WO2007083071A2

WO2007083071A2 - Process for preparing a precursor of an aromatic compound by biosynthesis

Info

Publication number: WO2007083071A2
Application number: PCT/FR2007/050674
Authority: WO
Inventors: Gilles Feron; Caroline Blin; Geneviève MAUVAIS
Original assignee: Institut National De La Recherche Agronomique - Inra
Priority date: 2006-01-23
Filing date: 2007-01-22
Publication date: 2007-07-26
Also published as: WO2007083071A3; FR2896513B1; FR2896513A1

Abstract

The present invention relates to the field of the enzymatic or biological synthesis of aromatic compounds. The invention relates more particularly to a process for obtaining an aromatic compound precursor, in particular a raspberry ketone precursor, by enzymatic bioconversion.

Description

PROCESS FOR THE PREPARATION OF A PRECURSOR OF AN AROMATIC COMPOUND BY BIOSYNTHESIS

FIELD OF THE INVENTION The present invention relates to the field of the synthesis of aromatic compounds enzymatically or biologically.

The invention relates more particularly to a process for obtaining a precursor of aromatic compounds, in particular a precursor of raspberry ketone, by enzymatic bioconversion.

In general, each natural flavor is made up of several hundred volatile compounds. However, among the many volatile compounds constituting a natural flavor, a volatile compound, or a small number of volatile compounds, alone confers the characteristic olfactory note of said flavor.

To date, more than 230 volatile compounds have been identified as constituting the raspberry flavor. However, the olfactory note of the natural raspberry flavor is conferred on it essentially, if not exclusively, by the compound 4- (4'-hydroxyphenyl) butan-2-one, which is also called para-hydroxyphenyl-butan-2-one or "Raspberry ketone" or "frambinone".

Raspberry ketone is an aromatic molecule that is highly sought after in the agri-food industry. In this area, raspberry ketone is not only used as a raspberry aroma. Raspberry ketone is also used in various aromatic mixtures, including aromatic mixtures of peach, pineapple, strawberry, kiwi or even cherry. Raspberry ketone is also used for its ability to provide a fruity note in certain aromatic mixtures in perfumery, cosmetics and pharmacy. Raspberry fruit is the only source for direct extraction of raspberry ketone. However, the ketone raspberry is present at very low concentrations in the fruit, of the order of 0.1 mg / l of raspberry juice. Consequently, the cost price of raspberry ketone obtained by direct extraction from the fruit is not compatible with the economic constraints related to the manufacture of industrial products, in particular food and cosmetics, which must be cost-effective. moderate when they are intended for wide public dissemination.

In order to overcome these technical and economic constraints, various methods of chemical synthesis of raspberry ketone have been described in the state of the art. A process for the chemical synthesis of raspberry ketone by condensation of phenol with methyl vinyl ketone has thus been described in the presence of a suitable catalyst. However, this chemical synthesis process has many disadvantages. Methyl vinyl ketone is a difficult compound to synthesize, and moreover is unstable. In addition, phenol is a toxic synthetic product that causes severe burns on the skin.

A process for the chemical synthesis of raspberry ketone has also been described in two steps, respectively (i) a step of synthesis of para-hydroxyphenyl-3-buten-2-one by condensation of parahydroxybenzaldehyde with ketone, followed (ii) by a step of catalytic hydrogenation of parahydroxyphenyl-3-buten-2-one to obtain parahydroxyphenyl-butan-2-one or raspberry ketone. The raspberry ketone obtained by such a process is classified as a "nature-identical" flavor, since it is chemically identical to natural raspberry ketone but has been obtained by chemical synthesis.

However, there is a need in the industry for natural flavorings, i.e. chemically defined flavoring substances which are obtained from natural sources, either by appropriate physical processes, including distillation and distillation. extraction by solvent, either by enzymatic or microbiological processes, from the material of plant or animal origin.

For the purpose of synthesizing a natural raspberry ketone aroma compound, a process comprising two biosynthetic steps, respectively (i) a step of condensation of 4-coumaroyl-CoA with malonyl-CoA, has previously been developed. and then (ii) a decarboxylation step of the intermediate compound produced in the previous step to obtain 4- (4'-hydroxy-phenyl) -but-3-ene-2-one. Then 4,4'-hydroxyphenyl) -but-3-ene-2-one is reduced to raspberry ketone by enzymatic catalysis in the presence of NADPH (Borejsza-wysocki and Hrazdina, 1994, Biosynthesis of p-hydroxyphenylbutan-2-one in Raspberry fruit and tissue cultures, Phytochemistry, Vol 35: 623). Despite its apparent simplicity, this method of raspberry ketone biosynthesis appears uneconomical, in particular because it requires the prior extraction of bioconversion enzymes from plant tissues and also because it requires the use of -Factors such as CoA SH, APP and NADPH.

It has also been described a process for obtaining raspberry ketone by bioconversion of 3- (4'-hydroxyphenyl) -1-methyl-propyl-beta-D-glucopyranoside, also called betuloside or rhododendrin, by successive actions of a beta glucosidase, and a secondary alcohol dehydrogenase (HRAS), as described in PCT application No. WO 99/49 069. ⁰ in this biosynthetic process, the starting material, which is the bétuloside compound, is extracted from the birch. Although this biosynthetic process appears to be technically satisfactory, its moderate-cost industrial use is uncertain, mainly because of the low availability of the starting material, and, to a lesser extent, also because of the need for it. to use purified enzymes from natural substances. There is therefore still a need in the state of the art for processes for obtaining aromatic compounds, in particular raspberry ketone, by total or partial biosynthesis, alternative to known methods. In particular, there is a need in the state of the art for new processes for the biosynthesis of aromatic compounds, especially raspberry ketone, which are of a moderate cost price. DETAILED DESCRIPTION OF THE INVENTION

According to the invention, there is provided a new process for the preparation by biosynthesis of para-substituted phenyl-3-buten-2-one aromatic precursors from readily accessible natural or synthetic starting materials.

Surprisingly, it has been shown according to the invention that the aromatic precursor compounds of the phenyl-3-buten-2-one type substituted in the para position can be obtained by means of a step of condensation, by enzymatic aldolization, of acetone with a benzaldehyde substituted in the para position.

Thus, surprisingly, it has been shown according to the invention, that the aldolisation reaction between acetone and a benzaldehyde substituted by a hydroxy or an alkoxy in the para position, which until now was performed exclusively by synthesis chemical, could be carried out microbiologically.

More specifically, it has been shown according to the invention that the aldolisation step of acetone with a benzaldehyde compound substituted in the para position by a hydroxyl or alkoxy group, in order to produce aromatic precursors of the phenyl-3-butene type. 2-one substituted in the para position by a hydroxy or alkoxy group, could be carried out by a deoxyribose-phosphate aldolase (DERA) catalysed enzymatic bioconversion step. The subject of the present invention is a process for the biosynthetic preparation of the compound of formula (I) below:

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, said process comprising the steps of: a) adding:

(i) a compound of the following formula (II):

wherein R1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, and (ii) acetone in a reaction medium comprising 2-deoxyribose-5-phosphate aldolase; and b) recovering the compound of formula (I) from the medium obtained at the end of step a).

Thus, it has been shown according to the invention that a particular aldolase, deoxyribose 5-phosphate aldolase, also called DERA, was capable of to convert a para-substituted benzaldehyde to a phenyl-3-buten-2-one having the same para-substituted substituent in the presence of acetone.

2-Deoxyribose-5-phosphate aldolase is a class I aldolase that is known to catalyze the condensation of acetaldehyde and D-glyceraldehyde-3-phosphate to produce 2-deoxyribose-5-phosphate.

DERA is classified according to international nomenclature in the class of aldolases EC 4.1.2.4. The specific catalytic activity of DERA can easily be verified by those skilled in the art, for example using the technique described by WONG et al. (2003, Bioorganic and Medicinal Chemistry, Vol 11: 43-52, Section "Experimental", subsection "DERA addition (aldol) assay")

In the compounds of formula (I) and (II), the group R 1 represents hydrogen or an unsubstituted linear or branched alkyl group having 1, 2, 3 or 4 carbon atoms.

According to a first preferred aspect, the compound of formula (II) is such that R 1 represents a hydrogen atom. In this particular embodiment, the compound of formula (II) is a para-hydroxybenzaldehyde compound which makes it possible, by means of the process of the invention, to prepare the compound of formula (I) consisting of parahydroxyphenyl-3-buten-2 -one, also referred to as 4-OH-benzylidene acetone, which is a precursor of raspberry ketone.

According to a second preferred aspect, the compound of formula (II) is such that the group R 1 represents a group -CH ₃ . According to this second particular aspect, the compound of formula (II) is paramethoxybenzaldehyde, which is also called anisaldehyde. According to this second particular aspect, the compound of formula (I) obtained according to the method of the invention is paramethoxyphenyl-3-buten-2-one, which is also called anisylidene acetone, which may also be a precursor compound of raspberry ketone.

The starting materials used in the biosynthetic process of the invention are easily accessible and inexpensive. For example, parahydroxybenzaldehyde can be obtained by extraction from a variety of biological sources. For example, parahydroxybenzaldehyde is produced by organisms such as certain Coleoptera such as Agabus melanarius, heteropterans such as Llyocoris cimicoides and Notonecta, and Lepidoptera such as Eldana saccharina. Also, parahydroxybenzaldehyde can be released, for example by simply heating, in the presence of emulsin, an aqueous suspension of hurrin, which is an amygdalin-like glucoside, the latter being present in particular in young shoots of sorghum (Sorghum). vulgare). Natural parahydroxybenzaldehyde can also be obtained by biodegradation of picteide, a stylbene present in the bark of spruce, as described by Shiotsu et al. (1989, Mukuzai, 35: 826).

Parahydroxybenzaldehyde can also be obtained by chemical synthesis, in particular according to the techniques described by Freudenberg and Gehrke (1951, Chem Ber, vol.84: 443-450) or Schoutissen (1935, Recl, Trav Chim, The Netherlands). , Vol. 54: 97-99). In addition, parahydroxybenzaldehyde is a compound that can be commonly found commercially.

A compound of formula (II) such as paramethoxybenzaldehyde or anisaldehyde can be obtained from natural sources such as oil extracted from star anise. Anisaldehyde is also a chemical compound that can be commonly found in commerce.

The acetone which is used in the implementation of the process of the invention can be obtained from natural sources, such as plants and trees. Acetone is also produced by various microorganisms. Acetone can be obtained by fermentation of glucose by cultures of bacterial cells of Clostridium acetobutylicum. Acetone is also a compound that is commonly found in commerce. Thus, thanks to the process of the invention, it is now possible to obtain a natural precursor of raspberry ketone. In addition, when this natural raspberry ketone precursor is subsequently converted to raspberry ketone microbiologically, the end product is then a raspberry ketone which consists of a natural flavor. According to a third preferred aspect, the above process is characterized in that in step a), a quantity by weight of acetone in excess, based on the weight of compound (II), is added to the reaction medium. Preferably, for a given weight (P) of the compound of formula (II), at least two times the weight (P) of acetone is added to step a). In some embodiments of the process, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 are added to step a). 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 times the weight (P) of acetone. By way of illustration, in the examples a weight (P) of 0.5 g / l of compound of formula (II) is used for 16 g / l of acetone, ie 32 times the weight (P).

In some embodiments of the method, step b) comprises the following steps: b1) performing at least one cycle of freezing and thawing of the medium obtained at the end of step a); b2) recovering the compound of formula (I) from the thawed medium obtained at the end of step b1).

Without wishing to be bound by any theory, the applicant believes that the cycle (s) of freezing and thawing lead to a change in the mobility of the solvent, which may be water, at the catalytic site of the enzyme. This change in the mobility of the solvent would lead to a change in the solubility and the accumulation of reaction intermediates, such as to promote the completion of the aldolization reaction of the compound of formula (II), and the final dehydration step leading to the production of the compound of formula (I).

It has been shown that the process of the invention can advantageously be carried out with a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase having at least 30% amino acid identity with the SEQ ID sequence polypeptide. # 1. The polypeptide of SEQ ID NO ^: 1 sequence consists of the amino acid sequence of 2-deoxyribose-5-phosphate aldolase expressed by Escherichia coli.

In general, it is known in the state of the art that an identity of at least 30% in amino acids, on a segment of at least 100 consecutive amino acids, between two polypeptides means a strong structural and functional relationship between these two polypeptides.

In addition, it has been shown in the examples that a reaction medium comprising a Bacillus subtilis 2-deoxyribose-5-phosphate aldolase having an identity in amino acids of 36% with the sequence SEQ ID No. 1 was able to catalyze the reaction. of aldolising the compound of formula (II) to produce the final compound of formula (I).

Bacillus subtilis 2-deoxyribose-5-phosphate aldolase has the amino acid sequence SEQ ID No. 2 according to the invention.

It has also been shown in the examples that a reaction medium comprising a Bacillus cereus 2-deoxyribose-5-phosphate aldolase having an amino acid identity of 38% with the sequence SEQ ID

No. 1 was able to catalyze the aldol reaction of the compound of formula (II) to produce the final compound of formula (I).

Bacillus cereus 2-deoxyribose-5-phosphate aldolase has the amino acid sequence SEQ ID NO: 3 according to the invention. For purposes of this disclosure, the term "amino acid sequence" may be used to denote either a polypeptide or a protein. The term "amino acid sequence" encompasses the protein material itself and is therefore not restricted to information concerning its sequence.

According to the invention, a first polypeptide having at least 30% amino acid identity with a second reference polypeptide, will have at least 30%, preferably at least 31%, 32%, 33%, 34%,

35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51% , 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,

87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%,

98%, 98.3% 98.6%, 99%, or at least 99.5% amino acid identity with said second reference polypeptide.

Also, for the purposes of this description, the expression

"Nucleotide sequence" can be used to denote either a polynucleotide or a nucleic acid. Expression

"nucleotide sequence" includes the genetic material itself and is therefore not restricted to information about its sequence.

The terms "nucleic acid", "polynucleotide", "oligonucleotide" or "nucleotide sequence" include RNA, DNA or cDNA sequences, or hybrid RNA / DNA sequences of more than one nucleotide, regardless of in the single-stranded form or in the form of duplex.

The term "nucleotide" refers to natural nucleotides (A, T, G, C and U).

For purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is base-paired. complementary to the second polynucleotide whose orientation is reversed. The complementary "bases" are A and T (or A and U), and C and G.

According to the invention, a first nucleic acid having at least 30% identity with a second reference nucleic acid, will have at least 30%, preferably at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52% 53% 54% 55% 56% 57% 58% 59% 60% 61% 62% 63% 64% 65% 66% 67% 68% 69 % 70% 71% 72% 73% 74% 75% 76% 77% 78% 79% 80% 81% 82% 83% 84% 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.3% 98, 6%, 99%, or at least 99.5% nucleotide identity with said second reference nucleic acid.

The "percent identity" between two amino acid sequences or between two nucleic acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.

The part of the amino acid sequence or of the nucleotide sequence in the comparison window can thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between the two sequences.

The percent identity is calculated by determining the number of positions at which an identical amino acid, or identical nucleotide base, is observed for the two sequences compared, and then dividing the number of positions at which there is identity between the two amino acids. , or between the two nucleobases, by the total number of positions in the comparison window, and then multiplying the result by one hundred to obtain the percentage amino acid or nucleotide identity of the two sequences between them. The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms.

Most preferably, the percentage of sequence identity is determined using the BLAST software, version 2.0, which implements the BLAST algorithm (for "Basic Local Alignment Search Tool"), as defined by Karlin and Altshul (Karlin, Samuel and Stephen F. Altschul (1990), Methods for assessing the statistical significance of molecular sequence by using general scoring schemes, Proc Natl Acad Sci USA 87: 2264-68, Karlin, Samuel and Stephen F. Altschul (1993), Applications and statistics for multiple high-scoring segments in molecular sequences, Proc Natl Acad Sci USA 90: 5873-7).

To determine the percentage of nucleotide identity between two polynucleotides, the BLASTN software is preferably used, based on the algorithm defined by Altschul et al. (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Zhang Jinghui, Zheng Zhang, Miller Webb, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs ", Nucleic Acids Res 25: 3389-3402), using the default settings.

To determine the percentage of amino acid identity between two polypeptides, use is preferably made of the BLASTP software, based on the algorithm defined by Altschul et al. (Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (1990), Basic Local Alignment Search Tool, J. Mol Biol 215: 403-10), using the parameters by default. To determine the percentage of amino acid identity between two polypeptides, use is preferably made of the BLASTP software, version 2.2.10, the principles of which are described by Tatusova et al. (Tatusova TA, Madden TL (1999) BLAST 2S, a new tool for comparing protein and nucleotide sequences, FEMS Microbiol.Lat.174: 247-250), with the parameters set as follows: (1) Matrix = BLOSUM62; (2) Gap Open = 11; (3) Gap Extension = 1; (4) X_dropoff = 15; (5) Expect = 10.000000; (6) Wordsize = 3; (7) Filter (T / F) default = T

Thus, step a) of the above process can be carried out using a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase having at least 30% amino acid identity with the sequence SEQ ID No. 1 of E. coli and encoded by a nucleic acid derived from (i) a prokaryotic organism, (ii) a eukaryotic organism, excluding filamentous fungi.

In particular, step a) of the above process may be carried out using a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase from a prokaryotic organism of a species selected from Escherichia coli, Bacillus, Pseudomonas, Streptomyces , Corynebacterium and Shewanella.

More particularly, step a) of the above process can be carried out using a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase encoded by a nucleic acid from a prokaryotic organism selected from Escherichia coli K12, Escherichia coli CFT073, Shigella flexneri, Salmonella enterica subsp. Enterica, Vibrio cholerae, Yersinia pestis, Yersinia pseudotubercolis, Photobacterium profundum, Vibrio fischeri, Erwinia carotovora, Actinobacillus pleuropneumoniae, Colwellia psychererythraea, Shewanella baltica, Shewanella amazonensis, Shewanella frigidimarina, Shewanella denitrificans, Chromohalobacter salexigens, Burkholderia pseudomallei, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. tomato, Idiomarina lawhiensis, Pseudomonas syringae pv. Syringae, Magnetospirillum magnetotacticum, Streptomyces coelicolor, Streptomyces avermitilis, Coxiella burnetii, Nocardioides sp. JS614, Paracoccus denitrificans, Mesorhizobium sp. BNC1, Silicibacter sp. TM 1040, Rhodobacter sphaeroides, Bacteroides thetaiotaomicron, Francisella tularensis, Bacteroides fragilis, Silicibacter pomeroyi, Burkholderia fungorum, Jannaschia sp. CCS 1, Porphyromonas gingivalis, Legionella pneumophila, Treponema denticola, Bacillus cereus, Geobacillus kaustophilus, Bacillus halodurans, Solibacter usitatus, Streptococcus suis, Bacillus licheniformis, Listeria monocytogenes, Deinococcus geothermalis, Chloroflexus aurantiacus, Rubrobacter xylanophilus, Desulfitobacterium hafniense, Arthrobacter sp. FB24, Bacillus clausii, Rhodospirillum rubrum, Staphylococcus haemolyticus, Corynebacterium glutamicum, Enterococcus faecium, Bacillus subtilis, Symbiobacterium thermophilum, Thermococcus kodakarensis and Exiguobacterium sp. 255-15.

In a particular embodiment of the above process, the reaction medium comprises a 2-deoxyribose-5-phosphate aldolase encoded by Bacilus subtilis, preferably 2-deoxyribose-5-phosphate aldolase of sequence SEQ ID No. ² . another particular embodiment of the above process, the reaction medium comprises a 2-deoxyribose-5-phosphate aldolase encoded by Bacilus cereus, preferably the 2-deoxyribose-5-phosphate aldolase sequence SEQ ID N ⁰ 3.

As mentioned previously, step a) of the above process can also be carried out using a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase encoded by a nucleic acid from a eukaryotic organism, for example a eukaryotic organism selected from Homo sapiens, Gallus gallus, Canis familiaris, Mus musculus, Caenorhabditis elegans, Anopheles gambiae, Danio rerio, Xenopus tropicalis, Plasmodium chabaudi, Plasmodium berghei, Plasmodium falciparum, Apis mellifera, Drosophila melanogaster, Entamoeba histolytica, Aspergillus fumigatus, Strongylocentrotus purpuratus, and Aspergillus nidulans.

For amino acid sequences and nucleotide sequences corresponding to the various prokaryotic organisms or eukaryotes specified above, those skilled in the art will advantageously refer to the left-hand column of Table 1 in which, for each prokaryotic or eukaryotic organism, the access reference to the corresponding sequences is indicated in the NCBI database. (for "National Center for Biotechnology Information").

For purposes of this disclosure, a "reaction medium" comprising a 2-deoxyribose-5-phosphate aldolase includes both (i) a reaction medium consisting of a culture of prokaryotic cells or eukaryotic cells producing a 2-deoxyribose-5 -phosphate aldolase, and (ii) a reaction medium consisting of an acellular composition comprising a 2-deoxyribose-5-phosphate aldolase.

In certain embodiments of a reaction medium of the first type (i), the cells are chosen from the prokaryotic or eukaryotic cells described above, which naturally produce a 2-deoxyribose-5-phosphate aldolase having at least 30% d amino acid identity with 2-deoxyribose-5-phosphate aldolase of E. coli of sequence SEQ ID N ⁰ 1.

In other embodiments of a reaction medium of the first type (i), the cells are chosen from recombinant cells into which has been inserted a nucleic acid coding for a 2-deoxyribose-5-phosphate aldolase having at least 30% identity in amino acids with 2-deoxyribose-5-phosphate aldolase E. coli sequence SEQ ID N ⁰ 1.

The invention also relates to the use of a nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase as defined above with a view to producing a 2-deoxyribose-5-phosphate aldolase for its implementation for the preparation of the reaction medium used in the process above.

From the amino acid sequences and / or nucleotide sequences of 2-deoxyribose-5-phosphate aldolases whose references are specified in Table 1, one skilled in the art is capable of detecting, isolating, cloning and characterizing any nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase, for example by synthesizing specific nucleotide probes or primers. To construct such nucleotide probes or primers, one skilled in the art can refer to any one of the many conventional techniques known in the state of the art.

According to a first aspect, the nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase consists of the nucleic acid encoding the amino acid sequence polypeptide SEQ ID N ⁰ 1, referenced as No. P0A6L0 in the base of NCBI data or in the SWISSPROT database.

According to a second aspect, the nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase consists of the nucleic acid encoding the amino acid sequence polypeptide SEQ ID N ⁰ 2, referenced under the number NP-391 821 in the NCBI database or in the SWISSPROT database.

In a third aspect, the nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase consists of the nucleic acid encoding the polypeptide of amino acid sequence SEQ ID N ⁰ 3 under the reference No. NP-978291 in the NCBI database or in the SWISSPROT database.

The invention also relates to a process for the production of one of the 2-deoxyribose-5-phosphate aldolases as defined above, said method comprising the steps of: i) inserting a nucleic acid encoding said polypeptide into an appropriate vector; ii) culturing, in a suitable culture medium, a host cell previously transformed or transfected with the recombinant vector of step a); iii) recovering the conditioned culture medium or lysing the host cell, for example by sonication, by osmotic shock, or with the aid of a French press; iv) separating and purifying from said culture medium or from the cell lysates obtained in step c), said polypeptide; v) where appropriate, characterizing the recombinant polypeptide produced.

In another aspect, a 2-deoxyribose-5-phosphate aldolase, produced by non-recombinant cells or by recombinant cells into which the corresponding nucleic acid has been inserted, can be purified by passage through an appropriate series of chromatography columns. according to the methods known to those skilled in the art, for example an immunoaffinity chromatography column on which the antibodies directed against this 2-deoxyribose-5-phosphate aldolase or against a fragment thereof have been immobilized beforehand.

In some cases, a 2-deoxyribose-5-phosphate aldolase according to the invention may also be prepared by conventional techniques of chemical synthesis indifferently in homogeneous solution or solid phase.

By way of illustration, a 2-deoxyribose-5-phosphate aldolase as defined according to the invention may be prepared by the homogeneous solution synthesis technique described by Houben Weyl (Houben Weyl, 1974, in Meuthode der Organischen Chemie, E. Wunsch Ed., Volume 15-1 and 15-

II,) or the solid phase synthesis technique described by

MERRIFIELD (Merrifield RB, 1965a, Nature, Vol 207 (996): 522-523, Merrifield RB., 1965b, Science, Vol 150 (693): 178-185).

In order to obtain recombinant cells expressing 2-deoxyribose-5-phosphate aldolase, a recombinant vector comprising a nucleic acid coding for a 2-deoxyribose-5-phosphate aldolase as defined above is advantageously used. For the purpose of the present invention, the term "vector" will be understood to mean a circular or linear DNA or RNA molecule which is indifferently in the form of a single-stranded or double-stranded form.

Expression vectors comprising, in addition to a nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase according to the invention, are preferred regulatory sequences making it possible to direct its transcription and / or its translation.

According to an advantageous embodiment, a recombinant vector according to the invention will comprise in particular the following elements: (1) regulatory elements of the expression of the nucleic acid to be inserted, such as promoters and enhancers;

(2) the coding sequence comprised in the nucleic acid according to the invention to be inserted in such a vector, said coding sequence being placed in phase with the regulation signals described in (1); and (3) appropriate transcription initiation and stopping sequences.

In addition, the recombinant vectors according to the invention may include one or more origins of replication in cell hosts in which their expression is sought, one or more selection markers.

By way of examples, the bacterial promoters may be the LacI, LacZ promoters, the T3 or T7 bacteriophage RNA polymerase promoters, the PR promoters, or the phage lambda PL promoters.

Promoters for eukaryotic cells will include the HSV thymidine kinase promoter or the mouse L-metallothionein promoter.

In general, for the choice of a suitable promoter, the skilled person can advantageously refer to the work of

SAMBROOK et al. (Sambrook, J. Fritsch, EF, and T. Maniatis, 1989. Molecular cloning: a laboratory manual, Coed Spring Harbor Laboratory, Spring Harbor Co, New York.) Or the techniques described by FULLER et al. (Fuller SA et al., 1996, Immunology in Current Protocols in Molecular Biology, Ausubel et al., Eds, John Wiley & Sons, Inc., USA). Particularly suitable vectors for an expression of the nucleic acids according to the invention in bacteria are, for example, the pQE70, pQE60 or pQE-9 vectors (marketed by QIAGEN), the pBluescript, Page script, pNH8A vectors; pNH16a, pNH18a, pNH46A (sold by the company Stratagene), the vectors pKK223-3, pKK233-3, pDR540 and pRIT5 (sold by the company Pharmacia).

Particularly suitable vectors for expression in eukaryotic cells are, for example, the pWLNEO, pSV2CAT, pOG44, pXT1 and pSG vectors (marketed by Stratagene), the pSVK3, pBPV, pMSG and pSVL vectors (marketed by Pharmacia).

A first vector preferably implemented in the context of the invention is the pcDNA3 vector marketed by Invitrogen. A second particularly preferred vector is the vector pBluescript SK (-), marketed by the company Stratagene.

The preferred bacterial vectors according to the invention are for example the pBR322 vectors (ATCC37017) or also vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotech, Madison, WI, USA).

Other commercialized vectors may also be mentioned, such as the vectors psiX174, pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene). It may also be baculovirus type vectors such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC N ⁰ CRL 1711) derived from Spodoptera frugiperda.

It can still be adenoviral vectors such as human adenovirus type 2 or 5.

In other embodiments of a reaction medium of the second type (ii), said reaction medium consists of an acellular composition comprising a 2-deoxyribose-5-phosphate aldolase according to the invention or a liquid composition comprising an enzymatic extract obtained from a prokaryotic or eukaryotic cell culture produces a 2-deoxyribose-5-phosphate aldolase.

For example, a 2-deoxyribose-5-phosphate aldolase according to the invention can be obtained, at varying degrees of purification, (i) from cells of any of the organisms referenced in Table 1 which produce naturally said enzyme, (ii) from recombinant cells into which a nucleic acid encoding said enzyme has been inserted, and which produces said enzyme.

By way of illustration, an acellular reaction medium comprising a 2-deoxyribose-5-phosphate aldolase may be prepared according to a process comprising the following steps:

1) culturing recombinant or non-recombinant cells producing said enzyme;

2) recovering the cell culture supernatant comprising the enzyme excreted by the cultured cells. 3) if necessary, enrich the medium obtained at the end of step b) in 2-deoxyribose-5-phosphate aldolase, by purification.

According to another alternative, an acellular reaction medium comprising a 2-deoxyribose-5-phosphate aldolase may be prepared according to a process comprising the following steps: 1) culturing recombinant or non-recombinant cells producing said enzyme;

2) preparing a cell extract from the cells cultured in step a); 3) if necessary, enrich the medium obtained at the end of step b) in 2-deoxyribose-5-phosphate aldolase, by purification.

In step 2) above, the cell extract may for example be prepared by grinding the cells cultured in step 1), and then centrifugation of the cell broyast thus obtained, in order to separate unwanted solid cellular particles and the liquid. extract which contains 2-deoxyribose-5-phosphate aldolase in suspension. The centrifugation may for example be carried out at 800 g for a suitable duration, for example ranging from 1 to 30 minutes, preferably from 15 to 25 minutes.

If necessary, one or more protease-inhibiting compounds can be added to the cell-free reaction mixture enriched in 2-deoxyribose-5-phosphate aldolase, in order to reduce or block the possible degradation of 2-deoxyribose-5-phosphate aldolase, which is susceptible to to occur during the execution of the process for the preparation by biosynthesis of the compound of formula (I) which is the subject of the invention. In step a) of the process for producing the compound of formula (I), when the reaction medium consists of a culture of cells, recombinant or non-recombinant, producing a DERA, the culture conditions are adapted according to the type of cells. prokaryotes or eukaryotes that are used. In general, prokaryotic or eukaryotic cell cultures having a cell density corresponding to at least a value of 2 OD, and more preferably at least 3 OD, are used, the absorbance value being measured at the wavelength of 600 nanometers.

The cell density value of the cell culture used in step a) of the process can be easily adapted by those skilled in the art depending on the type of bacterial cells used, including its growth rate in in vitro cultures and its ability to produce 2-deoxyribose-5-phosphate aldolase.

In step a) of the process, the acetone and the compound (II) are advantageously added to the cell culture substantially simultaneously over time. However, the order of addition of acetone and the compound of formula (II) to the cell cultures is irrelevant.

Step a) of the process according to the invention has a duration that can vary between 10 hours and 10 days, depending on the reaction medium used and the amounts of starting material added at the beginning of this step.

As illustrated in the examples, a high level of production of the compound of formula (I) is obtained from the first hours after the addition of the starting materials to the reaction medium containing a 2-deoxyribose-5-phosphate aldolase. A peak of compound of formula (II) is observed after about 6 hours to 30 hours following the addition of the starting materials of the reaction medium, in particular when using a cellular reaction medium. In other cases, a peak of the compound of formula (II) may be observed after several days, for example after 5 to 10 days following the addition of the starting materials to the reaction medium, in particular when a reaction medium is used. acellular.

In other embodiments, the process is carried out continuously.

Step a) of bioconversion of the starting products of compound of formula (I) is carried out, during the selected time, at a temperature ranging from 35 ^° C. to 39 ^° C., preferably from 36 ^° C. to 38 ^° C. Step a) is most preferably carried out at a temperature of about 37 ^° C.

As already mentioned, it is advantageous to add in the reaction medium a quantity by weight of acetone in excess, relative to the weight of compound (II). In step b), when the reaction medium consists of an acellular medium, said reaction medium is recovered by any known technique, for example by simple aspiration.

In step c), when the reaction medium consists of a cell culture, the culture supernatant can be recovered by any technique known to those skilled in the art. In particular, it can be recovered conventionally by centrifugation of the suspension of prokaryotic or eukaryotic cells in the culture medium and recovery of the centrifugation supernatant. In the embodiments of the process in which step b) comprises a step b1) and a step b2) advantageously in step b1) advantageously 1 to 5 cycles of freezing and thawing, and preferably 1 to 3 cycles freezing and thawing the culture supernatant obtained in step c). The method of the invention can be successfully implemented by performing, in step b1), a single cycle of freezing and thawing of the culture supernatant obtained at the end of step a).

Preferably, the culture supernatant is first frozen at a temperature of at least -1 ^{0 °} C., better still at least -2 ^° C. Preferably, a slow defrosting phase is carried out for a period of time. at least 5 hours.

Preferably, the frozen supernatant is slowly defrosted, for example by placing the frozen supernatant at a temperature of at least + ^{40 °} C., for example at a temperature of + 8 ^° C., for a duration of up to 5 hours. at 48 hours, better from 12 hours to 36 hours, for example during a period of 18 hours to 24 hours.

Step b1) of carrying out at least one cycle of freezing and then thawing the culture supernatant obtained at the end of step c) is essential to obtain the compound (I). At the end of step b) of the method, or in step b2) according to the embodiments, the compound (I) contained in the culture supernatant after thawing can be recovered by any technique known to man of the particularly by any liquid / liquid or liquid / gas extraction process appropriate.

The compound of formula (I), in step b), or in step b2) of the process can be recovered, in particular by a liquid / liquid extraction, for example in an organic phase, such as diethyl ether or esters such as ethyl acetate, or in any polar solvent immiscible with appropriate water. Advantageously, when the compound of formula (I) is recovered by a liquid / liquid extraction process, in step b) of the process, an acceptable solvent is advantageously used to obtain a final product under a process. This form is compatible with the regulations governing the placing on the market or the incorporation of natural flavorings into food and cosmetic products.

The recovery of the compound of formula (I) can also be carried out from the thawed culture supernatant by any purification technique by chromatography, including reverse phase chromatography. Thanks to the process of the invention, it is now easy to obtain an aromatic precursor of the phenyl-3-buten-2-one type substituted in the para position by a group R 1 as defined above, by simple bioconversion, and from starting materials that can be obtained from natural sources, including vegetable. In particular, thanks to the process of the invention, it is possible to obtain parahydroxyphenyl-3-buten-2-one, which is a precursor of the raspberry ketone compound, from parahydroxybenzaldehyde in the presence of acetone.

Other precursors of raspberry ketone can also be obtained by implementing the process of the invention with other starting materials such as paramethoxybenzaldehyde or anisaldehyde. In the latter case, the process comprises an additional step of converting the compound (II) anisaldehyde to parahydroxyphenyl-3-buten-2-one, via a demethoxylation or demethylation step. This demethoxylation or demethylation step can be carried out by any technique known to those skilled in the art.

For example, the technique described by Lopretti et al. (Lopretti M et al (1998) Demethoxylation of lignin-model compounds with enzyme extracts from Gloeophilum trabeum, Proc Biochem 33: 657-661). The technique described by Lopretti et al. includes the use of enzymatic extracts of fungous fungi for the demethoxylation of phenolic compounds. In practice, an appropriate quantity of an enzymatic extract obtained from a filamentous fungus, for example Gloeophilum trabeum, is incubated at a temperature of 28 ^° C. in the presence of the methoxylated compound to be converted, and then the compound is recovered. final demethoxylated.

It is also possible to implement a technique similar to that described by Bossert et al. (Bossert et al (1989) Anaerobic Oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium J. Bact 171: 2956-2962), which comprises a step of incubating a methylhydroxylase, at the temperature of 25 ^° C. and under anaerobic conditions, followed by a step of recovering the demethoxylated final compound.

As already mentioned previously in the present description, the compounds (I) constitute precursors of aromatic compounds. In particular, the compound (I) in which R 1 is hydrogen, that is to say parahydroxyphenyl-3-buten-2-one constitutes a direct precursor of the raspberry ketone compound.

In general, raspberry ketone is obtained from para-hydroxyphenyl-3-buten-2-one by a dehydrogenation reaction. by chemical or enzymatic reduction of the compound of formula (I) which is obtained at the end of step b), or at the end of step b2) of the process described above.

Consequently, the subject of the present invention is also a process for obtaining a compound of formula (III) below:

wherein R 1 represents hydrogen or an unsubstituted linear or branched alkyl group having from 1 to 4 carbon atoms, said process comprising the steps of: a) obtaining a compound of formula (I) from a compound of formula (II) by carrying out the process as described above, b) synthesizing the compound of formula (III) by chemical or enzymatic reduction of the compound of formula (I) obtained in step a).

The aromatic compound of formula (III), including raspberry ketone, can be obtained, in stage b) of the above process, by conventional chemical reduction, in particular by means of any suitable method using a catalyst. hydrogenation, for example a catalyst based on nickel or palladium.

According to a second aspect of the process above, the reduction of the compound of formula (I) to obtain the compound of formula (III) can be carried out enzymatically, for example according to the technique described in French patent application N ⁰ FR 2,729,386 and in the application

N ⁰ PCT WO 96 / 21,739. According to this technique, the compound of formula (I) is incubated in the presence of a yeast cell culture Saccharomyces cerevisiae, or a protein or enzymatic extract of Saccharomyces cerevisiae. Then, the culture supernatant of Saccharomyces cerevisiae cells, or the reaction medium comprising the enzymatic extract of Saccharomyces cerevisiae, is recovered. Then, the final aromatic compound of formula (III) is recovered, for example by liquid / liquid or liquid / gas extraction or purification by chromatography, including reverse phase chromatography or gas chromatography.

For example, to carry out step b) of synthesis of the compound of formula (III) by enzymatic reduction of the compound of formula (I) of the above process, a person skilled in the art will advantageously refer to the technique described in the application N ⁰ PCT WO 96 / 21,739. According to this technique, 1 kg of baker's yeast is first added to a container in the open air comprising, with stirring, 2.5 liters of water and 100 g of D-glucose. The suspension is vigorously stirred at the temperature of 3O ⁰ C to 35 ⁰ C. After starting the fermentation of glucose drop was added dropwise over 10 minutes a solution of compound (I) then, after the appropriate incubation time for example 48 hours, the compound of formula (III) is extracted.

The extraction of the compound (III) can be classically a liquid / liquid extraction, for example using methyltertiobutylether.

If desired, the solid residue obtained after evaporation of the solvent can be subjected to a final purification step of the compound of formula (III) by crystallization of the compound of formula (III) in an ethyl acetate solution to which add hexane drop by drop. If desired, the compound of formula (III) can be recrystallized, for example in a mixture of water and methanol. In place of a cell culture of Saccharomyces cerevisiae, it is possible to use a yeast protein extract such as that marketed by SIGMA under the reference Y-2875, as described in PCT application N ⁰ WO 96 /21.739. When the above process is carried out using a compound of formula (I) in which the group R 1 means hydrogen, ie the compound parahydroxybenzaldehyde, while the compound of formula (III ) is parahydroxyphenyl-butan-2-one or raspberry ketone. With the same process, raspberry ketone is also obtained as final product using paramethoxybenzaldehyde or anisaldehyde as starting material.

The subject of the present invention is also a kit for the preparation by biosynthesis of the compound of formula (I) below:

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, said kit comprising: a) a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase; b) a compound of formula (II) below:

wherein R 1 represents hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, and c) optionally, acetone; and d) the chemical or enzymatic reagent or reagents necessary for the conversion of a compound of formula (I) into a compound of formula (III).

Preferentially, in the kit above, the reaction medium, the compound of formula (II), and optionally acetone, are presented in separate containers. As already described in detail previously in the present description, the reaction medium may consist of (i) either recombinant or non-recombinant cells, which produce a 2-deoxyribose-5-phosphate aldolase according to the invention, ( or ii) an acellular composition comprising a 2-deoxyribose-5-phosphate aldolase according to the invention.

A reaction medium of the type (i) above may consist of a liquid medium comprising recombinant or non-recombinant cells in culture. It may be a cell preculture medium, which requires the implementation of a cell growth step prior to the addition of the compound of formula (II) and acetone. It can also be a reaction medium in which the density of the cells are in sufficient number or density to immediately start the process of the invention, by adding the appropriate amounts of the compound of formula (II) and acetone. . Alternatively, a reaction medium of the type (i) above, as contained in a kit according to the invention, may consist of a solid composition comprising the cells, recombinant or non-recombinant, in freeze-dried form. The implementation of the method of the invention, using a kit comprising such a reaction medium requires the addition of a suitable volume of culture medium to the solid composition, and, if appropriate, a preliminary step cell pre-culture, in order to provide a directly usable reaction medium for the implementation of the method according to the invention. In other embodiments of a kit above, a reaction medium of the type (ii) may consist of a liquid composition comprising a 2-deoxyribose-5-phosphate aldolase according to the invention, in suspension.

Alternatively, the reaction medium of the type (ii) may consist of a solid composition, for example a solid composition comprising 2-deoxyribose-5-phosphate aldolase according to the invention, in frozen or freeze-dried form.

Preferentially, the kit above is characterized in that the compound of formula (II) consists of a compound of formula (II) in which R 1 represents a hydrogen atom.

According to a second preferred aspect of said kit, the compound of formula (II) consists of a compound of formula (II) in which R 1 represents the group -CH ₃ .

The subject of the invention is also a kit for preparing a compound of formula (III) as defined in the present description, from a compound of formula (II) as defined in the present description, said kit comprising: a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase; b) a compound of formula (II) below:

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, c) optionally, acetone, and d) the chemical or enzymatic reagent (s) necessary for the conversion of a compound of formula (I) to a compound of formula (III).

Preferentially, in the kit above, the reaction medium, the compound of formula (II), optionally acetone, and the reagent or reagents necessary for the conversion of a compound of formula (II) into a compound of formula (III), are presented in separate containers.

The reaction medium consists of any of the reaction mediums that can be used for the kit described above. Thus, the reaction medium may consist of (i) either recombinant or non-recombinant cells, which produce a 2-deoxyribose-5-phosphate aldolase according to the invention, or (ii) an acellular composition comprising a 2-deoxyribose -5-phosphate aldolase according to the invention.

According to a particular embodiment of the kit above, said kit further comprises an enzymatic reagent chosen from Saccharomyces cerevisiae cells and an enzymatic extract of Saccharomyces cerevisiae.

According to a second preferred embodiment of the kit above, said kit comprises a chemical reagent consisting of a hydrogenation catalyst, for example a nickel-based catalyst or a palladium-based catalyst. The present invention is further illustrated, without being limited, by the following figures and examples. FIGURES

Figure 1 illustrates the production of 4-OH-Benzylidene acetone from 4-OH-benzaldehyde and acetone, in the presence of a reaction medium comprising E. coli cells (ATCC Accession No. 86963).

- On the abscissa: culture time, expressed in hours. The period between the -5h time and the Oh time consists of a cell preculture time.

- On the ordinate, on the scale on the left of the figure: concentration of 4-OH-benzaldehyde (^) or 4-OH-Benzylidene acetone (B), expressed in mg / l.

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers. Figure 2 illustrates the production of Anisylidene acetone from Anisaldehyde and acetone, in the presence of a reaction medium comprising E. coli cells (ATCC Accession No. 86963).

- On the abscissa: culture time, expressed in hours. The period between the -5h time and the Oh time consists of a cell preculture time. - In ordinates, on the scale on the left of the figure: concentration of Anisaldehyde (^), expressed in mg / l.

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers, or concentration of Anisylidene acetone (B) , expressed in mg / l.

Figure 3 illustrates the production of 4-OH-Benzylidene acetone from 4-OH-benzaldehyde and acetone in the presence of a reaction medium comprising E. coli K12 cells. - On the abscissa: culture time, expressed in hours. The period between the -5h time and the Oh time consists of a cell preculture time.

- In ordinates, on the scale on the left of the figure: concentration of 4-

OH-benzaldehyde ()), expressed in mg / l. - In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers, or concentration of 4-OH-Benzylidene acetone (B) expressed in mg / l.

Figure 4 illustrates the production of 4-OH-Benzylidene acetone from 4-OH-benzaldehyde and acetone in the presence of a reaction medium comprising Bacillus subtilis cells (ATCC Reference No. 31324).

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers. Figure 5 illustrates the production of 4-OH-benzylidene acetone from 4-OH-benzaldehyde and acetone, in the presence of a reaction medium comprising Bacillus subtilis cells (CIP reference No. 52.65).

- On the abscissa: culture time, expressed in hours. The period between the -5h time and the Oh time consists of a cell preculture time. - In ordinates, on the scale on the left of the figure: concentration of 4-

OH-benzaldehyde ()), expressed in mg / l.

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers, or concentration of 4-OH-benzilidene acetone (B) expressed in mg / l.

Figure 6 illustrates the production of 4-OH-benzylidene acetone from 4-OH-benzaldehyde and acetone, in the presence of a reaction medium comprising Bacillus subtilis 168 cells (ATCC Accession No. 23857).

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers. Figure 7 illustrates the production of 4-OH-Benzylidene acetone from 4-OH-benzaldehyde and acetone in the presence of a reaction medium comprising Bacillus cereus cells (CIP Part No. 54.9).

OH-benzaldehyde (^) or 4-OH-benzylidene acetone (B), expressed in mg / l.

- In ordinates, on the scale on the right of the figure: density of the bacterial cells (A), expressed in Units of Optical Density (OD) at the wavelength of 600 nanometers.

FIG. 8 illustrates the production of 4-OH-benzylidene acetone from 4-OH-benzaldehyde and acetone, in the presence of a reaction medium comprising an enzymatic extract of deoxyribose 5-phosphate aldolase from Escherichia coli (ATCC Reference No. 86963).

- On the abscissa: incubation time, expressed in days.

- On the ordinate, on the scale on the left of the figure: concentration of 4-OH-benzaldehyde (B) expressed in mg / l. The upper curve (u) illustrates the evolution of the concentration of 4-OH-benzaldehyde in the presence of a reaction medium comprising 1 mg, expressed as an amount of protein, of cell extract. The lower curve (u) illustrates the evolution of the concentration of 4-OH-benzaldehyde in the presence of a reaction medium comprising 5 mg, expressed as an amount of protein, of cell extract.

- In ordinates, on the scale on the right of the figure: concentration of 4-OH-

Benzylidene acetone (•), expressed in mg / l. The lower curve (•) illustrates the evolution of the 4-OH-benzylidene acetone concentration in the presence of a reaction medium comprising 1 mg, expressed as an amount of protein, of cell extract. The upper (•) curve illustrates the evolution of the concentration of 4-OH-benzylidene acetone in the presence of a reaction medium comprising 5 mg, expressed as an amount of protein, of cell extract.

EXAMPLES

A. MATERIAL AND METHODS A.1. Bacterial strains.

The following bacterial strains were used: (a) Escherichia coli (ATCC Reference No. 86963); (b) Escherichia coli K12 (ATCC accession No. 29425) (c) Bacillus subtilis (ATCC reference No. 31324, DSM reference No. 704) (d) Bacillus subtillis (Pasteur Institute collection -IPC reference No. 52.65) e) Bacillus subtilis 168 (ATCC Reference No. 23857); and f) Bacillus cereus (Institut Pasteur Collection - CIP Reference No. 54.9)

A.2. Media and culture conditions Composition of preculture medium (OSP 1 liter) BioValley and Sigma products for salts: meat peptone (4.3 g), NaCl (6.4 g), the pH is adjusted to 7 (NaOH or HCl) .

Composition of the medium of culture (QSR 1 liter) products Bio Balley and Sigma for the salts: Extract of yeast (2g), FeSO ₄ - 7H ₂ O (0,001g), CaCl ₂ (O ₁ OIg), CuSO-5H ₂ O (0.01 g), MgSO ₄ (0.8 g), ZnSO ₄ -H ₂ O (0.01 g), (NH ₄₎ ₂ SO ₄ (5 g), K ₂ HPO ₄ -3H ₂ O (1.32 g) MnSO ₄ -H ₂ O (0.1 g) The pH is adjusted to 7 (NaOH or HCl) and the medium is autoclaved at 121 ^° C. for 20 minutes When the temperature of the medium has dropped to 60 ^° C. we add 2-Desoxyadenosine (Fluka) at a final concentration of 10 mM All cultures and precultures are carried out on the preceding media at 30 ^° C. in a 5 liter Erlenmeyer flask containing 500 ml of medium and capped with carded cotton. The stirring is 130 agitations per minute.The precultures are carried out for 16 hours up to an OD of 4 to 4.5 for the two strains.The culture medium is seeded with this preculture at a rate of 5%. 12h of preculture (OD of 2 to about 3,5, according to the strain udiée), the precursors (acetone (Sigma)) at 2% and the phenolic compound (Sigma) at 0.5 g / l in the form of a mother ethanolic solution). The order of adding does not matter. A 5 ml sample is taken every 4 hours. OD is measured at 600 nm.

A3. Preparation of a cell extract comprising a 2-deoxyribose-5-phosphate aldolase

The cell extract was obtained from cells of the Escherichia coli strain referenced ATCC under No. 86963. For this strain of E. coli, the production of 2-deoxyribose-5-phosphate aldolase is induced in the presence of substrate.

The first step therefore consists of a step of induction of the production of 2-deoxyribose-5-phosphate aldolase by the cells in culture, lasting for 24 hours.

After 24 hours of preculture of E. coli with an OD of 3.2, the cells are centrifuged, washed 5 times in phosphate buffer pH 7.4 and then crushed with the French press in the same buffer supplemented with benzamidine (1 mM), EDTA (2 mM), PMSF (1 mM) and DTT (1 mM). After grinding, the extracts are centrifuged at 8000xg for 15 minutes and the supernatant is preserved.

The production tests for compounds of formula (I) are carried out with this cell extract, according to the following protocol:

in 10 mM Triethanolamine buffer pH 8.1 + 1 mM EDTA, the compound of formula (II) and acetone are added, as well as the enzymatic extract obtained as described above.

the reaction takes place under nitrogen at 37 ^° C. Regular additions of PMSF (1 mM) are carried out in order to limit the action of the proteases.

the samples containing the final products of the enzymatic reaction are extracted as described below.

A. 3. Extraction and dosage:

Measuring biomass:

5 ml of culture are filtered on 0.45 μm filter previously weighed. The filter is allowed to dry at 60 ^° C. for 24 hours before final weighing. When necessary, a viability measurement is carried out by spreading on the corresponding culture medium supplemented with agar

(15g / l).

Extraction of volatile compounds

The samples are centrifuged at 3000g for 15 minutes. The supernatant is then recovered and frozen at -2O ⁰ C. After slow thawing (one night between ⁴⁰ ^° C. and 80 ^° C.) and after addition of 40 μl of a 1 g / l solution of 1,4-dimethoxybenzene (internal standard), the supernatant is extracted twice successively with 5 ml. of ethyl acetate (Carlo Erba). The two extracts are then combined, dried over anhydrous sodium sulphate and then filtered on glass cotton and concentrated under a stream of nitrogen to a volume of 500 μl. Determination of volatile compounds

I μl of extract are injected onto a DB5 column (30m × 0.319mm), thickness 0.25 μm (reference J & W US 1 477866H). Helium or hydrogen vector gas (40.5 cm / second velocity at 40 ^° C.). Temperature gradient from 40 to 240 ° C / min.

The compounds are quantified with respect to the internal standard. A.4. Reaction Witnesses

It has been verified that no production of a final compound of formula (I) has been detected, under the reaction conditions described above, with any of the following control compositions:

- Acetone 2% single + culture medium + E. coli (ATCC 86963);

- 4-OH-benzaldehyde alone (0.5 g / l) + culture medium + E. coli (ATCC 86963); - Acetone (2%) + 4-OH-benzaldehyde (0.5 g / l) alone + culture medium;

- Culture alone of E. coli (ATCC 86963);

- DERA alone (origin Lactobacillus plantarum;

B. RESULTS

Example 1 Production of a compound of formula (I) using a reaction medium comprising cells producing a 2-deoxyribose-5-phosphate aldolase.

The results of FIG. 1 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-benzaldehyde and acetone, using a reaction medium comprising E. coli cells (ATCC 86963), in significant amount (160 mg / ml).

The results of FIG. 2 show that, thanks to the process of the invention, Anisylidene acetone is produced from anisaldehyde and acetone, using a reaction medium comprising E. coli cells (ATCC 86963 ), in significant amount (6 to 10 mg / ml)

The results of FIG. 3 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-benzaldehyde and acetone, using a reaction medium comprising E-cells. coli K12, in significant amount (approximately 4 mg / ml).

The results of FIG. 4 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-

Benzaldehyde and acetone, using a reaction medium comprising Bacillus subtilis cells (ATCC 86963), in a significant amount (about 25 mg / ml).

The results of FIG. 5 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-

Benzaldehyde and acetone, using a reaction medium comprising Bacillus subtilis cells (CIP 52.65), in significant amount (about 3.5 mg / ml).

The results of FIG. 6 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-

Benzaldehyde and acetone, using a reaction medium comprising Bacillus subtilis 168 cells, in a significant amount (about 30 mg / ml).

The results of FIG. 7 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-

Benzaldehyde and acetone, using a reaction medium comprising Bacillus cereus cells (CIP 54.9), in significant amount (about 30 mg / ml). Example 2 Production of a compound of formula (I) using a reaction medium comprising an enzymatic extract of E. coli cells producing a 2-deoxyribose-5-phosphate aldolase. The results of FIG. 8 show that, thanks to the process of the invention, 4-OH-benzylidene acetone is produced from 4-OH-benzaldehyde and acetone, using a reaction medium comprising an enzymatic extract obtained from E. coli cells (ATCC 86963), in significant amount. More particularly, the results of FIG. 8 show that with an amount of enzymatic extract comprising 1 mg of total protein, a final concentration of 4-OH-benzylidene acetone of about 2 mg / ml can be obtained after a period of 5 days of the enzymatic reaction.

Also, the results of FIG. 8 show that with an amount of enzymatic extract comprising 5 mg of total proteins, a final concentration of 4-OH-benzylidene acetone of about 5 mg / ml can be obtained after a period of 5 days of the enzymatic reaction.

Table 1

TABLE 1 (continued)

BACTERIA

TABLE 1 (continued)

BACTERIA

TABLE 1 (continued)

BACTERIA

TABLE 1 (continued)

Ref Organization Score% Identity% Positive% Gap Sequence Length (a.a.) (Access N °)

BACTERIA

TABLE 1 (continued)

TABLE 1 (continued)

BACTERIA

TABLE 1 (continued)

Claims

1. Process for the biosynthetic preparation of the compound of formula (I) below:

(i) a compound of the following formula (II):

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, and

(ii) acetone in a reaction medium comprising 2-deoxyribose-5-phosphate aldolase; and b) recovering the compound of formula (I) from the medium obtained at the end of step a).

2. Method according to claim 1, characterized in that the compound of formula (II) is such that R 1 represents a hydrogen atom.

3. Method according to claim 1, characterized in that the compound of formula (II) is such that Ri represents a group -CH ₃ .

4. Method according to any one of claims 1 to 3, characterized in that in step a), is added to the reaction medium an amount by weight of acetone in excess, relative to the weight of compound (II ).

5. Method according to one of claims 1 to 4, characterized in that step b) comprises the following steps: b1) carry out at least one cycle of freezing and thawing of the medium obtained at the end of step a) ; b2) recovering the compound of formula (I) from the thawed medium obtained at the end of step b1).

6. Method according to claim 5, characterized in that, for each cycle of freezing and thawing of step b1), the thawing phase consists of a slow defrosting of a duration of at least 5 hours.

7. Method according to one of claims 1 to 6, characterized in that in step b2), the compound of formula (I) is recovered by extraction with a solvent selected from diethyl ether ethyl acetate or any polar solvent immiscible with water.

8. Method according to one of claims 1 to 7, characterized in that the 2-deoxyribose-5-phosphate aldolase has an amino acid sequence having at least 30% identity with the amino acid sequence SEQ ID # 1

9. Process according to claim 8, characterized in that 2-deoxyribose-5-phosphate aldolase has an amino acid sequence selected from the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

10. Method according to one of claims 1 to 9, characterized in that the 2-deoxyribose-5-phosphate aldolase is encoded by a nucleic acid from a prokaryotic organism of a species selected from Escherichia coli, Bacillus, Pseudomonas , Streptomyces, Corynebacterium and Shewanella.

11. Method according to one of claims 1 to 9, characterized in that the 2-deoxyribose-5-phosphate aldolase is encoded by a nucleic acid from a prokaryotic organism selected from Escherichia coli K12, Escherichia coli CFT073, Shigella flexneri Salmonella enterica subsp. Enterica, Vi brio cholerae, Yersinia pestis, Yersinia pseudotubercolis, Photobacterium profundum, Vibrio fischeri, Erwinia carotovora, Actinobacillus pleuropneumoniae, Colwellia psychererythraea, Shewanella baltica, Shewanella amazonensis, Shewanella frigidimarina, Shewanella denitrificans, Chromohalobacter salexigens, Burkholderia pseudomallei, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. tomato, Idiomarina lawhiensis, Pseudomonas syringae pv. Syringae, Magnetospirillum magnetotacticum, Streptomyces coelicolor, Streptomyces avermitilis, Coxiella burnetii, Nocardioides sp. JS614, Paracoccus denitrificans, Mesorhizobium sp. BNC1, Silicibacter sp. TM 1040, Rhodobacter sphaeroides, Bacteroides thetaiotaomicron, Francisella tularensis, Bacteroides fragilis, Silicibacter pomeroyi, Burkholderia fungorum, Jannaschia sp. CCS 1, Porphyromonas gingivalis, Legionella pneumophila, Treponema denticola, Bacillus cereus, Geobacillus kaustophilus, Bacillus halodurans, Solibacter usitatus, Streptococcus suis, Bacillus licheniformis, Listeria monocytogenes, Deinococcus geothermalis, Chloroflexus aurantiacus, Rubrobacter xylanophilus, Desulfitobacterium hafniense, Arthrobacter sp. FB24, Bacillus clausii, Rhodospirillum rubrum, Staphylococcus haemolyticus, Corynebacterium glutamicum, Enterococcus faecium, Bacillus subtilis, Symbiobacterium thermophilum, Thermococcus kodakarensis and Exiguobacterium sp. 255-15.

12. Method according to one of claims 1 to 9, characterized in that 2-deoxyribose-5-phosphate aldolase is encoded by a nucleic acid from a eukaryotic organism belonging to a species selected from Homo sapiens, Gallus gallus, Canis familiaris, Mus musculus, Caenorhabditis elegans, Anopheles gambiae, Danio rerio, Xenopus tropicalis, Plasmodium chabaudi, Plasmodium berghei, Plasmodium falciparum, Apis mellifera, Drosophila melanogaster, Entamoeba histolytica, Aspergillus fumigatus, Strongylocentrotus purpuratus, and Aspergillus nidulans.

13. Method according to one of claims 1 to 12, characterized in that in step a), the reaction medium consists of a culture of prokaryotic or eukaryotic cells producing a 2-deoxyribose-5-phosphate aldolase.

14. The method of claim 13, characterized in that the prokaryotic or eukaryotic cells consist of cells naturally producing a 2-deoxyribose-5-phosphate aldolase.

15. The method according to claim 13, characterized in that the prokaryotic or eukaryotic cells consist of recombinant cells into which a nucleic acid encoding a 2-deoxyribose-5-phosphate aldolase has been inserted.

16. Method according to one of claims 1 to 12, characterized in that the reaction medium consists of an acellular composition comprising a 2-deoxyribose-5-phosphate aldolase.

17. Process according to claim 16, characterized in that the reaction medium consists of a liquid composition comprising an enzymatic extract obtained from a culture of prokaryotic or eukaryotic cells producing a 2-deoxyribose-5-phosphate aldolase.

18. Method according to one of claims 1 and 3 to 17, characterized in that when the group R1 of the compound (II) means -CH ₃ , the process comprises an additional step c) conversion of said compound (II) into p hydroxyphenyl-3-buten-2-one.

19. Process for obtaining a compound of formula (III) below:

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, said process comprising the steps of: a) obtaining a compound of formula (I) from a compound of formula (II) by carrying out the process according to any one of claims 1 to 18; b) synthesizing the compound of formula (III) by chemical or enzymatic reduction of the compound of formula (I) obtained in step a).

20. Process according to claim 19, characterized in that the compound of formula (II) has a group R 1 which signifies hydrogen or a group -CH ₃ , and in that the compound of formula (III) is the hydroxyphenyl-butan-2-one.

21. Kit for the biosynthetic preparation of the compound of formula (I) below:

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, and c) optionally, acetone.

22. Kit according to claim 21, characterized in that it comprises a compound of formula (II) in which R 1 represents a hydrogen atom.

23. Kit according to claim 21, characterized in that it comprises a compound of formula (II) in which R 1 represents -CH ₃ .

24. Kit according to one of claims 21 to 23, characterized in that the reaction medium consists of a composition comprising prokaryotic or eukaryotic cells producing a 2-deoxyribose-5-phosphate aldolase.

25. Kit according to one of claims 21 to 23, characterized in that the reaction medium consists of an acellular composition comprising a 2-deoxyribose-5-phosphate aldolase.

26. Kit for preparing a compound of formula (III) as defined in claim 19, from a compound of formula (II) as defined in claim 1, said kit comprising: a) a reaction medium comprising a 2-deoxyribose-5-phosphate aldolase; b) a compound of the following formula (II)

wherein R 1 is hydrogen or an unsubstituted linear or branched alkyl group having 1 to 4 carbon atoms, c) optionally, acetone, and d) the chemical or enzymatic reagent (s) necessary for the conversion of a compound of formula (I) in a compound of formula (III)

27. Kit according to claim 26, characterized in that the reaction medium consists of a culture of prokaryotic or eukaryotic cells producing a 2-deoxyribose-5-phosphate aldolase.

28. Kit according to claim 26, characterized in that the reaction medium consists of a liquid composition comprising a 2-deoxyribose-5-phosphate aldolase.

29. Kit according to any one of claims 26 to 28, characterized in that it comprises an enzymatic reagent selected from Saccharomyces cerevisiae cells and an enzymatic extract of Saccharomyces cerevisiae.

30. Kit according to any one of claims 26 to 28, characterized in that it comprises a chemical reagent consisting of a hydrogenation catalyst.