Enzymes with tyrosine ammonia lyase capability and uses thereof

Abstract

The present invention is in the field of enzyme and microorganism comprising such a modified enzyme to produce flavors, in particular raspberry ketone or zingerone. The enzymes of the invention are used for producing p-coumaric acid (PCA) from a tyrosine substrate, in particular from L-tyrosine. The invention also relates to microorganisms expressing such enzymes with Tyrosine Ammonia Lyase for producing flavors.

Description

DescriptionTitle: Enzymes with tyrosine ammonia lyase capability and uses thereofTechnical field

[0001] The present invention is in the field of enzyme and microorganism comprising such a modified enzyme to produce flavors, in particular raspberry ketone or zingerone. The enzymes of the invention are used for producing p- coumaric acid (PCA) from a tyrosine substrate, in particular from L-tyrosine. The invention also relates to microorganisms expressing such enzymes with Tyrosine Ammonia Lyase for producing flavors.Prior art

[0002] The bioproduction of "natural" flavors and fragrances has been an important area of research for the industry for many years in order to meet the needs of consumers who are increasingly concerned about being environmentally responsible. Synthetic biology, in particular through the use of microorganisms, allows this natural production, but the yields are not always sufficient for large-scale production.

[0003] The flavor of raspberry (Rubus idaeus) is linked to more than 200 compounds, but raspberry ketone (also known under the name raspinone or frambinone, these terms being interchangeably used herein), a naturally occurring phenolic compound, is the most impactful compound, defining its characteristic flavor (Klesk et al., 2004, J. Agric. Food Chem. 52, 5155- 61 ; Larsen et al., 1991 , Acta Agric. Scand. 41 , 447-54).

[0004] Since it is present only in small amounts in raspberries (1 -4 mg per kg of fruit), natural raspberry ketone is of great value (Larsen et al., 1991 ). However, because its natural availability is limited, its biotechnological production is highly desirable. However, raspberry flavor is a major ingredient for flavor and flagrance industry , and is thus very much in demand.

[0005] Raspberry ketone is a polyketide. Polyketides represent a large family of metabolites produced by plants. Some of them are widely used in medicine, agriculture, and cosmetic industry. Flavonoids, stilbenes, benzalacetones, are polyketides.

[0006] Zingerone, also called vanillylacetone, is a major flavor component of ginger (Zingiber officinale). Zingerone is similar in chemical structure to other flavor chemicals such as vanillin, eugenol or raspberry ketone. It is used as a flavor additive in spice oils and in perfumery to introduce spicy aromas.

[0007] The current method for synthesizing zingerone involves chemistry reaction between vanillin and acetone under basic conditions to form dehydro-zingerone. This reaction is followed by catalytic hydrogenation of the intermediate compound to form zingerone.

[0008] Thus, it is possible to produce synthetic flavors like zingerone or raspberry ketone, but biosynthesis is nowadays preferable and especially gives a natural molecule unlike the petrochemical production route.

[0009] In this context, the biosynthetic pathway of phenylpropanoid compounds, can be reconstituted within a microorganism through the insertion of heterologous genes encoding certain key enzymes of said pathway, thereby producing raspberry ketone or zingerone in recombinant microorganism. Phenylpropanoids are pivotal molecules involved in the biosynthesis of fragrances and aromas across a wide range of plants. The biosynthesis of these compounds can be efficiently achieved within a microorganism by expressing heterologous proteins from other organisms or homologous / heterologous proteins that have been engineered to enhance their catalytic properties or specificity. Expressing genes that encode these critical enzymes required for this pathway allow the provision of highly efficient flavorproducing microorganisms.

[0010] The raspberry ketone and zingerone production pathways both follow the phenylpropanoid production pathway in plants. Phenylpropanoids are key molecules for flavor production in most plants. The phenylpropanoid biosynthetic pathway can be reconstituted within a microorganism by inserting heterologous plant genes encoding some key enzymes of the pathway such as TyrosineAmmonia Lyase (“TAL”, catalyzing the transformation of Tyrosine into coumaric acid) and polyketide Synthases (“PKS”, catalyzing the transformation of cinnamoyl- CoA derivatives into benzalacetones. These benzalacetones can thereafter be reduced by reductase enzymes to synthesize aromatic compounds such as zingerone and raspberry ketone.

[0011] For producing zingerone, ferulic acid is converted into feruloyl-CoA by a 4- coumarate-ligase. Feruloyl-CoA is thereafter converted into vanillylidene acetone (or “VDA”) by a type III polyketide synthase with benzalacetone synthase (BAS) activity. VDA is then reduced into zingerone by a benzalacetone reductase enzyme (or “BAR”).

[0012] The production of raspberry ketone is made from p-coumaroyl-CoA by a 2- step reaction catalyzed by a type III polyketide synthase with an enzyme that has BAS activity. This BAS enzyme catalyzes the condensation of a p-coumaroyl-CoA molecule with a malonyl-CoA molecule into a 4-hydroxybenzalacetone (Abe et al., 2001 ). The 4-hydroxybenzalacetone molecule (or “4-HBA”) is then reduced to raspberry ketone by a benzalacetone reductase enzyme (or “BAR”). This pathway was recently implemented in the yeast Saccharomyces cerevisiae and resulted in the de novo production of raspberry ketone (Lee et al., 2016).

[0013] Among the phenylpropanoids, p-coumaric acid, is an aromatic compound with extensive applications in the food, cosmetics, and pharmaceutical industries due to its beneficial properties. Tyrosine Ammonia Lyases produce p-coumaric acid from tyrosine and are involved in the pathways for producing zingerone and raspberry ketone.

[0014] To facilitate the conversion of tyrosine into p-coumaric acid within a microbial system, the expression of a heterologous Tyrosine Ammonia Lyase (TAL) enzyme is the current state of the art. However, one of the issues that is encountered in the bioproduction of p-coumaric acid lies in the absence of highly active and substratespecific TAL enzymes. Identifying and providing an enzyme with an enhanced Tyrosine Ammonia Lyase like activity is a major challenge for industrial-scale production. The same challenge is present for identifying and providing a providing an enzyme with a Tyrosine Ammonia Lyase like activity that specifically transformstyrosine into p-coumaric acid. This challenge is exacerbated by the need to optimize key production parameters, including yield, titer, downstream purification processes, and the purity of the final product. Therefore, the provision of new TAL enzymes with enhanced TAL activity and / or with specificity towards tyrosine is critical for the efficient biosynthesis of phenylpropanoids in microorganisms.

[0015] So far, the use of microorganisms as production hosts has been developed to address these issues. It is now possible to engineer bacterial strains expressing TALs that are capable of synthesizing phenylpropanoid acids with better efficiency and specificity (Nijkamp et al., 2007; Limem et al., 2008; Vannelli et al., 2007). These engineered bacterial systems offer a sustainable and scalable approach to producing phenylpropanoid acids, providing significant advantages in terms of yield, cost-effectiveness, and environmental. Indeed, the heterologous expression of Tyrosine Ammonia Lyases (TAL) or Phenylalanine Ammonia Lyases (PAL) enzymes enables the direct catalytic deamination of L-tyrosine (L-Tyr) to p-coumaric acid (PCA) and L-phenylalanine (L-Phe) to cinnamic acid (CA), respectively. Incorporating these enzymes into a bacterial host is a critical modification for the microbial production of p-coumaric acid or cinnamic acid via a fermentation process.

[0016] Tyrosine Ammonia Lyase (TAL) enzymes were initially discovered in various plants, including sorghum, rice, wheat, and parsley, before being identified in microorganisms. The pioneering work of Kyndt et al. (2002) led to the isolation of the RcTAL gene from Rhodobacter capsulatus, where it was linked to the synthesis of the photoactive yellow protein (PYP) chromophore. This gene was subsequently heterologously expressed in Escherichia coli, marking a significant milestone in the study of TAL enzymes.

[0017] In subsequent years, several other TAL enzymes were identified and characterized, including RsTAL from Rhodobacter sphaeroides (Watts et al., 2006), Se-sam8 from Saccharothrix espanaensis (Berner et al., 2006), and RgTAL from Rhodotorula glutinis (Vannelli et al., 2007). Among these, RgTAL has been reported to exhibit superior catalytic activity compared to other bacterial and fungal TAL variants, making it a particularly promising candidate for biotechnological applications (Vannelli et al., 2007). The international patent applicationWO 2022 / 238645 discloses the use of a recombinant gene encoding a RgTAL, preferably modified with three point mutations in this TAL enzyme make it more effective: S9N; A11T; E518V.

[0018] However, one significant challenge with Tyrosine Ammonia Lyase (TAL) enzymes is their generally low catalytic activity towards L-tyrosine, which limits their effectiveness in industrial processes that demand high titers of the target molecule, p-coumaric acid. This low activity presents a bottleneck in achieving commercially viable production levels.

[0019] Additionally, the ability to provide a microbial strain that exclusively produces p-coumaric acid would greatly simplify the downstream processing, leading to a final product of higher purity and reducing the complexity and cost of purification of the final product.

[0020] Further, another significant challenge is that most TALs exhibit dual activity, functioning as both TAL and PAL, which underscores their versatility but also highlights the challenge of achieving high specificity for targeted phenylpropanoid production.

[0021] This statement is illustrated in the international patent application WO 2020 / 123286 discloses engineered TAL enzymes from TAL and PAL parent enzymes obtained from Stanieria cyanosphera, Chroogloeocystis siderophila, Flavobacterium johnsoniae, and Rhodotorula glutinis, for providing pharmaceutical compositions for the treatment of tyrosinemia, in which the engineered TAL comprise not only TAL activity but also may be active on phenylalanine.

[0022] Given these challenges, the identification and development of novel TAL enzymes with enhanced catalytic activity and / or high specificity for L-tyrosine, and being unable to transform phenylalanine into cinnamic acid, are crucial for advancing industrial applications. Such advancements would enable more efficient and scalable production of p-coumaric acid, meeting the demands of bio-industrial processes.

[0023] To sum up, current limitations of Tyrosine Ammonia Lyase (TAL) enzymes, particularly their non-specificity and low catalytic activity, fall short of therequirements needed for the industrial-scale bioproduction of p-coumaric acid. These shortcomings hinder the ability to achieve the high yields and titers necessary for a commercially viable and economically profitable process. To overcome these challenges, the discovery and development of more efficient TAL enzymes are imperative.

[0024] Moreover, for the downstream purification of p-coumaric acid to be more efficient and to obtain a final product of higher purity, it is essential to identify TALs with greater substrate specificity. Such advancements would not only streamline the production process but also significantly enhance the overall quality and economic feasibility of p-coumaric acid bioproduction at an industrial scale.

[0025] One object of the present invention is thus to provide new enzymes that have Tyrosine Ammonia Lyase activity or Tyrosine Ammonia Lyase-like activity, that have an enhanced catalytic activity as compared to wild-type TALs, in particular wild-type TAL enzyme from Rhodotorula glutinis.

[0026] Another object of the present invention is thus to provide new enzymes that have Tyrosine Ammonia Lyase activity or Tyrosine Ammonia Lyase-like activity, that have an enhanced specificity for tyrosine, in particular L-tyrosine, as compared to wild-type TALs, in particular wild-type TAL enzyme from Rhodotorula glutinis.

[0027] Another object of the present invention is to provide new enzymes that have Tyrosine Ammonia Lyase activity or Tyrosine Ammonia Lyase-like activity, and / or that have an enhanced specificity for tyrosine, in particular L-Tyrosine, as compared to wild-type TALs, in particular wild-type TAL enzyme from Rhodotorula glutinis, the new enzymes not having a Phenylalanine Ammonia Lyase activity or Phenylalanine Ammonia Lyase- like activity. In other words, the provided enzymes do not possess the capability to produce cinnamic acid starting from phenylalanine.

[0028] One of the aims of the invention is to provide new TAL enzymes and genetically modified microorganism comprising means for expressing such new TAL enzymes allowing a more efficient production of p-coumaric acid, in particular from a tyrosine, more particularly from L-tyrosine compound.

[0029] In particular, there is a need for an enzyme with a TAL activity that enhances the conversion of tyrosine, in particular of L-tyrosine, into p-coumaric acid, as compared to wild-type TAL enzyme from Rhodotorula glutinis. There is also a need for an enzyme with a TAL activity that enhances the conversion of tyrosine, in particular L-tyrosine, into p-coumaric acid, as compared to the wild-type TAL from Stanieria cyanosphera, or from Chroogloeocystis siderophila.Summary of the invention

[0030] In a first aspect of the present invention, it is provided an enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 95% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, for transforming a tyrosine substrate into p-coumaric acid.

[0031] In another aspect of the present invention, it is provided an enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 70% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, the enzyme of the invention being unable to transform L- phenylalanine (L-Phe) into cinnamic acid (CA), the enzyme of the invention being for transforming a tyrosine substrate into p-coumaric acid.

[0032] The enzymes that have the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3 have been identified as possessing a Tyrosine Ammonia Lyase activity. These three enzymes and their functional equivalents have the capability to transform tyrosine into p-coumaric acid, which was not known before. These three enzymes are further unable to transform phenylalanine into cinnamic acid.

[0033] When compared to wild-type TALs, in particular the TAL from Rhodotorula glutinis, or from Stanieria cyanosphera, or from Chroogloeocystis siderophila, these three enzymes possess a better catalytic activity on the transformation of tyrosine into p-coumaric acid. In other words, these enzymes produce more rapidly or to agreater extent p-coumaric acid under the same conditions as compared to wild-type TALs.

[0034] The enzymes that have the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3 are not able to transform L-phenylalanine (L-Phe) into cinnamic acid (CA), and thus do not possess a Phenylalanine Ammonia Lyase or Phenylalanine Ammonia Lyase-like activity. In other words, these three enzymes are unable to produce cinnamic acid. These enzymes thus do not possess a Phenylalanine Ammonia Lyase activity and are not able to transform L- phenylalanine into cinnamic acid. These three enzymes and their functional equivalents target specifically the transformation of tyrosine into p-coumaric acid.

[0035] The three enzymes and their functional equivalents identified by the inventors are thus more efficient for producing p-coumaric acid, and have an enhanced specificity for tyrosine, allowing the enhancement of the effectiveness of the bioproduction of flavors, in particular zingerone and raspberry ketone, in microorganisms.

[0036] In another aspect of the present invention, it is provided a recombinant microorganism, in particular a recombinant Pseudomonas putida, capable to express or expressing an enzyme having a TAL activity for transforming L-tyrosine into p-coumaric acid.

[0037] In another aspect, it is provided a genetically modified microorganism, in particular a recombinant microorganism, more particularly a recombinant Pseudomonas putida, capable to express or expressing a provided an enzyme which is has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

[0038] The recombinant microorganism, more particularly the recombinant Pseudomonas putida, is in particular provided for the production of p-coumaric acid, in particular from tyrosine, more particularly L-tyrosine.

[0039] The recombinant microorganism, more particularly the recombinant Pseudomonas putida, is in particular provided for the production of zingerone and / or raspberry ketone.

[0040] In another aspect, it is provided a process for the synthesis of p-coumaric acid, in particular from tyrosine, more particularly from L-tyrosine, by using an enzyme which is has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 95% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, and / or by using a genetically modified microorganism, in particular a recombinant microorganism, more particularly a recombinant Pseudomonas putida, capable to express or expressing an enzyme which is has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 95% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

[0041] In another aspect, it is provided a process for the synthesis of p-coumaric acid, in particular from tyrosine, more particularly from L-tyrosine, by using an enzyme which is has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 70% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, the enzyme of the invention being unable to transform L-phenylalanine (L-Phe) into cinnamic acid (CA), and / or by using a genetically modified microorganism, in particular a recombinant microorganism, more particularly a recombinant Pseudomonas putida, capable to express or expressing an enzyme which is has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 70% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, the enzyme of the invention being unable to transform L-phenylalanine (L-Phe) into cinnamic acid (CA).

[0042] The invention is defined in the independent claims. Dependent claims define preferred embodiments. The features set forth in the following paragraphs mayoptionally be implemented. They can be implemented independently of each other or in combination with each other.Short description of the drawingsThese and further aspects of the invention will be explained in greater detail by way of examples and with reference to the accompanying drawings in which:Fig. 1

[0043] Fig. 1 is a graph illustrating the concentration of p-Coumaric acid (pCA) in supernatants of cultured recombinant P. putida expressing wild-type TALs or selected enzymes that are tested for assessing their capability to possess a TAL activity. The abscissa axis represents the different recombinant P. putida, some of which incorporate TAL or TAL like enzymes according to the invention are defined according to table 2. The concentration of p-coumaric acid is represented on the ordinate axis (g. of p-coumaric acid per L. of culture medium).Fig. 2

[0044] Fig. 2 is a graph illustrating the concentration of p-Coumaric acid (pCA) and cinnamic acid in supernatants of cultured recombinant P. putida expressing wildtype TALs or selected enzymes that are tested for assessing their capability to possess a TAL activity but not PAL activity. The abscissa axis represents the different recombinant P. putida, some of which incorporate TAL or TAL like enzymes according to the invention are defined according to table 2. The concentration of p- coumaric acid and cinnamic acid is represented on the ordinate axis (g. of p- coumaric acid or cinnamic acid per L. of culture medium).Detailed description of embodiments of the invention

[0045] In a first aspect, it is provided an enzyme that has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 95% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3. These enzymesand their functional equivalents are for use in the transformation of a tyrosine substrate, in particular L-tyrosine, into p-coumaric acid.

[0046] The enzyme to be used according to the invention and their functional equivalents are able to catalyze the following reaction:Compound (I): L-tysosine - substrate compound (II): p-coumaric acid - product

[0047] The enzyme is a polypeptide that may comprise or consist in the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3. In an embodiment, the enzyme is a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

[0048] The enzyme having the sequence set forth in SEQ ID No. 1 is issued from Chroococcales cyanobacterium. Whole genome of Gloeocapsopsis sp., member of this species is referenced in the Genome Online Database (GOLD) under number Go0545253.

[0049] The enzyme having the sequence set forth in SEQ ID No. 2 is issued from Stanieria sp. Whole genome of this species is referenced in the Genome Online Database (GOLD) under number Go0408787.

[0050] The enzyme having the sequence set forth in SEQ ID No. 3 is issued from Cylindrospermum sp. The known genome of this species is referenced in the NCBI GenBank assembly under number AS M1469719v1 .

[0051] The enzymes and their functional equivalents have the capability to convert a tyrosine substrate, in particular L-tyrosine into p-coumaric acid. In a particular embodiment, the enzymes and their functional equivalents convert L-tyrosine into p-coumaric acid in a more efficient manner than a wild-type TAL, in particular a wildtype TAL from Rhodotorula glutinis (RgTAL), or from Chroogloeocystis siderophile (CsTAL), or from Flavobacterium johnsoniae (FjTAL), or from Stanieria cyanosphaera (ScTAL). In a particular embodiment of the invention, the enzymes and their functional equivalents convert L-tyrosine into p-coumaric acid in a more efficient manner than RgTAL, in particular RgTAL having the amino acid sequence set forth in SEQ ID No. 4.

[0052] In a particular embodiment of the invention, the enzymes to be used according to the invention and their functional equivalents have an improved catalytic activity on the transformation of L-tyrosine into p-coumaric acid as compared to the wild-type RgTAL having the amino acid sequence set forth in SEQ ID No. 4. In a more particular aspect, the enzymes to be used according to the invention and their functional equivalents improve at least 2, 3, 4, 5, 6 or 7 times more the transformation of a L-tyrosine into p-coumaric acid as compared to the catalytic activity of a wild-type TAL, in particular the wild type TAL of SEQ ID No. 4.

[0053] In a particular aspect of the invention, the enzymes to be used according to the invention and their functional equivalents have an improved catalytic activity on the transformation of L-tyrosine into p-coumaric acid as compared to the catalytic activity of a wild-type TAL and are not able to transform L-phenylalanine (L-Phe) into cinnamic acid (CA).

[0054] The enzymes to be used according to the invention and their functional equivalents possess a TAL capability. In other words, they have a catalytic activity on the transformation of L-tyrosine into p-coumaric acid. This capability can be determined by measuring by HPLC the total concentration of p-coumaric acid before and after a catalytic reaction of transforming L-tyrosine into p-coumaric acid.

[0055] In a particular embodiment of the invention, the enzymes to be used according to the invention and their functional equivalents are not able to transform L-phenylalanine (L-Phe) into cinnamic acid (CA). In other words, they are not able to exert a PAL activity, in particular when compared to the PAL activity exerted by a wild-type TAL, for example the TAL of SEQ ID No. 4. In an aspect, the enzymes to be used according to the invention and their functional equivalents are 2, 3, 4, 5, 6 or at least 7 times less efficient for transforming L-phenylalanine (L-Phe) intocinnamic acid (CA). as compared to the catalytic activity of a wild-type TAL, in particular the wild type TAL of SEQ ID No. 4.

[0056] As an example, the catalytic activity of the enzymes to be used according to the invention and their functional equivalents may be determined by measuring the catalytic constant kcat of the enzymes to be used according to the invention and their functional equivalents in a production reaction of a p-coumaric acid from L- tyrosine and / or in a production reaction of L-phenylalanine into cinnamic acid. It can be calculated from the maximum reaction rate and catalyst site concentration by methods known by the skilled artisan. The PAL catalytic activity of enzymes to be used according to the invention can be assessed by performing the experiments detailed in example 3 of the present description.

[0057] The functional equivalents of the enzyme having the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 may be obtained by genetic engineering of the enzyme from which the functional equivalent is derived, or from a gene or nucleic acid molecule encoding such an enzyme. One or more non-natural mutations can be introduced into the enzyme, for example by insertion, substitution or deletion of nucleotides encoding the enzyme, said mutations being obtained by transformation techniques or by gene editing techniques known to the skilled artisan.

[0058] Genetic modification techniques by transformation, mutagenesis or gene editing are described for example in "Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation", Journal of Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(2):83-99, and in Martinez-Garcia and de Lorenzo, "Pseudomonas putida in the quest of programmable chemistry", Current Opinion in Biotechnology, 59 :111 -121 , 2019.

[0059] By derived from an enzyme, it should be understood that the functional equivalent of the invention is a polypeptide that has a modified amino acid sequence as compared to the enzyme from which it is derived. The modification can be obtained by mutation, including by substitution (including conservative amino acid residue(s)) or by addition and / or deletion of amino acid residues or by secondary modification after translation or by deletion of portions of the wild-type proteins(s)resulting in fragments having a shortened size with respect to the wild-type protein of reference. Fragments of the enzymes having the amino acid sequence set forth in SEQ ID No. 1 , or SEQ ID No. 2 or SEQ ID No. 3, are encompassed within the present invention to the extent that they possess the capability to transform L- tyrosine into p-coumaric acid.

[0060] A functional equivalent of the enzyme having the amino sequence set forth in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 is a protein having a polypeptide sequence which is derived from the polypeptide sequence of SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3, which may comprise modification(s), i.e. substitution(s), insertion(s) and / or deletion(s) of one or more amino acids but which retains the TAL activity of the enzyme from which it is derived, and in particular the capability to convert tyrosine into p-coumaric acid as described above with at least the same catalytic activity. Preferably but not necessarily, the functional has at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% with the enzyme form which it is derived.

[0061] The TAL activity of a functional variants can be assessed by any method known to the skilled person, in particular as illustrated in the example of the invention, by expressing in a strain of Pseudomonas putida a recombinant gene encoding the functional variant, preferably cloned into a plasmid downstream of a promoter allowing its expression in the strain, and culturing the strain in the presence of L- tyrosine and assaying by HPLC the total concentration of p-coumaric acid, produced by the strain after 24h. The functional equivalent maintains at least the same TAL activity as compared to the TAL activity measured when the enzyme having the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID NO. 3 is expressed in the strain of Pseudomonas putida in the same experimental condition. The TAL activity is determined according to the amount or concentration of p- coumaric acid produced by the strain during a determined period.

[0062] The TAL activity of a protein that may be a functional equivalent may be determined by measuring the quantity of p-coumaric acid by HPLC in the following test:In a 200 pl medium comprising:- 60mM Tris-HCI pH 8,5- 1 mM Tyrosine0.5 pg / pL of the protein to be tested is added.The medium with the enzyme or functional equivalent is incubated during 24 hours at 30°C.After this 24h period, the enzymatic reaction is stopped by adding 50mM of HCI.The production of p-coumaric acid is measured by HPLC.An enzyme is considered to be a functional equivalent of the enzyme of SEQ ID No. 1 or SEQ Id No. 2 or SEQ ID No. 3 when the production of p-coumaric acid in presence of the tested protein is at least equal to the production of p-coumaric acid in presence of the enzyme of SEQ ID No. 1 or SEQ Id No. 2 or SEQ ID No. 3.

[0063] The PAL activity of a functional variants can be assessed by any method known to the skilled person, in particular as illustrated in the example of the invention, by expressing in a strain of Pseudomonas putida a recombinant gene encoding the functional variant, preferably cloned into a plasmid downstream of a promoter allowing its expression in the strain, and culturing the strain in the presence of L- phenylalanine and assaying by HPLC the total concentration of cinnamic acid, produced by the strain after 24h. The functional equivalent does not have a PAL activity when the amount or concentration of cinnamic acid produced by the strain during a determined period is 2, 3, 4, 5, 6 or at least 7 times less than the concentration of cinnamic acid produced by the strain expressing the wild type TAL of SEQ ID No. 4.

[0064] The PAL activity of an enzyme that may be a functional variant as defined herein may be determined by measuring the quantity of cinnamic acid by HPLC in the following test:In a 200 pl medium comprising:- 60mM Tris-HCI pH 8,5- 1 mM phenylalanine0.5 pg / pL of the protein to be tested is added.The medium with the enzyme or functional equivalent is incubated during 24 hours at 30°C.After this 24h period, the enzymatic reaction is stopped by adding 50mM of HCI.The production of cinnamic acid is measured by HPLC.An enzyme is considered to be a functional equivalent of the enzyme of SEQ ID No. 1 or SEQ Id No. 2 or SEQ ID No. 3 when the production of cinnamic acid in presence of the tested enzyme is at least inferior to the production of cinnamic acid in presence of the enzyme of SEQ ID No. 4.

[0065] Preferably, a functional equivalent of the enzyme having the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 corresponds to a polypeptide sequence having at least 70%, 80%, 85%, 90%, 95% and most particularly, at least 98% identity with one of the sequences selected from SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3. A percentage identity refers to the percentage of identical residues in a nucleotide or amino acid sequence on a given fragment after alignment and comparison with a reference sequence. For the comparison, an alignment algorithm is used and the sequences to be compared are entered with the corresponding parameters of the algorithm. The default parameters of the algorithm can be use. In a particular embodiment, for a nucleic acid or polypeptide sequence comparison and determination of a percent identity, the blastn or blastp algorithm as described in https: / / blast.ncbi.nlm.nih.gov / Blast.cgiavec is used, in particular with default parameters. In particular, the functional variant refers to a polypeptide that has an amino acid sequence that differs from one of the sequences selected from SEQ ID NO: 1 or SEQ ID No. 2 or SEQ ID No. 3 by less than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 substitutions, insertions or deletions. In another particular embodiment, the functional variant refers to a polypeptide that has an amino acid sequence that differs from one of the sequences selected from SEQ ID NO: 1 or SEQ ID No. 2 or SEQ ID No. 3 by fewer than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 substitutions, the substitutions preferably being conservative substitutions. The term "conservative substitution" as used herein refers to the replacement of one amino acid residue with another, without altering the conformation or enzymatic activity of thepolypeptide so modified, including, but not limited to, the replacement of one amino acid with another having similar properties (such as, for example, polarity, hydrogen bonding potential, acidity, basicity, shape, hydrophobicity, aromaticity and the like). Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine).

[0066] In a more particular embodiment, the enzyme to be used according to the invention is a polypeptide that consists in the amino acid sequence set forth in SEQ ID No. 1.

[0067] In a more particular embodiment, the enzyme to be used according to the invention is a polypeptide that consists in the amino acid sequence set forth in SEQ ID No. 2.

[0068] In a more particular embodiment, the enzyme to be used according to the invention is a polypeptide that consists in the amino acid sequence set forth in SEQ ID No. 3.

[0069] In a particular embodiment, the enzyme or its functional equivalent to be used according to the invention is recombinant.

[0070] In another embodiment, it is provided a nucleic acid molecule encoding an enzyme to be used according to the invention or their functional equivalents. In a preferred embodiment, the nucleic acid encodes an enzyme or a functional equivalent according to any embodiment disclosed herein. In a particular embodiment, the nucleic acid molecule that encodes an enzyme of a functional equivalent according to the invention has the sequence set forth in SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7. In a particular embodiment, the nucleic acid molecule that encodes an enzyme or a functional equivalent according to the invention is a plasmid, allowing expressing of the enzyme or the functional equivalent in a cell, in particular in a bacteria.In an embodiment, the enzyme is encoded by the nucleotide sequence set forth in SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7. In an embodiment, the enzyme is encoded within a vector, in particular a plasmid, or from a gene inserted into the chromosome under the control of a constitutive promoter.

[0072] As used herein, a vector is a nucleic acid molecule used as a vehicle to transfer a genetic material into a cell, and in a preferred embodiment allows the expression of a gene encoding the enzyme or a functional equivalent inserted within the vector. The term vector encompasses plasmids, viruses, cosmids and artificial chromosomes. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (a transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene ( / .e. the gene encoding the enzyme of the functional equivalent of the invention) in a target cell (i.e. a genetically engineered Pseudomonas putida), and generally have control sequences.

[0073] In another embodiment of the invention, the applicant has developed a bacterial strain, in particular a strain of Pseudomonas putida, capable of expressing an enzyme with an improved TAL activity and / or with a better specificity for L- tyrosine to more efficiently produce a p-coumaric acid from L-tyrosine.

[0074] According to another particular embodiment, the genetically modified Pseudomonas putida strain comprises an additional recombinant gene encoding a polypeptide with TAL activity. In particular, the modified strain may comprise a recombinant gene encoding an enzyme whose sequence corresponds to the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID NO: 3 or by a sequence having at least 70%, 80%, 85%, 90%, 95% and most particularly, at least 98% identity with the sequence SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID NO: 3. More particularly, the genetically modified Pseudomonas putida strain comprises an additional recombinant gene encoding an enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functionalequivalent thereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3. The genetically modified strain is particularly efficient for producing p-coumaric acid, in particular from a tyrosine substrate, in particular from L-tyrosine. Thus, the genetically modified strain may be used for transforming L-tyrosine into p-coumaric acid.

[0075] According to a particular embodiment, the genetically modified strain, in particular strain of Pseudomonas putida, comprises an additional recombinant gene encoding a polypeptide comprising or having or consisting of the nucleotide sequence set forth in SEQ ID No. 1 .

[0076] According to a particular embodiment, the genetically modified strain, in particular strain of Pseudomonas putida, comprises an additional recombinant gene encoding a polypeptide comprising or having or consisting of the nucleotide sequence set forth in SEQ ID No. 2.

[0077] According to a particular embodiment, the genetically modified strain, in particular strain of Pseudomonas putida, comprises an additional recombinant gene encoding a polypeptide comprising or having or consisting of the nucleotide sequence set forth in SEQ ID No. 3.

[0078] According to a particular embodiment, the genetically modified Pseudomonas putida strain comprises a nucleic acid, in particular vector, more particularly a plasmid, comprising or having or consisting of the nucleotide sequence set forth in SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7.

[0079] According to this particular embodiment, the genetically modified strain is capable of converting L-tyrosine into p-coumaric acid via the enzyme or its functional equivalent.

[0080] Thus, an object of the invention relates to a genetically modified strain, in particular a strain of Pseudomonas putida, characterized in that it is capable of expressing or expresses a recombinant gene encoding an enzyme capable of producing p-coumaric acid from L-tyrosine.

[0081] In a preferred embodiment, the Pseudomonas putida strain according to the present application is capable of producing zingerone and / or raspberry ketone.

[0082] In another preferred embodiment, said genetically modified strain, in particular genetically modified Pseudomonas putida strain, is characterized in that it expresses a recombinant gene encoding an enzyme or a functional equivalent according to the invention and at least one gene encoding a benzalacetone reductase, in particular selected from the group consisting of the NADPH-dependent 2-alkenal reductase of Arabidopsis thaliana (Uniprot Q39172, updated June 2, 202, the ene-reductase from Zingiber officinale (Uniprot A0A096LNF0, updated on April 7, 2021 ), the NADPH-dependent curcumin reductase, also called CurA from Pseudomonas Putida (Uniprot Q88K17, updated on December 2, 2020) NADP- dependent alkenal double bond reductase, also known as DBR from Olimarabidopsis pumila (Uniprot A0A1 C9CX65, updated August 12, 2020), NADP(+)-dependent 2-alkenal reductase, also known as DBR from Nicotiana tabacum (Uniprot Q9SLN8 ; EC 1. 3.1.102), or the NADP(+)-dependent 2-alkenal reductase, also known as Red from Capsicum annuum (Uniprot A0A1 U8GFY1 , updated 10 February 2021 ).

[0083] In a particular embodiment, the invention relates to a genetically modified strain, in particular a genetically modified Pseudomonas putida strain, characterized in that it expresses a recombinant gene encoding a functional equivalent of a previously described benzalacetone synthase. A benzalacetone synthase may be one disclosed in EP 4410972.

[0084] Wild type strains of Pseudomonas putida KT2440 are available for example in the NBRC Strain Bank (National Institute of Technology and Evaluation Biological Resource center https: / / www.nite.go.jp / en / nbrc / , NBRC100650).

[0085] Furthermore, the strain may be issued from strains of Pseudomonas putida or Pseudomonas taiwanensis that have already been optimized for tyrosine production (Calero et al., ACS Synth Biol. 2016 Jul 15;5(7):741 -53; Wierckx et al, Appl Environ Microbiol. 2005 Dec;71 (12):8221 -7, Appl Environ Microbiol. 71 (12):8221 -7; Wynands et al, 2018; Otto et al 2019, Front Bioeng Biotechnol Nov 20;7:312).

[0086] As used in this description, the terms "genetically modified (Pseudomonas putida) strain," "modified (Pseudomonas putida) strain," or "genetically modified strain," are considered synonymous with each other.

[0087] In particular, "genetically modified strain" is understood to mean a strain that comprises either (i) at least one recombinant nucleic acid, or transgene, stably integrated into its genome, and / or present on a vector, e.g., a plasmid vector, or (ii) one or more unnatural mutations by nucleotide insertion, substitution, or deletion, said mutations being obtained by transformation techniques or by gene editing techniques known to the skilled person. In a particular embodiment, a genetically modified strain is a strain having stably integrated into its genome at least one exogenous nucleic acid, i.e. not naturally present in P. putida, for example a nucleic acid from another species.

[0088] For the purposes of the present invention, "recombinant gene encoding a benzalacetone synthase " means an exogenous nucleic acid comprising at least a portion encoding a benzalacetone synthase according to the invention as described above. In addition to the region encoding the benzalacetone synthase, the recombinant gene may be under the control of a promoter enabling its expression in the strain, preferably a promoter enabling its expression in the P. putida strain.

[0089] In one embodiment, which may be combined with the preceding ones, the recombinant gene encoding benzalacetone synthase as described above is placed under the control of a heterologous promoter, in particular a constitutive or inducible promoter, for example selected from among the ptrc, xyls / pm or araC / pBAD promoters, which allows the recombinant gene encoding benzalacetone synthase to be overexpressed in the genetically modified strain according to the invention.

[0090] The techniques of genetic modification by transformation, mutagenesis or gene editing are well known to the person skilled in the art and are described, for example, in "Molecular cloning: a laboratory manual", J. Sambrook, ed. Cold Spring Harbor, "Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation," Journal of Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(2):83-99. and in Martinez-Garcia and de Lorenzo,"Pseudomonas putida in the quest of programmable chemistry," Current Opinion in Biotechnology, 59: 111 -121 , 2019.

[0091] In one embodiment, a genetically engineered strain may comprise an expression-modifying nucleic acid, preferably overexpressing the expression of one or more genes naturally expressed in Pseudomonas putida.

[0092] Overexpression of a gene is understood as a higher expression of said gene in a genetically modified strain compared to the same strain in which the gene is expressed only under the control of the natural promoter. Overexpression can be achieved by inserting one or more copies of the gene directly into the genome of the strain, preferably under the control of a strong promoter, or also by cloning into plasmids, in particular multicopy plasmids, preferably also under the control of a strong promoter.

[0093] In another particular mode, a genetically modified strain may comprise a nucleic acid encoding one or more enzymes not naturally expressed in Pseudomonas putida.

[0094] In another particular mode, a genetically modified strain may comprise a nucleic acid encoding one or more enzymes naturally expressed in Pseudomonas putida, the encoded one or more enzymes being optimized version of the naturally expressed enzymes.

[0095] Most advantageously, the Pseudomonas putida strain may be capable of expressing a benzalacetone synthase and efficiently producing 4- hydroxybenzalacetone.

[0096] Most advantageously, the Applicant has developed a Pseudomonas putida strain capable of expressing a benzalacetone reductase and efficiently producing raspberry ketone. Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing a raspberry ketone from 4- hydroxybenzalacetone.

[0097] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing 4-hydroxybenzalacetone from 4-coumaroyl- CoA.

[0098] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing raspberry ketone from 4-coumaroyl-CoA.

[0099] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing a zingerone from 4-(4-Hydroxy-3- methoxyphenyl)but-3-en-2-one.

[0100] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2- one from ferulic acid.

[0101] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing zingerone from ferulic acid.

[0102] In a particular embodiment of the invention, there is provided a strain overproducing p-coumaric acid, and / or 4-hydroxybenzalacetone and / or raspberry ketone or 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one and / or zingerone. By "overproducing strain" in the sense of the present disclosure is meant a modified Pseudomonas putida strain capable of producing p-coumaric acid, 4- hydroxybenzalacetone and / or a raspberry ketone or 4-(4-Hydroxy-3- methoxyphenyl)but-3-en-2-one and / or zingerone in a higher amount when compared to a wild type Pseudomonas putida strain.

[0103] Preferably, the overproducing strain according to the present disclosure is capable of producing 2, 3, 4, 5 or 6 times more 4-hydroxybenzalacetone and / or raspberry ketone or 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one and / or zingerone compared to a wild-type Pseudomonas putida strain or a Pseudomonas putida strain expressing a wild type TAL.

[0104] Preferably, the overproducing strain is capable of converting all of the L- tyrosine in situ or added to the culture medium, into p-coumaric acid , and then p- coumaric acid into 4-hydroxybenzalacetone or 4-(4-Hydroxy-3-methoxyphenyl)but- 3-en-2-one, and preferably converting the 4-hydroxybenzalacetone to raspberry ketone or of 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one to zingerone.

[0105] According to another particular embodiment, the strain according to the present application is capable of producing raspberry ketone from 4-hydroxybenzalacetone or zingerone from 4-(4-Hydroxy-3-methoxyphenyl)but-3-en- 2 -one (in situ synthesis). The strain may thus further comprise at least one additional recombinant gene enabling the synthesis of raspberry ketone or zingerone, preferably a recombinant gene encoding a benzalacetone reductase.

[0106] By the term "additional recombinant gene" is meant in the sense of the present invention any recombinant gene present in the Pseudomonas putida strain in addition to the recombinant gene encoding a Tyrosine Ammonia Lyase according to the invention.

[0107] The additional recombinant gene may result from the insertion of a heterologous promoter, for example a strong promoter to overexpress an endogenous Pseudomonas putida gene, or a recombinant coding sequence encoding a protein not naturally expressed in Pseudomonas putida.

[0108] According to another particular embodiment that can be combined with the previous one, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding 4-coumarate-CoA ligase (4-CL). In particular, the modified strain may comprise a recombinant gene encoding a 4-CL (EC 6.2.1.12).

[0109] According to this particular embodiment, the genetically modified strain is capable of converting coumaric acid to p-coumaryl-coA via the 4-CL enzyme.

[0110] According to a preferred embodiment, the genetically modified Pseudomonas putida strain comprises one or several additional recombinant gene(s), namely one, two or the three following additional recombinant genes:- a recombinant gene encoding a 4-coumarate-CoA ligase (4-CL), and / or- a recombinant gene coding for a benzalacetone synthase (BAS), and / or- a recombinant gene coding for a benzalacetone reductase (BAR).

[0111] The 4-CL, BAS and BAR enzymes are all enzymes involved in the synthesis of phenylpropanoid compounds, that allow the production of raspberry ketone and zingerone. According to this preferred embodiment, the modified strain is capable of producing a multitude of phenylpropanoid compounds, namely p-coumaric acid,p-coumaroyl-coA, 4-hydroxybenzalacetone and raspberry ketone or 4-(4-Hydroxy- 3-methoxyphenyl)but-3-en-2-one and zingerone, in particular starting from L- tyrosine.

[0112] Another object of the invention relates to the use of an enzyme of functional equivalent thereof according to the invention for producing 4-hydroxybenzalacetone, or raspberry ketone, or 4-hydroxybenzalacetone and raspberry ketone.

[0113] Another object of the invention relates to the use of an enzyme of functional equivalent thereof according to the invention for producing 4-(4-Hydroxy-3- methoxyphenyl)but-3-en-2-one, or zingerone, or 4-(4-Hydroxy-3- methoxyphenyl)but-3-en-2-one and zingerone.

[0114] According to a particular embodiment of the invention, it is provided a genetically modified strain of Pseudomonas putida as defined above, for use in the synthesis of 4-hydroxybenzalacetone, or raspberry ketone, or 4- hydroxybenzalacetone and raspberry ketone.

[0115] According to a particular embodiment of the invention, it is provided a genetically modified strain of Pseudomonas putida as defined above, for use in the synthesis of 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one, or zingerone, or 4-(4- Hydroxy-3-methoxyphenyl)but-3-en-2-one and zingerone.

[0116] Another object of the invention relates to a process for the synthesis of 4- hydroxybenzalacetone, or raspberry ketone, or 4-hydroxybenzalacetone and raspberry ketone, the process comprising a step of transforming L-tyrosine into p- coumaric acid by an enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

[0117] The process according to the invention comprises the implementation of a step of growth of a genetically modified strain of Pseudomonas putida as defined above in a culture medium under conditions allowing the expression of the recombinant gene encoding the enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalentthereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

[0118] Preferably, the culture conditions are the culture conditions conventionally used in a fermenter for growth of Pseudomonas putida.

[0119] In a particular embodiment, the method according to the present disclosure is characterized in that the culture medium comprises L-Tyrosine.

[0120] In another embodiment, the process according to the present disclosure comprises carrying out a step of growing a genetically modified strain of Pseudomonas putida as defined above in a culture medium under conditions that allow for the expression of the recombinant gene encoding the enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3 and one or several additional recombinant gene(s) necessary to synthesize raspberry ketone or zingerone starting from L-Tyrosine.

[0121] According to a particular embodiment, the process comprises a step of growing a genetically modified strain of Pseudomonas putida comprising a recombinant gene encoding the enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent thereof which is a polypeptide that has at least 90% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3 according to the invention and comprising the one, two or the three following additional recombinant genes encoding:- a 4-coumarate-CoA ligase (4-CL), and / or- a benzalacetone synthase (BAS), and / or- a benzalacetone reductase (BAR).

[0122] According to a particular embodiment, the synthesis process according to the invention allows the production of raspberry ketone or zingerone in large quantities, for example in yields of at least 100 mg / L, preferably at least 500 mg / L, more preferably at least 1 g / L, 1 .5 g / L or 2 g / L and in particular in 2-fold, 3-fold, 4-fold, 5-fold or 6-fold production quantities in comparison with a wild-type Pseudomonas putida strain or one comprising the following additional recombinant genes encoding :- a 4-coumarate-CoA ligase (4-CL), and / or- a benzalacetone synthase (BAS), and / or- a benzalacetone reductase (BAR).

[0123] In one embodiment, the process converts at least 50%, preferably 60%, 70%, 80%, 90%, 95%, or even at least 99% of tyrosine synthesized in situ by the strain or added within the culture medium of the strain into p-coumaric acid.

[0124] The synthesis process according to the invention may also comprise a step of purification and / or recovery of the p-coumaric acid produced by the strain.

[0125] The synthesis process according to the invention may also comprise a step of producing raspberry ketone or zingerone.

[0126] The synthesis process according to the invention may also comprise a step of producing raspberry ketone or zingerone, and a step of purification and / or recovery of the raspberry ketone or zingerone produced by the strain.Examples

[0127] Example 1 : In silico study

[0128] In the literature, the inventors identified 4 enzymes described as TAL: RgTAL from Rhodotorula glutinis, CsTAL from Chroogloeocystis siderophila, FjTAL from Flavobacterium johnsoniae (Jendresen et al., 2015) and ScTAL from Stanieria cyanosphaera (Liu, Dellas, et Jenne 2020). While RgTAL exhibits both TAL and PAL activities, CsTAL, FjTAL and ScTAL have strictly the TAL activity.

[0129] Using the 3 protein sequences from CsTAL, FjTAL and ScTAL, a mathematic / biological model was applied to find other protein sequences likely to have the same specificity and potentially enhanced activity. The HMMER mathematic model (Finn, Clements, et Eddy 2011 ) was used to characterize the common regions between these sequences, or in other words, create a profile that characterizes a given set of proteins. This profile was then used to search indatabases for matching protein sequences. The 500 sequences with the best similarity score with the searched profile were kept. In this particular case, it was estimated that these first 500 sequences had a percentage of homology of 60%- 70% or more with the reference sequences. Not restricting the homology percentage allows to provide diversity in terms of protein sequences while maintaining a good probability of having the desired enzymatic activity. From this list of protein candidate, the enzymes were “clustered”. Clustering is a technique for comparing sequences pairwise, allowing them to be grouped according to one or more proximity criteria defined by the user. Here, the ESM clustering technique was used. This technique resulted in 3 clusters. The 15 first sequences from the cluster containing CsTAL, FjTAL and ScTAL were selected to be cloned. Most of these dbTAL sequences have not been previously described as Tyrosine Ammonia Lyase.

[0130] Example 2: In vivo study of p-coumaric acid production

[0131] Materials and methods1 - Mutagenesis of gene coding for the TAL enzymeThe genes coding for the new TAL enzymes were cloned in a suicidal plasmid pk18mobsacB (Graf & Altenbuchner, 2011 ). The plasmid was assembled by Gibson Assembly (Gibson et al., 2009). The plasmid was first transformed in E. coli S17.1 .2- Bacterial transformationThe genes coding for the TAL enzymes were integrated chromosomally in the bacterial chassis of interest by homologous recombination. The suicidal plasmids of all TAL presented were transformed in a tyrosine over producing strain of Pseudomonas putida KT2440.3- Media and growth conditionsAll resulting strain of P. putida were cultivated in an optimized minimum medium from Sun et al. (2006).Cultures were done in 96-Well microplates containing 200pL of minimal medium. 2,5g / L of glucose and 2,5g / L of xylose and, in the condition of tyrosine, 5mM of tyrosine were added to the media. Cultures were inoculated at OD600 of 0,05 withovernight pre-cultures. Microwell plates were incubated at 30°C, under continuous shaking at 140 rpm for 30h.Fed-batch cultures were performed in 3L bioreactors (Bionet, FO-Baby). Cultures were inoculated at an initial OD600 of 0,025 with pre-culture to an OD of 0,7. pH was maintained at 7.0 by automatic addition of NH3OH (Roth, ref 4346.5) with a pH sensor (Hamilton, EasyFerm Bio PHI Arc 120). Temperature was controlled at 30°C. Dissolved oxygen tension was measured with a pO2 sensor (Hamilton, VisiFerm HO 225mm) and maintained at 20% air saturation during all cultivation period by modifying the impeller speed and air flow. To avoid foaming, the antifoam Struktol® J647 was added when necessary. The initial medium for fermentation cultures was the same as previously described supplemented with 4g / L of glucose and 1 g / L of xylose. Feeding was made with a 450g / L glucose, 150g / L xylose and 12,5g / L MgSO4 solution.The cultures were centrifugated and the supernatants were collected for HPLC analysis.4- HPLC AnalysisDetermination of the p-CA concentration was done by HPLC (Shikimadzu) using Kinetex 5 pm F5 100A (Phenomenex) and a diode array detector. The samples were eluted with 0,1 % (v / v) formic acid and an acetonitrile gradient. The oven temperature was 40°C and the pump flow was 1 mL / min. The injected volume was 1 pL. The method was 32 min long and the product was detected at 315nm. To determine the p - CA concentration, a standard curve was constructed with p - CA concentrations from 0.2 to 2mM. The supernatant was diluted 5 times for microwell essay and x times for Bioreactors essay.Table 1 - Acetonitrile

[0132] Results

[0133] Among the 15 dbTALs resulting from the in silico study, 12 dbTALs were successfully cloned in a tyrosine-overproducing strain of P. putida KT2440. The results of the plate cultures are presented Figure 1 for only a subset of the cloned dbTALs that have an improved TAL capability. None of the dbTALs produce cinnamic acid unlike RgTAL which produces 0,05g / L of cinnamic acid. This attests the precision of the EnzymeFinder profile for selecting tyrosine specific TALs. Among the 12 cloned dbTALs, in the absence of tyrosine in the culture medium, 5 dbTALs (including dbTAL2 from Chroococcidiopsis sp. TS-821 and dbTAL8 from Trichocoleus sp. FACHB-832) produced lower concentration compared to the TALs used for bioinformatics profile and 7 dbTALs (including the enzyme of SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 as defined in table 2 below) gave similar productions. In presence of 5mM (0,9g / L) tyrosine, only the enzymes having the sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 were able to consume all added tyrosine. Even the strain harboring ScTAL and CsTAL lacked consuming all available tyrosine. The strain harboring the enzyme of SEQ ID No. 2 produced 1 ,4 g / L of PCA and the one harboring the enzyme of SEQ ID No. 3 produced 1 ,2 g / L of PCA. Therefore, these two enzymes had the highest activities among all TAL tested, in particular a higher activity in comparison with RgTAL, ScTAL, FjTAL, and CsTAL, which respectively produce in same conditions approximately 0,87 g / L, 0,63 g / L, 0,3 g / L, and 0,75 g / L of PCA.Table 2: TAL enzymes according to the invention

[0134] In a 3L bioreactor, the enzymes of SEQ ID No. 2 and SEQ ID No. 3 strains respectively reached a production of 8.55±0.61 g / L of PCA with a yield of 6.03±0.36% and 7.33±0.24 g / L of pCA with a yield of 5.44±0.44% (n=3) after 42h of fermentation. To compare, RgTAL strain produced 5,87±0.24 g / L of pCAwith a yield of 5,15±0.39% and 1 ,26±0.13g / L of CA. When the enzymes of SEQ ID No. 2 and SEQ ID No. 3 were integrated into a strain allowing to overproduce even more tyrosine, they produced for 5 10.98 ±0.55g / L of PCA with a yield of 8.60±0.49% for the enzyme of SEQ ID No. 2, and 8.94±0.25g / L of PCA with a yield of 7.61 ±0.13% for the enzyme of SEQ ID No. 3 (n=3).These results clearly illustrate the higher efficiency of P. putida expressing an enzyme having the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 for producing p-coumaric acid from L-tyrosine as compared to P. putida strain transformed with wild-type RgTAL or with wild-type ScTAL or with wildtype FjTAL or with wild-type CsTAL. The addition of the enzymes having the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 into the strains allows Pseudomonas putida to consume almost all of L-tyrosine and produce more than 1 g / L of p-coumaric acid, way over the yield obtained with other strains. It can be seen that the enzymes to be used according to the invention consume almost all the tyrosine presents in the culture medium, unlike the other enzymes, and thatthe production of anthranilic acid is reduced. Anthranilic acid is an intermediate in the coumaric acid production pathway. The inventors have observed that, with the lack of efficiency of the prior art TAL enzymes, this compound accumulated in the culture medium. The reduction in the production of anthranilic acid supports the efficacy of the use of the enzymes according to the invention, since there is no further accumulation of anthranilic acid in the minimal medium condition when the enzymes to be used according to the invention are present. Further, when tyrosine (Fig. 1 ) or phenylalanine (Fig. 2) are added, the enzymes to be used according to the invention are effective enough to pull the production pathway in favor of coumaric acid production and thus reduce the production of this intermediate, thereby illustrating the effectiveness of the enzymes of the invention for improving the production of p-coumaric acid.

[0135] Example 3: In vivo study of p-coumaric acid and cinnamic acid production in presence of phenylalanine

[0136] Starting from the same strain of P. putida expressing a TAL that were cultivated in an optimized minimum medium in example 2, cultures were done in 96- Well microplates containing 200pL of minimal medium. 2,5g / L of glucose and 2,5g / L of xylose and 5mM of phenylalanine were added to the media. Cultures were inoculated at OD600 of 0,05 with overnight pre-cultures. Microwell plates were incubated at 30°C, under continuous shaking at 140 rpm for 30h.

[0137] The culture protocol is the same as the one described in example 2.

[0138] The cultures were centrifugated and the supernatants were collected for HPLC analysis for determining the concentration of p-coumaric acid and cinnamic acid, and their precursors, tyrosine and phenylalanine, respectively.

[0139] As illustrated in figure 2, even in presence of phenylalanine, the enzymes of SEQ ID No. 1 , SEQ ID No. 2 and SEQ ID No. 3 are those that produce PCA in greater yield. The prior art TALs efficiency in producing PCA is significantly impaired in the presence of phenylalanine. This limitation arises because phenylalanine acts as a competitive substrate or inhibitor for these enzymes, disrupting the intended enzymatic pathway. In contrast, the enzymes of the invention exhibit robust TALactivity that is not only unaffected by the presence of phenylalanine but also enables the highest production of PCA under these conditions.

[0140] The enzymes to be used according to the invention maintain TAL activity despite phenylalanine interference representing an unexpected technical advantage. This outcome based on the known properties of TAL enzymes in the prior art, which typically display susceptibility to interference by structurally similar compounds like phenylalanine, was not predictable.

[0141] Thus, the enzymes of the invention can be used in industrial and biotechnological processes, where phenylalanine is often present either as a contaminant or a co-substrate. By avoiding any significant effect of phenylalanine on their TAL activity, the enzymes for use according to the invention enable more efficient and reliable PCA production, reducing the need for costly substrate purification or process optimization, while the prior art enzymes are limited by their inability to overcome phenylalanine interference.

[0142] Table 3 below lists the amino acid and nucleotide sequences referred to in this application with respect to the enzymes of the invention, as well as the amino acid sequence from the organism R. glutinis.Table 3: Amino acid and nucleotide sequences referred to in this application

Claims

35Claims1. An enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent enzyme thereof in the capability to convert tyrosine into p-coumaric acid, which is a polypeptide that has at least 95% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, for transforming a tyrosine substrate, in particular L-tyrosine, into p- coumaric acid.

2. An enzyme which is a polypeptide that has at least 70% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, the enzyme being unable to transform L-Phenylalanine into cinnamic acid, for transforming a tyrosine substrate, in particular L-tyrosine, into p-coumaric acid.

3. The enzyme according to claim 1 or 2 which is a recombinant enzyme, in particular expressed in a microorganism, more particularly in Pseudomonas putida.

4. The enzyme according to claim 1 or 2, which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3.

5. The enzyme according to claim 1 or 2, which has at least the same catalytic constant kcat, on the transformation of tyrosine into p-coumaric acid as compared to a TAL having the amino acid sequence set forth in SEQ ID No. 4, in particular wherein the catalytic activity of the enzyme on the transformation of tyrosine into p- coumaric acid is determined by measuring by HPLC the total concentration of p- coumaric acid before and after a catalytic reaction of transforming tyrosine into p- coumaric acid.

6. The enzyme according to claim 1 , which is at least twice less efficient for transforming Phenylalanine into cinnamic acid as compared to a TAL having the amino acid sequence set forth in SEQ ID No. 4.-.

7. A nucleic acid molecule encoding an enzyme which has the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, or a functional equivalent enzyme thereof which is a polypeptide that has at least 70% identity with the amino acid sequence set forth in SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3, in particular36 a nucleic acid molecule comprising or consisting of the nucleotide sequence set forth in SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7.

8. A genetically modified microorganism, in particular a Pseudomonas putida strain, capable of expressing or expressing a recombinant gene encoding an enzyme according to claim 1 or 2, and / or being transformed with a nucleic acid molecule according to claim 7.

9. The genetically modified microorganism according to claim 8, which is capable to produce p-coumaric acid, in particular from tyrosine.

10. The genetically modified microorganism according to claim 7 or 8, which further comprises:- a gene encoding a 4-coumarate-ligase, and / or- a gene encoding a benzalacetone synthase (BAS), and / or- a gene encoding a benzalacetone reductase (BAR), in particular comprising comprises a gene encoding a 4-coumarate-ligase, a gene encoding a BAS and a gene encoding a BAR11. Use of an enzyme according to claim 1 or 2, for producing p-coumaric acid, in particular starting from a tyrosine substrate.

12. The use according to claim 11 , for producing a Raspberry ketone compound or a zingerone compound.

13. A process for producing p-coumaric acid, in particular from tyrosine, comprising a step of using an enzyme according to claim 1 or 2.

14. A process for producing p-coumaric acid, in particular from tyrosine, comprising a step of using an enzyme according to any one of claims 1 to 6, or a genetically modified microorganism according to any one of claims 8 to 10, the process comprising a step of culturing the microorganism according to any one of claims 8 to 10.

15. A process for producing raspberry ketone or zingerone, comprising a step of using an enzyme according to any one of claims 1 to 6, or a genetically modified microorganism according to any one of claims 8 to 10, the process comprising astep of culturing a genetically modified Pseudomonas putida strain according to any one of claims 8 to 10 for transforming a tyrosine substrate, in particular L-tyrosine, into p-coumaric acid.

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