccharomyces bailii ), millet wine fission yeast ( Schizosaccharomyces pombe ), Brussels yeast ( Dekkera brucelensis ), intermediate Dike yeast ( Dekkera intermedia ), Kusterbreed yeast ( Brettanomycces custersii ), intermediate Brett yeast ( Brettanomycces intermedius ), heat-resistant Kluyveromycin ( Kluyveromyces themotolerens ), Spherical red thallus yeast ( Torulaspora globosa ) and smooth round red thallus yeast ( Torulaspora glabrata ).

[0145] Even more specifically, the yeast belongs to the genus *Saccharomyces*, with *Saccharomyces cerevisiae* being the preferred species.

[0146] The host cell may also preferably be bacteria, particularly selected from the phyla Proteobacteria, Actinomycetes, and Firmicutes, and more preferably from the genera Escherichia, Pseudomonas, and Streptomyces. Streptomyces Corynebacterium spp. Corynebacterium ), Bacillus spp. ( Bacillus ) and Lactobacillus genus ( Lactobacillus Even more specifically, the bacteria are from the genus Escherichia, preferably the species Escherichia coli.

[0147] According to one implementation, the host cell contains at least one copy of a nucleic acid molecule as defined above and is integrated into its genome.

[0148] According to one implementation, the host cell contains a single copy of a nucleic acid molecule as defined above and integrates it into its genome.

[0149] When the host cell is a yeast cell, one or more copies of the nucleic acid molecule can be integrated into various loci, preferably into the URA3, JLP1, LEU2, or TRP1 loci of the yeast cell genome. When the host cell is a yeast cell and multiple copies of the nucleic acid molecule are integrated, the copies can be integrated into the same locus or into different loci, preferably into any combination of the URA3, JLP1, LEU2, and / or TRP1 loci.

[0150] Advantageously, the codons used in the nucleic acid molecule have been adapted to achieve optimal expression in the selected host cell (see the section above on the nucleic acid molecule of the present invention).

[0151] Therefore, those skilled in the art know which techniques to use to modify the codons of nucleic acid molecules. For example, the codons can be modified by in vitro site-directed mutagenesis using a sample of nucleic acid molecules for which the codons to be modified, and then amplified by polymerase chain reaction (PCR). Alternatively, nucleic acid molecules can be synthesized directly in vitro using the optimized codons.

[0152] Host cells can be cultured / grown in small and large aerobic or anaerobic bioreactors, in flasks, or in petri dishes. Culture can be carried out at temperatures, pH, culture media, and oxygen levels suitable for the specific host cells.

[0153] The present invention also relates to compositions comprising, consisting primarily of, or composed of mutant polypeptides according to the invention, nucleic acid molecules according to the invention, vectors according to the invention, host cells according to the invention, or any combination thereof.

[0154] Advantageously, a mutant polypeptide is any mutant polypeptide defined in the “Mutant Polypeptides” section above.

[0155] Advantageously, a nucleic acid molecule is any nucleic acid molecule as defined in the “Nucleic Acid Molecules” section above.

[0156] Advantageously, a carrier is any carrier as defined in the “Carrier” section above.

[0157] Advantageously, the host cell is any host cell as defined above.

[0158] Methods and uses Method for producing mutant peptides according to the present invention The present invention also relates to a method for producing the mutant polypeptide of the present invention, preferably any mutant polypeptide having phloroglucinol synthase activity as defined above.

[0159] According to one embodiment, a method for producing the mutant peptide as defined above includes at least the following steps: (i) Introducing the nucleic acid molecule or vector as described above into a suitable host cell, in accordance with the foregoing description; and (ii) The host cells obtained in step (i) are cultured in vitro under conditions that allow for the growth of the host cells and / or the expression of the nucleic acid molecules, thereby producing the mutant polypeptide.

[0160] According to another embodiment, a method for producing a polypeptide having phloroglucinol synthase activity as defined above includes at least the following steps: (i) The mutant polypeptide is produced by culturing a host cell (e.g., the host cell described above) expressing the mutant polypeptide in vitro, wherein the culture conditions allow the growth of the host cell and / or the expression of nucleic acid molecules contained in the host cell.

[0161] According to one embodiment, a method for producing the mutant polypeptide according to the invention includes at least one additional step selected from the following steps: (α) Recovering cells expressing the mutant polypeptide obtained after the culture step; and (β) Purify the mutant polypeptide from the cells recovered in step (α).

[0162] Methods / uses for producing phloroglucinol The present invention further relates to the use of mutant peptides according to the invention, nucleic acid molecules according to the invention, vectors according to the invention, host cells according to the invention, compositions according to the invention, or any combination thereof for the production of phloroglucinol.

[0163] Advantageously, a mutant polypeptide is any mutant polypeptide defined in the “Mutant Polypeptides” section above.

[0164] Advantageously, a nucleic acid molecule is any nucleic acid molecule as defined in the “Nucleic Acid Molecules” section above.

[0165] Advantageously, a carrier is any carrier as defined in the “Carrier” section above.

[0166] Advantageously, the host cell and / or composition is any host cell or composition as defined in the “Host Cell-Composition” section above.

[0167] The present invention also relates to a method for producing phloroglucinol.

[0168] According to one embodiment M1, a method for producing phloroglucinol includes at least the following steps: (i) Obtaining cells by performing one of the methods described above; (ii) Contact the cells obtained in step (i) with a suitable substrate; (iii) Incubate the mixture obtained in step (ii) under conditions suitable for the production of phloroglucinol; (iv) Optionally, recover the reaction medium containing phloroglucinol obtained after step (iii); and (v) Optionally, phloroglucinol is purified from the reaction medium of step (iv).

[0169] According to another embodiment M2, the method for producing phloroglucinol includes the following steps: a) Contact the host cell expressing the mutant polypeptide as defined above (e.g., the host cell as defined above) with a suitable substrate; b) Culture the host cells of step a) in vitro, wherein the culture conditions allow the growth of the host cells and / or the expression of nucleic acid molecules contained in the host cells, thereby producing phloroglucinol; c) Optionally, recover the phloroglucinol-containing culture medium obtained after step b); and d) Optionally, phloroglucinol is purified from the culture medium of step c).

[0170] For the purposes of methods M1 and M2, the substrate is a carbon source. Advantageously, the carbon source is a pure carbon source or an industrial byproduct (e.g., molasses or green syrup, for example, from the sugar industry). Preferably, the substrate in the pure carbon source or industrial byproduct is a monosaccharide, such as glucose (or dextrose), fructose, galactose, mannose, sucrose, lactose, or maltose; a complex sugar, such as a monosaccharide, disaccharide, or trisaccharide, or a polysaccharide, such as starch; an alcohol, such as ethanol; an acid; a fatty acid and its ester derivatives; or a mixture of sugars, alcohols, acids, and / or fatty acids or their ester derivatives.

[0171] Preferably, the substrate is glucose. Alternatively, the substrate is ethanol.

[0172] According to another embodiment M3, the method for producing phloroglucinol includes at least the following steps: (i) Contact at least one polypeptide obtained in step (β) of the above method with malonyl-CoA (MLC); (ii) Incubate the mixture obtained in step (i) under conditions suitable for the production of phloroglucinol; (iii) Optionally, the reaction medium containing phloroglucinol obtained after step (ii) is recovered; and (iv) Optionally, phloroglucinol is purified from the reaction medium of step (iii).

[0173] According to another embodiment M4, the method for producing phloroglucinol includes at least the following steps: a) Contact the mutant polypeptide as defined above with malonyl-CoA (MLC); b) Incubate the mixture obtained in step a) under conditions suitable for the production of phloroglucinol; c) Optionally, recover the reaction medium containing phloroglucinol obtained after step b); and d) Optionally, pyrogallol is purified from the reaction medium of step (iii).

[0174] According to a preferred embodiment, the purification of phloroglucinol is carried out using any technique known to those skilled in the art, including (but not limited to) liquid-liquid extraction or crystallization.

[0175] The following examples are intended to illustrate the invention and are not intended to limit it.

[0176] The enzymes encoded by the PHLD gene of *Pseudomonas fluorescens* (Zha et al., 2006) and the PKS1 gene of *Brownia fringeii* (Meslet-Cladière et al., 2013) were used as controls.

[0177] sequence The amino acid sequences of the wild-type and mutant peptides cited in this article are summarized in Table 4 below.

[0178] Table 4: PHLD Sequences The nucleic acid sequences encoding wild-type and mutant peptides cited in this article are summarized in Table 5 below.

[0179] Table 5: Nucleic acid sequences encoding PHLD The sequences cited in this article, excluding wild-type and mutant peptides, are summarized in Table 6 below.

[0180] Table 6: Other sequences Attached Figure Description

[0181] Figure 1 The homology model of PhlD_Tpu in the form of a homodimer is shown. The two chains are represented in black and gray, respectively.

[0182] Figure 2 The map of the YCplac22_PGK_CYC_TEF_ADHCYC plasmid (low copy) is shown.

[0183] Figure 3 The PCR products of site-directed mutagenesis are shown.

[0184] Figure 4 The spectrum of pET-26b-PhlD-Tpu is shown.

[0185] Figure 5 This is a graph showing the 100 µM malonyl-CoA (MLC) (black) and CoA (CoA) (gray) standard solutions separated by HPLC.

[0186] Figure 6 The Tm of purified PhlD_Tpu M27 (gray) and PhlD_Tpu WT (black) as determined by nanoDSF is shown. Detailed Implementation

[0187] Example 1. Example 1: Design of a novel and comprehensive strategy for the production of optimized phloroglucinol synthase - Materials and methods 1.1. Molecular Modeling Methods 1.1.1. PhlD_Tpu Modeling and PhlD_Tpu: Ligand Complex Modeling A 3D model of the Phld_Tpu homodimer was constructed using comparative modeling combination. The model was derived from Mycobacterium tuberculosis (…). Mycobacterium tuberculosis The crystal structure of type III polyketide synthase PKS18 was used as a template. To obtain a reasonable model, approximately 1500 structures were calculated. The modeling structure with the lowest energy was selected to represent a fully folded and stable conformation. The final main-chain RMSD between the modeling structure and the template structure was 0.9 Å.

[0188] Next, we modeled the complex of PhlD_Tpu with malonyl-CoA (MLC) substrate. The docking site of MLC was determined based on structural analysis of other available PKS III structures co-crystallized with MLC from the PDB database (e.g., PDB accession number: 1EE0).

[0189] Two hypothetical transient binding modes of Phlo were modeled based on literature data. The first binding mode was determined based on the product binding site located at the dimer interface, a site previously reported in the crystal structures of other PKS structures (PDB accession number: 4B0N, PKS-I from *Bryophyta fringeii*, with malonic acid as the product, Meslet-Cladière et al., 2013). In that case, protein docking was simultaneously applied to generate a symmetrical complex structure starting from the monomer docked with MLC. In the absence of MLC, another binding mode was generated, where Phlo is located near the catalytic triplet (C167, H305, N338).

[0190] 1.1.2 Model Analysis and Simulation Molecular dynamics simulations were performed on all systems in the presence of dominant solvents.

[0191] We further analyzed molecular dynamics simulations using atomic fluctuation cross-correlation (Lange O. et al., 2006) to understand the coupled motions of different residues in the molecular dynamics simulation trajectory and to identify potential allosteric communication pathways.

[0192] The simulations were also analyzed to identify residues involved in substrate and product binding. The energy binding contribution of each residue in the substrate (MLC) and product (Phlo) was calculated using the MM-GBSA method. Calculations were performed using the Python script MMGBSA.py (Miller B. et al., 2012) from the AmberTools package. For each PhlD_Tpu recombination system, the first 5 nanoseconds (ns) of each trajectory (from two independent simulations) were concatenated to produce a total of 10 ns trajectories, which were then used for binding energy calculations.

[0193] The PROSS design method (Goldenzweig A. et al., 2016) was applied to identify design sites. For this purpose, we used multiple sequence alignment of enzymes in the PKSIII family to identify variable regions. Highly conserved regions were excluded because they are involved in protein folding and function. Furthermore, regions near ligand binding and protein-protein interfaces were also excluded (residues 1-5, 8, 23, 37, 43, 57-59, 62-63, 65-66, 68-69, 70-72, 75, 93-97, 99-100, 103-104, 107-108, 110, 128-130, 132, 134-149, 155-169, 172, 175-17). 6, 179-180, 182-187, 194-195, 205-208, 210-218, 222, 245-251, 253-269, 271-277, 284, 291, 302, 304-314, 330, 333, 337-338, 340-341, 350, 358, 364, 366, 371-377, 379, 386-388). Once the locations on the protein surface are identified, depth screening is performed to identify the nature / type (type of residue mutation) of the mutation at each location, which is expected to improve the enzyme's thermostability compared to the wild type. The nature / type of mutations included at each location used for depth screening is determined based on the residue variability at that location in multiple sequence alignments of the PKSIII family. Based on this, we designed a PhlD_Tpu mutant library.

[0194] To investigate the release of Phlo from its active site, we used stochastic accelerated molecular dynamics simulations to calculate the Phlo release pathway. The amino acids that come into contact with Phlo during its release were identified.

[0195] Molecular dynamics simulations of the PhlD_Tpu dimer were analyzed, identifying 34 residues that could be further targeted for mutagenesis. Several residues were also identified for introducing disulfide bonds.

[0196] Finally, we used ancestral sequence reconstruction (ASR), which has proven to be a useful method for engineering proteins with enhanced properties, such as improved activity and / or stability. Here, we used the ASR method to reconstruct the PhlD ancestral sequence of the Actinobacteria subtree.

[0197] Homologous sequences of PhlD_Tpu belonging to the phylum Actinobacteria were retrieved from the Uniprot database (Uniprot database version 2022_02) using EFI-EST (Zallot, R. et al., 2019). In addition to the 2369 sequences obtained using EFI-EST, four sequences previously identified from natural diversity and nine sequences described in patent WO2019002799A1 were added to the sequence set. Next, redundant sequences were removed using CD-Hit software at a 90% sequence identity threshold. Finally, sequences with less than 40% identity to Phld_Tpu were removed, resulting in a final set of 52 sequences. Following the same protocol, five homologous sequences from the phylum Cyanobacteria were selected and used as outgroups for phylogenetic tree root determination (described below).

[0198] The 57 sequences were then aligned using MAFFT (Katoh K. et al., 2002), with N- and C-termini pruned as they showed low sequence conservation. Phylogenetic trees were constructed using the resulting multiple sequence alignments (MSA) and iq-tree software (Minh, B. et al., 2020). A total of 168 substitution models were tested, and the best model was selected based on Bayesian information criteria (LG+F+I+G4) and used for phylogenetic tree construction. The resulting unrooted tree was ultimately rooted by selecting cyanobacterial sequences as outgroups.

[0199] Using a phylogenetic tree, ancestral sequences corresponding to each tree node were reconstructed using GRASP (Foley G. et al., 2022). Sequences were inferred using the edge reconstruction method, which yields the probability distribution for each residue of a single ancestor. Sequences corresponding to the three nodes (ancestors) closest to PhlD_Tpu were analyzed. Mutations located at the N-terminus or with low posterior probability (p<0.7) were restored to wild-type amino acids.

[0200] 1.2. Experimental Methods 1.2.1. Site-directed mutagenesis method Site-directed mutagenesis was employed to generate single mutants at the selected target sites using standard methods. In short, reverse PCR (using non-overlapping primers, one of which contains the desired mutation) was used to introduce the point mutation, resulting in the amplification of linear, double-stranded, mutant products. For each target residue, a forward primer containing the desired amino acid substitutions was used in a PCR reaction containing a ClonAMP HiFi PCR Premix (a pre-assembled mixture of enzyme, optimization buffer, and dNTPs), enabling rapid and simple setup of the PCR reaction. The template used for mutagenesis was PhlD_Tpu (wild-type (WT)), cloned in the YCplac22_PGK_CYC_TEF_ADH yeast expression vector (low copy) controlled by the pTEF1 promoter and possessing an ADH terminator, as shown below. Figure 2 As shown. It is a shuttle vector containing two replication origins to facilitate its reproduction in yeast and Escherichia coli. The Trp1 gene serves as a auxotrophic marker for selection in yeast via TRP, while the ampicillin resistance gene allows selection in Escherichia coli.

[0201] It is important to note that the PhlD_Tpu mutant was expressed using the YCplac22_PGK_CYC_TEF_ADH vector, which does not contain the ACC1 gene. This means that these mutants rely solely on malonyl-CoA produced by ACC1, encoded only by the ACC gene present in the yeast genome. PCR products observed by 0.8% agarose gel electrophoresis showed a band at 7.2 kb (5 kb = vector + 2 kb insert). Figure 3 kb = kilobases.

[0202] Following PCR amplification, the sample was incubated at 37°C for 1 hour or overnight using a methylation-sensitive endonuclease. Dpn The mutagenic plasmid was digested, treated with polynucleotide kinase (T4 PNK) to add a phosphate group, and then ligated. The resulting ligation product was transformed into *Escherichia coli* TOP 10 cells, and positive clones were selected after overnight incubation at 37°C. Plasmids were then extracted from the positive clones using the QIAprep® Spin Miniprep Kit (Qiagen) and sequenced by Sanger sequencing (Eurofins). The confirmed PhlD_Tpu variant was transformed into competent *Saccharomyces cerevisiae* cells prepared according to the protocol of Gietz et al., 2014 (LiAc / SS vector DNA / PEG method).

[0203] 1.2.2. Generation of multi-site yeast mutant libraries A custom library with eight target sites was designed for PhlD_Tpu, and the amino acids to be substituted at each site were rationally defined based on experimental knowledge of site-directed mutagenesis. The custom-designed library was obtained from the commercial supplier GenScript. The target gene PhlD_Tpu, cloned in plasmid YCplac22_PGK_CYC_TEF_ADH, was sent to GenScript, and their in-house technology was used to prepare a custom-designed combinatorial library. The library was delivered as a clonable plasmid and directly used for transformation into competent Saccharomyces cerevisiae cells prepared using the LiAc / SS vector DNA / PEG method, followed by high-throughput screening.

[0204] 1.2.3. Determination of phloroglucinol production in yeast mutants Following transformation, clones were selected based on yeast auxotrophs for tryptophan. Phlo production was evaluated at two different scales: low-throughput and high-throughput. For site-saturated mutagenesis (SSM) libraries, a low-throughput scale was performed manually, where transformed clones were selected, inoculated into 96-well plates (DWPs), and incubated at 30°C for 24 hours. Using a multichannel pipette, 10 µL of the grown culture was transferred to 500 µL of fresh Trp_dropout CS medium (1.7 g / L yeast nitrogen source without amino acids and ammonium sulfate, 2.03 g / L ammonium chloride, 0.74 g / L fully synthetic deletion mixture - Trp Formedium, and 2% glucose) in 96-well plates (3 DWPs for sampling over three days), and Phlo production was monitored at different time points (day 1, day 2, and day 3) using colorimetric reagents as described below.

[0205] On the other hand, for custom libraries, Phlo production is performed at a high-throughput scale. For this purpose, transformed colonies grown on QTrays (QPix2XT, Molecular Devices) were picked and inoculated into standard 96-well plates pre-filled with 200 µL of CS medium using a liquid handling robot (Nimbus, Hamilton). After incubation at 30°C, 10 µL of overnight culture was transferred to 500 µL of fresh Trp_dropout CS medium in 96-well plates using an automated liquid handling system. Protein induction was not required because the promoter used for expression was constitutive (pTEF1 promoter). On day 3, prior to Phlo measurement, the OD 600 nm density of cells in all cultures was measured in microplates. The amount of Phlo produced in the supernatant was measured after centrifugation at 3700 rpm for 10 minutes. 150 µL of 500 mg / L 4-hydroxy-3-methoxycinnamicaldehyde (CAS: 458-36-6) in HCl (37% (w / w)) / ethanol solution (v:v 1:3) was added to 100 µL of supernatant. Simultaneously, standard curves were prepared in parallel for different Phlo concentrations ranging from 5 mg / L to 125 mg / L. The solution mixture was incubated at room temperature for 30–40 minutes, followed by absorbance measurement at 540 nm using a microplate reader. Results from days 1 and 2 were excluded because Phlo production was difficult to measure during this period; only results from day 3 were considered.

[0206] 1.2.4. Subcloning into an Escherichia coli expression plasmid and overexpressing it. The selected PhlD_Tpu sequence was amplified from a yeast expression plasmid and fused with a 6xHis tag encoded at the C-terminus. This fusion was then inserted into the pET26b(+) plasmid, located at... Xho I and Nde I between restriction sites (e.g.) Figure 4 (as shown), and by Lac The I promoter controls its expression. *Escherichia coli* BL21(DE3) competent cells were transformed with the recombinant plasmid and cultured in 500 mL of LB medium (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract) with shaking at 37°C. When the OD 600 nm reached 0.4 to 0.5, expression of the recombinant plasmid was initiated by adding 0.1 mM IPTG. Cells were then grown overnight at 20°C until they reached an OD 600 nm of approximately 1.5 to 1.6.

[0207] 1.2.5. Purification Transformed Escherichia coli BL21(DE3) cells, as described in Section 1.2.4 above, were lysed using Fast-Prep for three cycles at a speed setting of 6 m / sec, with each cycle lasting 45 seconds. The supernatant was filtered through a 0.45 µm membrane and then loaded onto a 1 mL Talon® resin column. The column was first washed with 10 column volumes of sodium phosphate buffer (50 mM, pH 7; 100 mM NaCl) and then with 10 column volumes of sodium phosphate buffer containing 10 mM imidazole (3% Buffer B, 50 mM phosphate buffer, 500 mM imidazole, pH 8.5; 100 mM NaCl). Protein elution was performed using 150 mM imidazole in sodium phosphate buffer (50 mM, pH 7, 100 mM NaCl). The collected fractions were combined and percolated using a 30 kDa percolator (Amicon® Ultra, Merck) with 5 volumes of sodium phosphate buffer (50 mM, pH 7, 100 mM NaCl), then the volume was reduced to 500 µL. The concentrate was added to a gel filtration column (S200 TRICORN 10 / 300 Superdex 200). Elution was performed using an isocratic gradient of sodium phosphate buffer (50 mM, pH 7, 100 mM NaCl). The purity of the obtained fractions was checked by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Gels, Bio-RADS).

[0208] 1.2.6. Determination of kinetic parameters To obtain the kinetic parameters of PhlD_Tpu, the concentrations of consumed MLC and generated CoA were monitored by HPLC. Experiments were run for 120 seconds, and only the linear portion of CoA change over time was used to calculate the initial velocity. Experiments were performed using a series of MLC concentrations (5 to 100 µM) in sodium phosphate buffer (50 mM, pH 7) at 30°C, along with 0.5 µM of purified enzyme. At 10, 20, 30, 50, 60, and 120 seconds, 100 µL of the reaction mixture was quenched with 10 µL of 10% (v / v) formic acid and stored at room temperature prior to HPLC analysis. Under these conditions, the generated CoA increased linearly over time up to 50 seconds. The HPLC conditions used to monitor MLC consumption and CoA generation are listed in Table 7.

[0209] Table 7: HPLC conditions for the separation and quantification of MLC and CoA. CoA and MLC were well separated, with retention times of 4.60 minutes and 6.78 minutes, respectively. Figure 5As shown.

[0210] 1.2.3. Determination of phloroglucinol production in Escherichia coli Precultured Escherichia coli BL21(DE3) cells, transformed as described in Section 1.2.4 above, were seeded into M9 medium containing kanamycin (2% glucose; inorganic salts (Na2HPO4 18 g / L, KH2PO4 3.13 g / L, NaCl 0.53 g / L, NH4Cl 2.11 g / L); MgSO4 2 mM; CaCl2 0.2 mM; trace metals 1x (4.5 mg / L ZnSO4, 1 mg / L H3BO3, 0.3 mg / L CuSO4, 1 mg / L MnCl2, 15 mg / L Na2 EDTA, 0.3 mg / L CoCl, 0.4 mg / L Na2MoO4 and FeSO4); kanamycin 50 µg / mL), and incubated overnight at 20°C after induction with 0.1 mM IPTG when the OD 600 reached 0.7. Then, the concentration of extracellular Phlo was measured using the colorimetric reagents described in Section 1.2.3 above.

[0211] 2. Example 2: Characterization of the obtained mutant peptide - Results 2.1 Yeast Research The computational experiments performed according to Example 1 allowed for the prediction of the three-dimensional structure of the functional homodimer of PhlD_Tpu and the revelation of molecular determinants involved in substrate and product recognition. Using these original and complementary computer-aided strategies, combined with a literature review, multiple amino acid positions were predicted as targets for mutagenesis to improve key enzyme properties, including catalytic activity and stability. A series of yeast mutants were then constructed and screened for improved Phlo production. The best yeast mutants were ultimately characterized. In this section, we describe the identified active mutants. A summary of the mutants and their Phlo production activities measured in yeast is provided in Table 9.

[0212] Next, using the best yeast mutants M21 (A124P, A154P, S177A, S236P, A270S, A298D) and variants of the deletion mutation A270S (A124P, A154P, S177A, S236P, A298D; named M26) as new templates, the best mutations identified for positions 268 and 271 were introduced to create new yeast mutants, which in some cases showed an increase in Phlo production, up to 86% compared to PhlD_Tpu (Table 8).

[0213] Of all the yeast mutants tested, the M27 mutant, containing seven mutations (A124P, A154P, S177A, S236P, A298D, E268Q, and E271R), was one of the best Phlo producers, with a relative activity of approximately 190% compared to WT. The stability and enzyme kinetics of the M27 variant were further characterized in vitro and compared with WT.

[0214] The stability of M27 was studied using nano DSF experiments, which confirmed that M27 has better thermal stability and improved To. m (66.7°C), 5°C higher than the measurement of the natural sequence. Figure 6 ).

[0215] Evaluation of reaction kinetics showed that the k of yeast M27 cat It decreased by 1.3 times, while K M The catalytic efficiency of this variant is 1.6 times higher than that of PhlD_Tpu WT, which is twice as low as that of PhlD_Tpu WT (Tables 8 and 9).

[0216] Table 8: Dynamic parameters of PhlD_Tpu WT and M27 Table 9: A list of Phld_Tpu yeast mutants designed from libraries 1 to 12, exhibiting similar (≥90% Phlo yield percentage relative to WT) or higher phloroglucinol production activity. For each yeast mutant, the location, activity, and the protein scaffold in which the mutation was introduced are indicated. Relative activity refers to the ratio of the amount of phloroglucinol (g / L) obtained in the supernatant after 3 days of mutant culture to the amount of phloroglucinol (g / L) obtained in the supernatant after 3 days of WT culture under the same conditions. 2.2 Research on Escherichia coli Phlo production activity of Escherichia coli BL21(DE3) cells transformed with plasmids expressing Phld_Tpu mutants E271K or M27 was compared with Phlo production activity of Escherichia coli BL21(DE3) cells transformed with plasmids expressing Phld_Tpu WT or empty plasmids.

[0217] The results of two independent experiments comparing E271K and Phld_Tpu WT are shown in Table 10 below. They show that the E271K mutant of Escherichia coli BL21(DE3) exhibits significantly higher Phlo production activity than that of Escherichia coli BL21(DE3) Phld_Tpu WT.

[0218] Table 10: Phlo production activity in cells transformed with Escherichia coli BL21(DE3). In further experiments, the Phlo production activity of *Escherichia coli* BL21(DE3) M27 (A124P, A154P, S177A, S236P, E268Q, E271R, and A298D) was compared with that of *Escherichia coli* BL21(DE3) Phld_Tpu WT and *Escherichia coli* BL21(DE3) transformed with a plasmid expressing *P. fluoresceinii* Phd_PF. This also confirmed that the M27 mutant exhibited significantly higher Phlo production activity than Phld_Tpu WT, and this was also true in *Escherichia coli* (see Table 11 below).

[0219] Table 11: Phlo production activity in cells transformed with Escherichia coli BL21(DE3). References Achkar J et al., (2005) "Biosynthesis of phloroglucinol" J Am Chem Soc. 127:5332-5333. Foley,G.;Mora,A.;Ross,C. M.;Bottoms,S.;Sützl,L.;Lamprecht,M. L.;Zaugg,J.;Essebier,A.;Balderson,B.;Newell,R.;Thomson,R. E. S.;Kobe,B.;Barnard,R. T.;Guddat,L.;Schenk,G.;Carsten,J.;Gumulya,Y.;Rost,B.;Haltrich,D.;Sieber,V.;Gillam,E. M. J.;Bodén,M. Engineering Indel and Substitution Variants ofDiverse and Ancient Enzymes Using Graphical Representation of AncestralSequence Predictions(GRASP). PLOS Comput. Biol. 2022,18(10),e1010633. Gietz RD. Yeast transformation by the LiAc / SS carrier DNA / PEG method.Methods Mol Biol. 2014;1163:33-44. Goldenzweig,A.;Goldsmith,M.;Hill,S. E.;Gertman,O.;Laurino,P.;Ashani,Y.;Dym,O.;Unger,T.;Albeck,S.;Prilusky,J.;Lieberman,R. L.;Aharoni,A.;Silman,I.;Sussman,J. L.;Tawfik,D. S.;Fleishman,S. J. Automated Structure- andSequence-Based Design of Proteins for High Bacterial Expression andStability. Mol. Cell 2016,63(2),337–346. Katoh,K.;Misawa,K.;Kuma,K.;Miyata,T. MAFFT: A Novel Method for RapidMultiple Sequence Alignment Based on Fast Fourier Transform. Nucleic AcidsRes. 2002,30(14),3059–3066. Kimple ME,Brill AL,Pasker RL. Overview of affinity tags for proteinpurification. Curr Protoc Protein Sci. 2013 Sep 24;73:9.9.1-9.9.23. Lange,O. F.;Grubmüller,H. Generalized Correlation for BiomolecularDynamics. Proteins Struct. Funct. Bioinforma. 2006,62(4),1053–1061. Miller,B. R. I.;McGee,T. D. Jr.;Swails,J. M.;Homeyer,N.;Gohlke,H.;Roitberg,A. E. MMPBSA.Py: An Efficient Program for End-State Free EnergyCalculations. J. Chem. Theory Comput. 2012,8(9),3314–3321. Minh,B. Q.;Schmidt,H. A.;Chernomor,O.;Schrempf,D.;Woodhams,M. D.;vonHaeseler,A.;Lanfear,R. IQ-TREE 2: New Models and Efficient Methods forPhylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020,37(5),1530–1534. Needleman et Wunsch. J.Mol. Biol. 48,443-453,1970 Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584 WO 2013 / 045510 WO2019 / 002798 WO2019 / 002799 Zallot, R.; Oberg, N.; Gerlt, J. A. The EFI Web Resource for GenomicEnzymology Tools: Leveraging Protein, Genome, and Metagenome Databases toDiscover Novel Enzymes and Metabolic Pathways. Biochemistry 2019, 58(41), 4169–4182. Zha W, Rubin-Pitel SB and Zhao H. (2006) "Characterization of the substratespecificity of PHLD, a type III polyketide synthase from Pseudomonasfluorescens." J Biol Chem. 281:32036-32047.

Claims

1. A mutant polypeptide comprising the strain of *Pneumocystis jirovecii* as shown in SEQ ID NO:

1. Tsukamurella pulmonis The amino acid sequence of the phloroglucinol synthase has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, and has one or more substitutions selected from one or more amino acid residues of the following: a) Any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, any amino acid residue or combination of amino acid residues located at positions from position 268 to position 271 of SEQ ID NO: 1; or b) The amino acid residue located at position 49 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 49 of SEQ ID NO: 1; or c) The amino acid residue located at position 339 of SEQ ID NO: 1; or After optimal global alignment with SEQ ID NO: 1, the amino acid residue located at position 339 of SEQ ID NO: 1; or d) Amino acid residues located at positions 93 and 262 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 93 and 262 of SEQ ID NO: 1; or e) Amino acid residues located at positions 129 and 176 of SEQ ID NO: 1; or Following optimal global alignment with SEQ ID NO: 1, the amino acid residues located at positions corresponding to positions 129 and 176 of SEQ ID NO: 1; and f) Any combination thereof.

2. The mutant polypeptide of claim 1, comprising one or more substitutions of any amino acid residue or combination of amino acid residues located at positions 268 to 271 of SEQ ID NO: 1, or comprising one or more substitutions of any amino acid residue or combination of amino acid residues located at positions corresponding to positions 268 to 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO:

1.

3. The mutant polypeptide according to claim 2, comprising: a) Substitution of the glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or substitution of an amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, preferably wherein the glutamic acid residue at position 271 of SEQ ID NO: 1, or substitution of an amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is located at position 271 of SEQ ID NO:

1. The amino acid residue at position 271 of NO:1 is replaced by an amino acid residue selected from arginine (R), histidine (H), lysine (K), glutamine (Q), valine (V), methionine (M), phenylalanine (F), leucine (L), tyrosine (Y), and proline (P); preferably replaced by an amino acid residue selected from arginine (R), histidine (H), lysine (K), valine (V), methionine (M), and phenylalanine (F); more preferably replaced by an amino acid residue selected from arginine (R), lysine (K), valine (V), and methionine (M); even more preferably replaced by an amino acid residue selected from arginine (R), lysine (K), and valine (V); even more preferably replaced by an amino acid residue selected from arginine (R) and lysine (K); b) Substitution of the glutamic acid (E) residues at positions 268 and 271 of SEQ ID NO: 1, or substitution of the amino acid residues at positions corresponding to positions 268 and 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, preferably wherein: - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; or c) Substitution of amino acid residues at positions 124, 154, 177, 236, 268, 271, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 124, 154, 177, 236, 268, 271, and 298 of SEQ ID NO: 1, preferably wherein: - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by alanine (A); and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or d) Substitution of amino acid residues at positions 124, 154, 177, 236, 268, 270, 271, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 124, 154, 177, 236, 268, 270, 271, and 298 of SEQ ID NO: 1, preferably wherein: - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by alanine (A); and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamic acid (E) residue at position 268 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from Q, T, and S; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 270 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The glutamic acid (E) residue at position 271 of SEQ ID NO: 1, or the amino acid residue at position 271 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an amino acid residue selected from K, R, and V; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or e) Substitution of amino acid residues at positions 35, 60, 91, 123, 124, 154, 177, 236, 270, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 35, 60, 91, 123, 124, 154, 177, 236, 270, and 298 of SEQ ID NO: 1, preferably wherein: - The serine (S) residue at position 35 of SEQ ID NO: 1, or the amino acid residue at position 35 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a glycine (G) residue; and - The alanine (A) residue at position 60 of SEQ ID NO: 1, or the amino acid residue at position 60 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 91 of SEQ ID NO: 1, or the amino acid residue at position 91 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 123 of SEQ ID NO: 1, or the amino acid residue at position 123 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 270 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 298 of SEQ ID NO: 1, or the amino acid residue at position 298 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an aspartic (D) residue; or f) Substitution of amino acid residues at positions 35, 46, 60, 91, 123, 124, 154, 177, 191, 236, 238, 270, 290, and 298 of SEQ ID NO: 1, or substitution of amino acid residues at positions corresponding to positions 35, 46, 60, 91, 123, 124, 154, 177, 191, 236, 238, 270, 290, and 298 of SEQ ID NO: 1, preferably wherein: - The serine (S) residue at position 35 of SEQ ID NO: 1, or the amino acid residue at position 35 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a glycine (G) residue; and - The serine (S) residue at position 46 of SEQ ID NO: 1, or the amino acid residue at position 46 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The alanine (A) residue at position 60 of SEQ ID NO: 1, or the amino acid residue at position 60 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 91 of SEQ ID NO: 1, or the amino acid residue at position 91 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an arginine (R) residue; and - The alanine (A) residue at position 123 of SEQ ID NO: 1, or the amino acid residue at position 123 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The alanine (A) residue at position 124 of SEQ ID NO: 1, or the amino acid residue at position 124 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The alanine (A) residue at position 154 of SEQ ID NO: 1, or the amino acid residue at position 154 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The serine (S) residue at position 177 of SEQ ID NO: 1, or the amino acid residue at position 177 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The methionine (M) residue at position 191 of SEQ ID NO: 1, or the amino acid residue at position 191 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a valine (V) residue; and - The serine (S) residue at position 236 of SEQ ID NO: 1, or the amino acid residue at position 236 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a proline (P) residue; and - The glutamine (Q) residue at position 238 of SEQ ID NO: 1, or the amino acid residue at position 238 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a lysine (K) residue; and - The alanine (A) residue at position 270 of SEQ ID NO: 1, or the amino acid residue at position 268 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by a serine (S) residue; and - The aspartic acid (D) residue at position 290 of SEQ ID NO: 1, or the amino acid at position 290 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1, is replaced by an alanine (A) residue; and - The alanine (A) residue at position 298 of SEQ ID NO:1, or the amino acid residue at position 298 of SEQ ID NO:1 after optimal global alignment with SEQ ID NO:1, is replaced by an aspartic (D) residue.

4. The mutant polypeptide according to any one of claims 1 to 3, comprising the amino acid sequence defined in SEQ ID NO: 2: in: a) Amino acid residue X 271 It is any amino acid residue selected from arginine (R), histidine (H), lysine (K), glutamine (Q), valine (V), methionine (M), phenylalanine (F), leucine (L), tyrosine (Y), and proline (P); preferably amino acid residue X. 271 Selected from arginine (R), histidine (H), lysine (K), valine (V), methionine (M), and phenylalanine (F); more preferably, amino acid residue X. 271 Selected from arginine (R), lysine (K), valine (V), and methionine (M), or even more preferably, amino acid residue X. 271 Selected from arginine (R), lysine (K), and valine (V); and b) Amino acid residue X 35 X 46 X 49 X 60 X 91 X 93 X 123 X 124 X 129 X 154 X 176 X 177 X 191 X 236 X 238 X 262 X 268 X 270 X 290 X 298 and X 339 Independently selected from any amino acid; Among them, the following are preferred: i. Amino acid residue X 35 It is an amino acid residue S or G, preferably G; ii. Amino acid residue X 46 It is an amino acid residue S or A, preferably A; iii. Amino acid residue X 49 It is any amino acid residue selected from R, A, and S, preferably X. 49 It can be any amino acid residue A or S; iv. Amino acid residue X 60 It is amino acid residue A or R, preferably R; v. Amino acid residue X 91 It is amino acid residue A or R, preferably R; vi. Amino acid residue X 93 It is an amino acid residue P or C, preferably C; vii. Amino acid residue X 123 It is amino acid residue A or S, preferably S; viii. Amino acid residue X 124 It is amino acid residue A or P, preferably P; ix. Amino acid residue X 129 It is amino acid residue Q or M, preferably M; x. Amino acid residue X 154 It is amino acid residue A or P, preferably P; xi. Amino acid residue X 176 It is any amino acid residue selected from V, I, and L; preferably X. 176 It can be L or I, preferably I; xii. Amino acid residue X 177 It is an amino acid residue S or A, preferably A; xiii. Amino acid residue X 191 It is an amino acid residue M or V, preferably V; xiv. Amino acid residue X 236 It is an amino acid residue S or P, preferably P; xv. amino acid residue X 238 It is amino acid residue Q or K, preferably K; xvi. Amino acid residue X 262 It is amino acid residue A or C, preferably C; xvii. Amino acid residue X 268 It is any amino acid residue selected from E, Q, T, S, C, G, K, M, and R; preferably X. 268 It is any amino acid residue selected from Q, T, S, C, G, K, M, and R; preferably X. 268 It is any amino acid residue selected from Q, T, and S; xviii. Amino acid residue X 270 It is amino acid residue A or S, preferably S; xix. Amino acid residue X 290 It is amino acid residue D or A, preferably A; xx. Amino acid residue X 298 It is amino acid residue A or D, preferably D; xxi. Amino acid residue X 339 It is any amino acid residue selected from M, I, Q, and T; preferably X. 339 It is any amino acid residue selected from I, Q, and T, preferably X. 339 For I; or xxii. Any combination thereof.

5. The mutant polypeptide according to any one of the preceding claims, comprising the amino acid sequence shown in SEQ ID NO: 1, having one or a group of substitutions selected from: 1a. A124P, A154P, S177A, S236P, E268Q, E271R and A298D (mutant peptide M27); 2a. A124P, A154P, S177A, S236P, E268T, E271K and A298D (mutant peptide M28); 3a. A124P, A154P, S177A, S236P, E268S, A270S, E271K and A298D; 4a. A124P, A154P, S177A, S236P, E268Q, E271K and A298D; 5a. A124P, A154P, S177A, S236P, E268S, E271R and A298D; 6a. E268Q and E271K; 7a. E268T and E271K; 8a. E268T and E271R; 9a. A124P, A154P, S177A, S236P, E268Q, A270S, E271K and A298D; 10a. A124P, A154P, S177A, S236P, E268T, A270S, E271R and A298D; 11a. A124P, A154P, S177A, S236P, E268T, A270S, E271V and A298D; 12a. A124P, A154P, S177A, S236P, E268S, E271K and A298D; 13a. Q129M and V176I; 14a. E268Q and E271R (mutant peptide M29); 15a. A124P, A154P, S177A, S236P, E268Q, A270S, E271R and A298D; 16a. A124P, A154P, S177A, S236P, E268S, A270S, E271R and A298D; 17a. E271K; 18a. S35G, A60R, A91R, A123S, A124P, A154P, S177A, S236P, A270S and A298D (mutant peptide M22); 19a. E268S and E271K; 20a. P93C and A262C; 21a. E268T and E271V; 22a. A124P, A154P, S177A, S236P, E268T, A270S, E271K and A298D; 23a. E271R; 24a. S35G, S46A, A60R, A91R, A123S, A124P, A154P, S177A, M191V, S236P, Q238K, A270S, D290A and A298D (mutant peptide M23); 25a. R49A; and 26a. M339I.

6. The mutant polypeptide according to any one of the preceding claims, comprising, being primarily composed of, or consisting of an amino acid sequence selected from SEQ ID NO: 3-28.

7. The mutant polypeptide according to any one of the preceding claims, further comprising one or more additional mutations, such as mutations selected from substitution, deletion and insertion; preferably, wherein the one or more additional mutations are silent mutations or conserved mutations, particularly when the one or more additional mutations are substitutions.

8. A nucleic acid molecule comprising a nucleic acid sequence encoding a mutant polypeptide according to any one of claims 1 to 7, preferably wherein: a) Nucleic acid molecules are isolated; or b) The nucleic acid molecule further contains a promoter that controls the expression of the nucleic acid sequence; or c) The isolated nucleic acid molecule further contains a transcription terminator that controls the expression of the nucleic acid sequence; or d) The nucleic acid sequence is further optimized for expression in host cells, particularly in yeast or bacteria; or e) Any combination of a) to d); Preferably, the nucleic acid molecule comprises, is mainly composed of, or is composed of a nucleic acid sequence, wherein the nucleic acid sequence has at least 80% identity with a nucleic acid sequence selected from SEQ ID NO: 46-72, more preferably at least 85% identity, and even more preferably at least 90% identity.

9. The nucleic acid molecule according to claim 8, wherein: a) When the nucleic acid molecule further includes a promoter that controls the expression of the nucleic acid sequence, said promoter is an exogenous promoter, particularly a yeast promoter; or b) When the isolated nucleic acid molecule further contains a transcription terminator that controls the expression of the nucleic acid sequence, the transcription terminator is an exogenous terminator, such as the yeast terminator; or c) The nucleic acid sequence was further optimized for expression in yeast.

10. A vector comprising at least one nucleic acid molecule according to any one of claims 8 to 9, or expressing a mutant polypeptide according to any one of claims 1 to 7, wherein the vector is preferably a plasmid.

11. A host cell comprising or expressing a mutant polypeptide according to any one of claims 1 to 7, or comprising a nucleic acid molecule according to any one of claims 8 or 9, or comprising a vector according to claim 10, or any combination thereof.

12. The host cell according to claim 11, wherein it is yeast or bacteria; preferably selected from the genus *Saccharomyces* (…). Saccharomyces ), Candida ( Candida ), Ashu pseudocystis ( Ashbya ), Decker yeast ( Dekkera ), Pichia pastoris ( Pichia (Hansenula polymorpha) Hansenula ), Debali yeast ( Debaryomyces ), *Corydalis* genus ( Clavispora ), Lode yeast ( Lodderomyces ), Yeastra genus ( Yarrowia ), Zygosaccharomyces ( Zigosaccharomyces ), genus *Fissionyomyces* ( Schizosaccharomyces ), Rhodotorula spp. ( Torulaspora Kluyveromyces ( ) Kluyveromyces ), Brett's yeast ( Brettanomycces Cryptococcus ( Cryptococcus ) and Malassezia ( Malassezia Yeast selected from the species *Saccharomyces cerevisiae*; more preferably, yeast selected from the species *Saccharomyces cerevisiae*. Saccharomyces cerevisiae ), Saccharomyces boulardii ( Saccharomyces boulardii ), Douglas yeast ( Saccharomyces douglasii Pasteur yeast ( Saccharomyces bayanus ), Bydion conjugated yeast ( Zigosaccharomyces bailii ), Saccharomyces cerevisiae ( Schizosaccharomyces pombe ), Brussels yeast ( Dekkera brucelensis ), intermediate Dike yeast ( Dekkera intermedia ), Kusterbreed yeast ( Brettanomycces custersii ), intermediate Brett yeast ( Brettanomycces intermedius ), heat-resistant Kluyveromycin ( Kluyveromyces themotolerens ), Spherical red thallus yeast ( Torulaspora globosa ) and smooth round red thallus yeast ( Torulaspora glabrata Yeasts, or yeasts selected from the genus Escherichia ( Escherichia ), Pseudomonas spp. Pseudomonas Streptomyces ( Streptomyces Corynebacterium spp. Corynebacterium ), Bacillus spp. ( Bacillus ) and Lactobacillus genus ( Lactobacillus ( ) bacteria; even more preferably yeasts of the genus Yeast or bacteria of the genus Escherichia, preferably yeasts of the species Saccharomyces cerevisiae or bacteria of the species Escherichia coli.

13. A composition comprising a mutant polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to any one of claims 8 or 9, a vector according to claim 10, a host cell according to claim 11 or 12, or any combination thereof.

14. Use of the mutant polypeptide according to any one of claims 1 to 7, the nucleic acid molecule according to any one of claims 8 or 9, the vector according to claim 10, the host cell according to claim 11 or 12, or any combination thereof, for the production of phloroglucinol.

15. A method for producing phloroglucinol, comprising the following steps: a) Contact a host cell expressing the mutant polypeptide according to any one of claims 1 to 7 with a suitable substrate; b) The host cells of step a) are cultured in vitro under conditions that allow for the expression of nucleic acid molecules contained in the host cells, thereby producing phloroglucinol; c) Optionally, recover the phloroglucinol-containing culture medium obtained after step b); and d) Optionally, phloroglucinol is purified from the culture medium of step c).

16. A method for producing phloroglucinol, comprising the following steps: a) Contacting the mutant polypeptide according to any one of claims 1 to 7 with malonyl-CoA; b) Incubate the mixture obtained in step a) under conditions suitable for the production of phloroglucinol; c) Optionally, recover the reaction medium containing phloroglucinol obtained after step b); and d) Optionally, purify phloroglucinol from the reaction medium of step c).

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