WO1994001564A1 - Yeast strain for the co-expression of a plant cytochrome p450 mono-oxygenase activity and an endogenous or heterologous nadph-cytochrome p450-reductase, and use thereof in bioconversion - Google Patents

Abstract

A yeast strain for the co-expression of a plant cytochrome P450 mono-oxygenase activity and an endogenous or heterologous NADPH-cytochrome P450-reductase, wherein one of the NADPH-cytochrome P450-reductase or cytochrome P450 genes is integrated into the chromosome of said strain, and the strain is transformed by a vector having an expression cassette for the other gene. The use of said yeast strain, as well as a cDNA sequence coding for cytochrome P450 CA4H of the Jerusalem artichoke Helianthus tuberosus or Arabidopsis thaliana, for bioconversion purposes, is also disclosed.

Description

 "YEAST STRAIN ALLOWING THE CO-EXPRESSION OF A MONO-OXYGENASE ACTIVITY OF PLANT CYTOCHROME P450 AND AN ENDOGENOUS OR HETEROLOGOUS NADPH- CYTOCHROME P450-REDUCTASE AND ITS USE FOR BIOCONVERSION PURPOSES" The present invention relates to a strain of yeast allowing the co-expression of a microsomal cytochrome P450 mono-oxygenase activity of the plant and of an endogenous or heterologous NADPH-cytochrome P450-reductase.

The present invention also relates to a method of co-expression in a yeast of a mono-oxygenase activity of plant microsomal cytochrome P450 and of endogenous or heterologous NADPH-cytochrome P450-reductase.

The present invention also relates to the use for bioconversion purposes of a yeast strain according to the invention. More specifically, the present invention relates to a bioconversion process intended for specific monoxygenation, in particular for stereospecific hydroxylation, of a substrate compound of a cytochrome P450.

The present invention finally relates to a cDNA sequence coding for cytochrome P450 catalyzing the hydroxylase of trans-cinnamate or or CA4H in the tubers of Jerusalem artichoke or in seedlings of Arabidopsis thaliana.

Cytochromes P450 are proteins with mono-oxygenase activity which constitute one of the largest superfamilies of genes known in higher eukaryotes. They are able to oxidize a very wide variety of generally hydrophobic substrates using molecular oxygen dissolved in the cytoplasm, as well as reducing equivalents provided by NADPH-cytochrome P450-reductase. These reactions are most often characterized by the insertion of an oxygen atom in CH, C = C, N = N bonds, either by the oxidation of a heteroatom or even in rarer cases by a reduction nitro groups or dehalogenation (Guengerich and Macdonald, 1990, FASEB J. 4, pp 2453-2459).

Cytochromes P450 are membrane proteins most often associated with the endoplasmic reticulum, some forms being mitochondrial. Their intervention in humans and animals in the early stages of the metabolism of xenobiotic substances such as drugs or pollutants, as well as their involvement in important pathways of endogenous metabolism of membrane and hormonal steroids, bile acids, pheromones , vitamin B, fatty acids, prostaglandins have made cytochromes P450 an object of keen interest (Nebert and Gonzales, 1987, Ann. Rev. Biochem. 56, pp 945-993).

The intense research efforts have resulted in the development of a large number of biochemical, immunological and genetic tools which have made it possible to isolate approximately 200 DNA sequences (genomics or cDNA) to date.

Relative to the rapidly increasing number of known sequences of many isoforms of cytochrome P450 in humans and animals, no DNA sequence from a plant cytochrome P450 with a well-defined function has been isolated.

In the context of the present invention, the cDNA sequence of a plant cytochrome P450, CA4H, which catalyzes the hydroxylation at position 4 of trans-cinnamate, is claimed. The product thus formed is trans-coumarate.

In general, research progress in the field of plant P450 cytochromes has been delayed due to their low content in plant tissues and the great difficulties encountered in their purification. Oxidations of endogenous substrates in plants, catalyzed by P450 cytochromes, include hydroxylations of aliphatic and aromatic compounds, epoxidation, demethylation and intramolecular rearrangements. Forty enzyme activities catalyzed by P450 cytochromes have been characterized so far in plants. They are involved in the metabolism of phenylpropanoids, terpenes, lipids and also participate in the synthesis of phytoalexins, anthocyanins, tannins, flavors, hormones, membrane sterols, alkaloids and cutins and suberins. Given their participation in the synthesis of this large number of plant metabolites, some of which are of great industrial interest as food additives or natural pigments, the cloning and coordinated overexpression of cytochromes P450 in a microorganism suitable for carrying out bioconversion reactions are of great interest and much sought after.

The dealkylation of xenobiotics has also been reported (Fonné et al., 1988, Plant Sci., 55, pp 9-20). There is a clear revival of interest in agronomy for plant P450 cytochromes since they are involved in the first stages of the metabolism of certain pesticides or herbicides such as diclofop or chlortoluron (Zimmerlin and Durst, 1990, Phytochemistry, 29, pp 1729-1732, Fonne-Pfister and Kreuz, 1990, Phytochemistry 29, pp 2793-2797) which gives them selective resistance to these products. Mastering and understanding their function is therefore of primary importance for the agricultural industry.

Due to its key position in the secondary metabolism of the plant, cinnamate 4-hydroxylase to P450, which is the common starting point for the synthesis of a very large number of metabolic pathways, the end products of which have crucial roles either as a structural support for the cell (lignins, phenols in the wall), either as a support for surface protection (suberins), or as a defense substance for the plant cell against pathogens (flavonoids, isoflavonoids, stilbenes, phenolic acids, tannins ), or as UV radiation filters. In addition to these vital functions, a number of compounds from these metabolic pathways are highly sought after in the food industry as a natural flavor or color. For example, we can cite vanillin, the first stage of biosynthesis in the pod of Vanilla planifolia is the transformation of trans-cinnamate into trans-coumarate. The world market for vanillin is currently estimated at 5,000 tonnes / year.

It has been described in the literature the expression in yeast of a mammalian cytochrome P450 (rat, beef) and of endogenous or rat NADPH-cytochrome P450 reductase on non-integrative vectors.

Neither the possibility of expression, transport and maturation of membrane proteins of plant origin in a yeast cell context, nor the functionality of a coupling between plant cytochrome P450 and NADPH-P450 yeast reductase could not be established before the present invention.

The present invention relates to a yeast strain allowing the co-expression of a mono-oxygenase activity of plant cytochrome P450 and an endogenous or heterologous NADPH-cytochrome P450-reductase, characterized in that one of the genes NADPH-cytochrome P450-reductase or cytochrome P450 is integrated into the chromosome of said strain, and the strain is transformed by a vector carrying an expression cassette for the other gene. Said vector may or may not be an integration vector.

When said vector is an integration vector in the chromosome of the strain, the two genes are therefore in fact integrated in the genome of the strain.

The present invention therefore in particular relates to a yeast strain allowing the co-expression of the mono-oxygenase activity of plant cytochrome P450 and of an endogenous or heterologous NADPH-cytochrome P450-reductase, characterized in that the gene NADPH-cytochrome P450-reductase is integrated into the chromosomal genome of said strain and the strain is transformed by a vector carrying an expression cassette for the plant cytochrome P450 gene.

Preferably, a microsomal cytochrome P450 gene will be used.

The term “microsomal” is understood here to mean the fractions of intracellular membranes, in particular of the endoplasmic reticulum, rough or smooth, of the Golgi apparatus.

In a particular embodiment, said vector is not an integration vector but a replicative vector, in particular a plasmid vector.

In the present patent application, the term "heterologous gene" means a gene originating from an organism other than a yeast. Preferably, the cytochrome P450 gene is integrated into the chromosome of a strain. More preferably, the two genes of cytochrome P450 and of NADPH-cytochrome-P450 respectively are integrated into the chromosomal genome of the strain.

The microsomal cytochrome P450 according to the invention is more particularly cited as the protein catalyzing the transcinnamate hydroxylase, also called the CA4H protein, in particular originating from Jerusalem artichoke tubers Heliantus tuberosus or young seedlings of Arabidopsis thaliana.

Suitably, said gene of said cytochrome P450 is the cDNA sequence coding for said cytochrome P450. Said gene of said cytochrome P450 may be a hybrid gene.

The term “hybrid gene” is understood to mean a gene which may result from homologous recombinations between two cytochrome P450 genes of different origins, in particular from different organisms, or which may result from fusions of parts of cytochrome P450 genes from different origins, in particular d 'different organisms, at least one being of plant origin.

In particular, the cDNA sequence encoding CA4H as shown in FIG. 1 is cited.

In a particular embodiment, the NADPH-cytochrome P450-reductase is the endogenous yeast NADPH-cytochrome P450-reductase.

In another embodiment, said NADPH-cytochrome P450-reductase gene consists of the cDNA sequence coding for heterologous NADPH-cytochrome P450-reductase. Gene Cloning of NADPH-cytochrome P450 reductase heterologous, especially of plants, was described in the French patent application No. ^β 92 04491 and the patent application PCT / FR93 / 00367, the 2nd International Symposium on Cytochrome P450 of Microorganisms and Plants de Tokyo - June 13-17, 1993, in the communication "Yeast as a Cloning and Expression Host for Plant P450s and Reductases" by Denis Pompon, C. Mignotte, S. Régnier, P. Urban, G. Truan, and Mr. Kazmaier.

This efficient process for cloning a DNA sequence coding for a plant NADPH-Cytochrome P45 Reductase comprises the following steps: 1- A yeast strain transformed with plasmids from a total cDNA library of said plant is subjected , and having a NADPH-Cytochrome P450 activity deficiency phenotype

Endogenous reductase, to treatment with a cytochrome P450 d inhibitor, in particular ketoconazole, at a dose which makes the absence of NADPH-Cytochrome P450 activity lethal

Endogenous reductase, namely 10 to 50 μg / ml of culture medium, then 2- The surviving clones are recovered and the plasmids are extracted therefrom allowing the expression of a NADPH-Cytochrome P450 Reductase activity which corresponds to the NADPH-Cytochrome P450 of said plant. Mention may in particular be made, as appropriate, as a gene for

NADPH-cytochrome P450-reductase, genes of plant origin, in particular the genes of NADPH-cytochrome P450-reductase from Arabidopsis thaliana or Jerusalem artichoke Helianthus tuberosus.

The cDNA sequences encoding these genes of plant origin have been represented in French patent applications No. 92 04491 and

PCT / FR No. 93/00367. The sequences ATR1 (Helianthus tuberosus) and ATR2 (Arabidopsis thaliana) were introduced into the EMBL bank under the following references: For ATR1: Accession number Y66016

Identification number ATATR1G; For ATR2:

Accession number X66017 Identification number ATATR2M. In a particular embodiment, when the NADPH-cytochrome P450-reductase gene is heterologous, it is of the same genus and of the same species as the heterologous cytochrome P450.

In order to obtain the most stable integration possible, preferably the gene (s) for NADPH-cytochrome P450-reductase and / or cytochrome P450 is (or are) integrated (s) in the locus of NADPH - endogenous cytochrome P450-reductase on dredge allele of the chromosome.

According to the invention, the yeast strain can be chosen in particular from the genera Saccharomvces. Hansenula. Kluvveromvces. Pichia and Yarrowia. Mention is more particularly made of yeasts of the genus Saccharomvces cerevisiae.

Advantageously, to allow good modulation and regulation of the expression of cytochrome P-450 in said vector, the cytochrome P450 gene is placed under the control of an inducible promoter. In a particular embodiment, the vector pYEDP60 described below was used. Advantageously also, the natural promoter of NADPH-cytochrome P450-reductase was replaced, which was placed under the control of an inducible heterologous promoter.

Advantageously also, the NADPH-cytochrome P450-reductase gene and the cytochrome P450 gene are placed under the control of the same inducible promoter, in particular, the GAL10 CYC1 promoter inducible in galactose medium.

In one particular embodiment, said strain is obtained from the PES 1-3-U strain deposited at the National Collection of Cultures of Microorganisms (CNCM) under the number 1-1187. This strain has been described in French patent applications No. 92 04491 and PCT / FR No. 93/00367.

This strain expresses, when used on a medium containing galactose, a level of NADPH-cytochrome P450-reductase which is 20 to 30 times higher than the level present in the parental strain containing the natural promoter. Conversely, in the absence of galactose, the level of reductase present is extremely low, in fact undetectable, that is to say at least 100 times lower than the level present in the parental strain.

Intermediate levels of expression between these two extremes are possible using limited durations of galactose induction (0 to 12 hours).

In the detailed embodiment which follows, said strain is the WRCA strain obtained by transformation of the PES 1-3-U strain with the vector pCA4H / YeDP60. Preferably, to obtain the maximum expression levels, the strain co-expresses NADPH-cytochrome P450-reductase and cytochrome P450 in an optimum NADPH-cytochrome P450-reductase / cytochrome P450 molar ratio between 1/3 and 1/10 , especially 1/5.

The variation in the respective expression levels can be obtained by varying the number of copies of the respective genes and / or by using different and more or less strong promoters and / or by shifting the induction time of the respective promoters.

In order to obtain large quantities of a molecule of industrial interest by a bioconversion process, the choice of the microorganism best suited for the expression of the cytochrome P450 / P450 reductase complex is essential. In this regard, according to the present invention, we uses the yeast Saccharomvces cereviae. recombined in order to give the heterologous P450 an optimal environment for the efficient expression of its activity.

According to the present invention, particular attention has been paid to the high-level coexpression of CA4H and of the associated P450 reductase. Since P450 reductase has a faster catalytic cycle than P450 cytochromes, it is important to respect a particular molar ratio essential for proper functioning in yeast. In fact, a too low heterologous reductase / P450 molar ratio leads to a low specific activity due to the reductase defect. A too high heterologous reductase / P450 molar ratio leads to the destruction of a large fraction of P450 due to the strong increase in abortive cycles. This is why, in order to best respect an optimal reductase / P450 molar ratio, a recombinant yeast was used according to the invention in which the natural promoter of the endogenous P450 reductase gene has been replaced by the inducible promoter GAL10 / CYC1. Since the sequence of C4AH on plasmid is under the control of the same promoter, it can reasonably be assumed that the final molar ratio is given by the number of copies of the plasmid compared to the single copy of the reductase gene.

According to the present invention, a recombinant yeast strain has been constructed which ensures an optimal level for the expression of the first plant cytochrome P450 with known function. More specifically, for a level of expression comparable to that found in the literature of 100 picomoles per mg of microsomal proteins, the specific activity is of the order of 400 min-1, the highest value ever measured for a cytochrome P450 microsomal. This activity also remains stable over time. In particular, according to the present invention, a recombinant yeast strain is therefore described which, on the productivity criteria, is entirely suitable for the bioconversion of trans-cinnamate into tr-coumarate by the Jerusalem artichoke CA4H.

The present invention therefore also provides a method of co-expression in a yeast of the mono-oxygenase activity of plant microsomal cytochrome P450, characterized in that a strain according to the invention is cultivated in an appropriate culture medium.

The present invention also relates to the use for bioconversion purposes of a yeast strain according to the invention. The present invention relates, in particular, to a bioconversion process intended for the specific monoxidation of a substrate compound of a cytochrome P450, characterized in that a medium containing said compound or a precursor is converted with one of the strains according to the invention expressing the monoxygenase activity of said cytochrome P450 and in that the monoxygenated product is recovered.

Preferably, the culture medium of the strain is used as the conversion medium. However, it is possible to provide, when the mass of yeast initially cultivated is sufficient, to carry out the conversion in a medium which is not a culture medium.

In general, the substrate is contained in the medium, it enters the cells where it is converted, then is released into the medium. However, in certain cases, the medium may contain only a precursor of the substrate, the substrate only appearing inside after transformation of the precursor.

The mono-oxidation can be, for example, a stereospecific hydroxylation, in particular when the cytochrome P450 is CA4H, in which case the substrate compound is trans-cinnamate and the trans-coumarate is recovered. Finally, the present invention also relates to the cDNA sequences coding respectively for the cytochrome P450 CA4H of Arabidopsis thaliana and the cytochrome P450 CA4H of Helianthus tuberosus. in particular the cDNA sequences as represented in FIGS. 1 and 12 or the cDNA sequences hybridizing under weakly stringent conditions at 50 ° C. and / or having at least 60% homology with one of the sequences previously described, or the same reformatted sequences including only the open reading phase.

Other characteristics and advantages of the present invention will appear in the light of the detailed description of the embodiment (s) which follows, illustrated by the appended drawings in which:

FIG. 1 represents the cDNA of the cytochrome P450 gene of Jerusalem artichoke Helianthus tuberosus CA4H,

FIG. 2 represents the cloning diagram of the reformatted cDNA comprising only the open reading phase of the Jerusalem artichoke CA4H in the vector of pYED P60, - Figure 3 shows, schematically, the strain of lecure WRCA simultaneously overexpressing in galactose medium an exogenous CA4H and the endogenous NADPH-cytochrome P450-reductase (hereinafter abbreviated as "P450-reductase"), - Figure 4 represents the spectrum difference in absorption in the presence of carbon monoxide of a microsomal fraction of WRCA strain,

FIG. 5 represents the type I perburbation spectrum following the addition of trans-cinnamic acid to the microsomes from the induced culture of the WRCA strain,

FIG. 6 represents a chromatogram for the detection of trans-cinnamate after conversion of the trans-cinnamate by a culture of WRCA in galactose medium,

FIG. 7 represents the variation in the amplitude of absorption in a type I perturbation spectrum as a function of the trans-cinnamate added in increasing amounts to oxidized microsomes originating from an induced culture of the WRCA strain,

FIG. 8 represents the coupling efficiency between the yeast NADPH P450-reductase and the Jerusalem artichoke CA4H in the microsomes of the WRCA strain,

- Figure 9 shows a representation according to Lineweaver-Burk giving the inverse of the CA4H activities measured as a function of the inverse of the concentration of added trans-cinnamate allowing the determination of the maximum rate of conversion of CA4H in yeast microsomes as well as the Km,

FIG. 10 represents the kinetics of p-coumarate formation on WRCA microsomes,

FIG. 11 represents the kinetics of p-coumarate formation from trans-cinnamate on whole cells containing the Jerusalem artichoke CA4H,

- Figure 12 shows the complete cDNA of the CA4H gene of Arabidopsis thaliana.

- Figures 13 A, 13B and 13C respectively represent the 3 steps (A, B, C) necessary for the construction of the integrative vector pYEDPllO. In FIG. 13A is illustrated the PCR amplification of the 5 'and 3' non-coding regions which border the open reading phase of the genomic locus of the NADPH cytochrome P450 reductase yeast. Are also illustrated the restriction sites created at the edge of the amplified fragments. In FIG. 13B is shown the replacement of the PGK terminator by the 3 ′ non-coding region of a yeast NADPH cytochrome P450 reductase gene in the vector pYEDPS 1. In FIG. 13C is shown the replacement of the origin of replication 2 microns of yeast by the 5 ′ non-coding region of the yeast NADPH cytochrome P450 reductase gene in the vector pYEDPlOO. The integrative vector thus obtained is called "pYEDPl lO",

FIG. 14 represents the physical structure of the Yred locus in the different strains,

FIG. 15 represents a summary table in which the resistances of the different strains to ketoconazole are reported, the NADPH cytochrome P450 reductase activities measured (in nmoles reduced cyte / min / mg of microsomal proteins) on microsome preparations, as well as ability to convert tr-cinnamate to coumarate. +++ assessment indicates that the bioconversion reaction is complete in 30 minutes,

- Figures 16A to 16D show HPLC separations of the metabolites obtained after the incubation of the Cil, C21, Cl 00 and 21/21 strains in the presence of 10 μM of tr-cinnamate. The paracetamol present on the chromatograms is added to the samples analyzed as an internal control of the extraction efficiency.

I) ISOLATION of cDNA ENCODING FOR P450 CYTOCHROMES OF THE COUMARATE 4-HYDROXYIASE TYPE (E.C. 1.14. 3.1) OF PLANT ORIGIN

1.1) Isolation of the cvtochrome P450 cDNA catalyzing the hydroxylation of tr-cinnamate in Jerusalem artichoke tubers (CA4H = cinnamate4-hvdroxylase.ECl14.13.1). The CA4H cloning procedure uses conventional methods of molecular biology known to those skilled in the art. The purification protocol, as well as obtaining a specific anti-CA4H antibody using the purified protein, is described in the scientific literature (Gabriac et al. Archives of Biochemistry and Biophysics, 1991, Vol 288 , pp 302-309) and is not repeated in detail here. Briefly, slices of Jerusalem artichoke tubers, incubated in a medium containing manganese to induce the synthesis of the enzymatic activity of CA4H, constitutes the starting material for the preparation of the microsomal fraction. It follows a first stage of enrichment in CA4H by a phase partition in triton X-114, and two stages of chromatography on DEAE-Trisacryl and on Hydroxylapatite. The protein thus purified, which shows an apparent molecular weight of 57KD in SDS-Acryla ide gel, was used for the manufacture of polyclonal antibodies. _. These antibodies react selectively with a 57 KD polypeptide in Western blot and also strongly inhibit CA4H activity in microsomes of different plant species. Their use to screen a Jerusalem artichoke cDNA library in phage lambda gt11, obtained from tubers induced for the overexpression of CA4H, made it possible to isolate a cDNA fragment of 1130 bp having sequences characteristic of cytochrome P450 bacterial, fungal or animal. This fragment was then used as a probe to screen a second Jerusalem artichoke cDNA library in the phage lambda gt10 constructed by priming with oligodT. This made it possible to isolate a clone of 1608 bp which presents all the sequences characteristics of a P450 and the complete sequence is shown in figure 1. This cDNA can be identified as that of the Jerusalem artichoke CA4H for the following reasons: a) the isoelectric pH of the purified protein and that calculated of the polypeptide derived from the cDNA, are both close to pH 9.0 b) the anti-CA4H antibody recognizes in the expression library a fusion polypeptide having sequences characteristic of P450 c) the 5 'terminal part of the polypeptide deduced from the cDNA isolated by immunological logic screening with a specific antibody CA4H is completely identical to the N-terminal sequence of purified CA4H.

We therefore have the first nucleotide sequence coding for a cytochrome P450 of plant origin of known function, determined to date.

1.2) Isolation of the cDNA of cytochrome P450 catalyzing the hydroxylation of tr-cinnamate in young seedlings of Arabidopsis thaliana. The feasibility of isolating a cDNA coding for CA4H in a total cDNA library of another plant species, and more particularly in a total cDNA library made from young seedlings of Arabidopsis thaliana, in using the coding region of Jerusalem artichoke CA4H as a probe for cross-species cross-hybridization, is based on two hypotheses: a) the cDNAs coding for a protein with identical function are sufficiently conserved in the plant kingdom to allow their detection by cross- interspecies hybridization with reduced stringency; b) CA4H being a cytochrome P450 ubiquitous in the plant rule, whose final metabolic products have crucial roles for the whole plant (structural support of cells, surface protection, defense against pathogens, protection against UN radiation), ADΝc encoding this enzyme must be enriched in a cDNA bank made from young seedlings in full growth phase. As starting material for inter-species cloning by cross-hybridization, a cDNA library of Arabidopsis thaliana made from young seedlings at the two-leaf stage, well known to those skilled in the art, was chosen. scientific literature (MINET et al., 1992, Plant 2, pp 417-422). The complexity of this library has been estimated at 2.5 × 10 ⁶ of independent clones.

100,000 clones of the original glycerolated stock of this cDNA library were spread on 20 LB / ampicillin petri dishes (12 cm × 12 cm) at the rate of 500 clones / dish. After 8 hours of incubation at 37 ° C., the bacterial colonies are transferred to a Hybond N membrane (Amersham) according to the conventional methods recommended by the manufacturer. The prehybridization, as well as the hybridization, were carried out under standard conditions well known to those skilled in the art, but at a temperature of 55 ° C. to take account of the use of a heterologous probe. 200 ng of the CA4H Jerusalem artichoke cDNA described in 1.1 were labeled with 32P using the random priming kit provided by Biolabs and 108 cmp are used during hybridizations. 12 clones showing a hybridization signal during the first round of screening were then subjected to 2 amplification cycles, resulting in two independent clones, pATCA4H2 and pATCA4H9, with very strong hybridization signal. The clone pATCA4H2 has a plant insert of approximately 1.8 kb, compatible with a complete cDNA coding for a cytochrome P450. This insert was then subcloned into the sequence vector pKSII provided by Stratagene. The entire sequence of the insert was read on both strands by the creation of progressive deletions utilal to exonuclease III by following the standard conditions described in the scientific literature. For the sequencing of double-stranded DNA, the T7 polymerase Sequencing Kit supplied by Pharmacia was used according to the recommended conditions. The 1,735 bp sequence contains an open reading phase of 505 amino acids, which has 85.9% homology with the Jerusalem artichoke CA4H sequence. The cDNA sequence coding for cinnamate 4-hydroxylase with cytochrome P450 from a different plant source was thus identified by cross-species cross-hybridization. The entire sequence is shown in Figure 12. II) CLONING IN REPLICATIVE OR INTEGRATIVE EXPRESSION VECTORS OF SACCHAROMYCES CEREVISIAE OF CDNA ENCODING PLANT P450 CYTOCHROMS AND PLANT REDUCED CYTOCHROME P450

In order to be able to very effectively coexpress the plant cytochrome P450 activity in Saccharomyces cerevisiae, the following tools have been used:

- the haploid strain of Saccharomyces cerevisiae PES 1-3U. This strain is completely isogenic with the parental strain W 303.1B, with the exception of the natural promoter of the endogenous P450 reductase gene, which has been advantageously replaced by the inducible promoter GAL10 / CYC1. In this way, the PES 1-3U strain overproduces its own P450 reductase when it is grown in galactose medium. Its obtaining and its detailed description is the subject of a patent application PCT / FR 91 08884 and is also published in the scientific literature (TRUAN et al., 1993, Gene 125, pp. 49-55);

- the replicative vector in Saccharomyces cerevisiae pYEDP 60. This vector allows expression under the control of the inducible promoter GAL10-CYC1 of heterologous DNA in yeast. The construction of pYEDP 60 has been described in the scientific literature (URBAN, CULLINET, POMPOM, 1990, Biochemistry, 72, pp. 463-472) and a schematic description is shown in Figure 3;

- the integrative vector in Saccharomyces cerevisiae pYEDP 100. Its construction is done in two stages which will be repeated in more detail here:

Step 1: PCR amplification of the 5 'and 3' regions flanking the yeast NADPH cytochrome P450 reductase gene, which will serve as homologous recombination terminals during targeted integration. The template used is the plasmid pGP1 which is characterized by the insertion of a Hind III fragment of approximately 9 kbp from the yeast genomic DNA containing the yeast NADPH cytochrome P450 reductase gene into the vector pUC 19 The oligonucleotides serving as primers for PCR amplification were designed in such a way that the amplified DNA fragments are bordered by restriction sites useful during future subcloning. For greater ease, the PCR fragments with blunt ends are abod subcloned into the vector pUC19, before recovering the DNA fragments with compatible ends by digestion. enzymatic of circular DNA. More precisely, the non-coding 5 'part (638 bp upstream of the initiating ATG) is recovered in the form of a Bgl II-Hind III fragment, the non-coding 3' part (410 bp downstream of the TAA containing the terminator YRed) in the form of an EcoRI-Pvu II fragment. Step 1 is schematically illustrated in FIG. 13A.

Step 2: Subcloning of the EcoRI-PvuII fragment into the expression vector pYEDP51.

The vector pYEDP51 (FIG. 13B) is a precursor of the published vector pYEDP60, which lacks the genetic marker ADE2. Double digestion with EcoRI and PvuII results in a linear DNA fragment of XXXX bp corresponding to the vector pYEDP51 from which part of the unique cloning sites (SmaI and SacI), the PGK terminator and part of the sequence sequences have been removed. pUC between the terminator and the PvuII site. After purification on agarose gel, the vector is recircularized by ligation in the presence of the EcoRI-PvuII fragment. The vector pYEDPlOO thus constructed is characterized in that the PGK terminator has been replaced by 410 bp of the 3 ′ non-coding region of the yeast NADPH cytochrome P450 reductase gene containing its terminator.

Step 3: Subcloning of the Bgl II-HindIII fragment into the expression vector pYEDP 100.

The double digestion with Bgl II and HindIII of the vector pYEDPlOO results in three linear fragments of 4,561 respectively M bp. The fragment of 4561 bp corresponds to the vector pYEDPlOO, except for the origin of autonomous replication of the yeast, which was excised during double digestion. This linear fragment is recircularized by ligation in the presence of the Bgl II-HindIII fragment, comprising 638 bp of the non-coding region of the NADPH cytochrome P450 reductase gene upstream of the promoter. The 5,200 bp pYEDPl lO vector thus constructed (see FIG. 13C) can be propagated in E coli thanks to the origin of bacterial replication, on the other hand it can no longer replicate in yeast. In addition, during a NotI digestion (two sites created by PCR) of the vector pYEDPllO releases a DNA fragment bordered by 5 'and 3' non-coding regions of the yeast NADPH cytochrome P450 reductase gene, which will serve as recombination terminals for homologous recombination. Between these two regions of homology are the genetic marker URA 3 and the promoter GAL10 / CYC1 sivi of unique restriction sites, useful during the cloning of the heterologous sequences to be expressed. The 4 plant cDNAs taken into consideration for subcloning into the yeast expression vectors pYEDP60 and pYEDPl 10 with a view to the coexpression of a plant cytochrome P450 activity in Saccharomyces cerevisiae are: - Jerusalem artichoke

- CA4H of Arabidopsis thaliana

The entire sequence of these two cDNAs is given in Figures 1 and 12:

- ATR1

- ATR2 These are two cDNAs coding for two NADPH cytochrome P450 reductase isolated by functional complementation in a library of Arabidopsis thaliana well described in the scientific literature (MINET et al., 1992, Plant J. 2, pp. 417-422). Their obtaining and their characterization are described in the patent application in France n ° 92 04491. The subcloning of open reading phases, with the exception of non-coding sequences, in order to give the best environment for efficient transcription, being done identically for the two vectors in question, we only repeat in detail here the cloning of the Jerusalem artichoke CA411 in the vector pYEDP60, illustrated in FIG. 2. The cloning method shown diagrammatically in FIG. 2 calls for a step d amplification by PCR, which by a judicious choice of oligonucleotides serving as primers for PCR, results in the selective amplification of the coding region of CA4H, rids the amplified cDNA of the 5 'and 3' non-coding borders and also introduces suitable restriction sites for further subcloning. After passage of the amplified cDNA fragment having blunt ends in pUC19 digested with SmaI, the plasmid pCA4H thus obtained is then digested with suitable restriction enzymes flanking the cDNA fragment, which were previously introduced by PCR. The yeast vector pYEDPβ O is then digested at the multicloning site located between the promoter GAL1 0 / CYC1 and the terminator PGK so as to generate a linear vector comprising ends compatible with the fragment of cDNA containing the coding region of CA4H. Then we proceed to recircularization in a reaction medium - nel comprising the linearized vector, the cDNA fragment containing CA4H and ligase. The recirculated vector thus obtained contains the sequences necessary for propagation in E. coli (origin of replication and the bla gene conferring resistance to ampicillin in E. coli), in propagation in S. cer (origin of fold - cation in S. cer and the URA3 and ADE2 genes making it possible to complement the auxotrophies of the host strain), as well as the regulatory signals allowing the induction of the expression of the cDNA coding for CA4H in ga¬ lactos medium (figure 2). All the methods used in order to carry out this cloning are standard methods and well described in reference books (Sambrook, Fritsch and Maniatis, 1989, Molecular Cloning, 2 ed., CSH Laboratory Press). The construction of the yeast vector pYEDP 60 has been described in the literature (Urban, Cullin and Pompon, 1990, Biochemistry, 72, pp 463-472).

The vectors thus obtained are: - pYEDP60 / CA4H (CA4H Jerusalem artichoke)

- pYEDP60 / CA4HAT (CA4H from Arabidopsis)

- pYEDP60 / ATRl

- pYEDP60 / ATR2

- pYEDP110 / CA4H - pYEDP110 / CA4HAT

- pYEDPHO / ATRl

- pYEDP110 / ATR2

I I I) CONSTRUCTION OF A SET OF STRAINS EXCLUSIVELY EXPRESSING CA4H OR NADPH ACTIVITIES

CYTOCHROME P450 REDUCTASE OF PLANT ORIGIN.

The vectors pYEDP110 / CA4H, pYEDPHO / ATR1, pYEDP110 / ATR2 are subjected to exhaustive digestion with NotI. The digestion mixture is then precipitated and used without any treatment for the transformation of the strain W303. 1B. Indeed, only the fragment carrying the heterologous sequences behind the GAL10 / CYC1 promoter can give the transformed strain a Ura 3 + phenotype after the expression cassette has been inserted into the yeast NADPH cytochrome P450 reductase locus using the 5 'and 3' non-coding borders for homologous recombination. The persistence of a small undigested vector fraction undetectable in agarose gel cannot lead to a ura + phenotype, the vector pYEDPl 10 being no longer capable of propagating in yeast in the absence of the 2 μ origin (see Figure 13C). Thus, one proceeds by homologous recombination to the replacement of the endogenous gene of NADPH cytochrome P450 reductase by the expression cassette carrying the heterologous gene of interest.

The transformation mixture is spread on ura- boxes, the clones capable of growing in the absence of uracil are then analyzed by genomic southern blot to verify whether there has been integration of the heterologous sequences in the right place. . A second control of the integration event consists in measuring the resistance of the recombinant strains to ketoconazole, an antifungal inhibitor of the yeast cytochrome P450 C14-demethylase. Indeed, it has been shown that the loss of the NADPH cytochrome P450 reductase gene induces the hypersensitivity phenotype to ketoconazole (Sutter and Loper, 1989, Biochem. Biophys. Res. Comm., 160, pp. 1257-1266) . Table 1 clearly illustrates that, when the Jerusalem artichoke CA4H gene has been integrated in place of the yeast NADPH cytochrome P450 reductase, the strain WCA41 becomes hypersensitive to ketoconazole. On the other hand, the strains WAT11 and WAT21, having acquired the cDNAs of ATR1 (WAT11) or ATR2 (WAT21) by homologous recombination are resistant to ketoconazole, but to a lesser degree than the parental strain W303. 1B, which reflects the less efficient coupling of plant cytochrome P450 reductases NADPH with yeast cytochrome P450 compared to endogenous cytochrome p450 reductase NADPH. This phenotype of resistance to ketoconazole is induced in galactose medium, which proves that the resistance is due to the activity of the heterologous NADPH cytochrome P450 reductase under the control of the promoter GAL10 / CYC1.

In summary, using the integrative vectors pYEDP110 / CA4H, pYEDPHO / ATR1, pYEDP / ATR2 to transform the strain W303.1B, the following strains were obtained: WAT11: This strain is characterized in that the yeast NADPH cytochrome P450 reductase gene is replaced by that of Arabidopsis. which is under the control of the promoter GAL10 / CYC1. Thus, this strain overexpresses the NADPH cytochrome P450 reductase ATI activity when it is cultured in galactose medium.

WAT21: This strain is characterized in that the yeast NADPH cytochrome P450 reductase gene is replaced by that of Arabidopsis. which is under the control of the promoter GAL10 / CYC1. Thus this strain overexpresses the NADPH cytochrome P450 reductase ATR2 activity when it is cultivated in galactose medium. WCA41: This strain is characterized in that the yeast NADPH cytochrome P450 reductase gene is replaced by that of the Jerusalem artichoke CA4H, which is under the control of the GAL10 / CYC1 promoter. Thus, this strain overexpresses CA4H when it is cultivated in galactose medium, but no activity is measurable in the absence of NADPH cytochrome P450 reductase capable of transferring the reducing equivalents to cytochrome P450, necessary for its activity.

A schematic illustration of the NADPH cytochrome P450 reductase locus of the parental strain, as well as of the recombinant strains constructed, is given in FIG. 14.

3.1) Transformation of WAT21 strains. WCA41. PES 1-3U: a) The strain WAT1 1 is transformed by the vector pYEDP110 / CA4H so as to simultaneously overexpress in galactose medium the CA4H of Jerusalem artichoke on plasmid and the NADPH cytochrome P450 reductase of Arabidopsis ATR1 to constitute an active hydroxylating complex d entirely vegetable origin. The replicative plasmid is maintained by selection of the Ura + or Ade + phenotype, provided by the replicative plasmid. This strain is subsequently named WAT11 / CA4H. The transformation is carried out according to a standard lithium chloride method (POMPON, 1988, Eur. J. Biochem., 177, pp. 285-293). b) The WAT21 strain is transformed by the vector pYEDP1 10 / CA4H so as to simultaneously overexpress in galactose medium the Jerusalem artichoke CA4H on plasmid and the NADPH cytochrome P450 reductase from Arabidopsis ATR2 to constitute an active hydroxylating complex of entirely vegetable origin. The replicative plasmid is maintained by selection of the Ura + or Ade + phenotype, provided by the replicative plasmid. This strain is subsequently named WAT21 / CA4H. The transformation is carried out according to a standard lithium chloride method (POMPON, 1988, Eur. J. Biochem., 177, pp. 285-293). c) The PES 1-3U strain is transformed by the vector pYEDP1 10 / CA4H so as to simultaneously overexpress in galactose medium the Jerusalem artichoke CA4H on plasmid and the endogenous NADPH cytochrome P450 reductase to constitute an active hydroxylating complex of mixed origin. This strain is subsequently named "WRCA". d) The strain WCA41 is transformed by the vector pYEDPHO / ATR1 so as to simultaneously overexpress in galactose medium the integrated Jerusalem artichoke CA4H and the NADPH cytochrome P450 reductase from Arabidopsis ATR1 on plasmid to constitute an active hydroxylating complex of entirely vegetable origin. The replicative plasmid is maintained by selection of the Ura + or Ade ⁺ phenotype, provided by the replicative plasmid. This strain is subsequently named "WCA41 / ATR1". The transformation is carried out according to a standard lithium chloride method (POMPON, 1988, Eur. J. Biochem., 177, pp. 285-293). e) The WCA41 strain is transformed by the vector pYEDPHO / ATR1 so as to simultaneously overexpress in galactose medium the integrated Jerusalem artichoke CA4H and the NADPH cytochrome P450 reductase from Arabidopsis ATR2 on plasmid to constitute an active hydroxylating complex of entirely vegetable origin. The replicative plasmid is maintained by selection of the Ura + or Ade + phenotype, provided by the replicative plasmid. This strain is subsequently named "WCA41 / ATR2". The transformation is carried out according to a standard method with lithium chloride (POMPON, 1988, Eur. J. Biochem., 177, pp. 285-293). IV) CONSTRUCTION OF STRAINS DP DIPLOID YEASTS STABLE COEXPRESSING THE ACTIVITIES CYTOCHROME P450 HETEROLOGUES AND NADPH CYTOCHROME P450 HOMOLOGATED OR HETEROLOGOUS REDUCTASES.

The transformed yeast strains described above must be maintained in a selective medium, in order to ensure the persistence of the plasmid which provides one of the compounds of the active hydroxylating complex. Advantageously, this selection pressure is overcome by the construction of strains which have stably integrated a copy of each of the cDNAs coding for CA4H, in particular Jerusalem artichoke and a NADPH cytochrome P450 reductase in particular from Arabidopsis. Such strains are easily obtained by crossing two haploid strains of opposite sexual sign. Like their parental strain, strains WAT11, WAT21 and WCA41 are of sexual sign (mating type) a. Thanks to the Ho plasmid technique applied according to the published method (Methods in Enzymology, vol. 194, pp. 132-146), the sexual sign is exchanged into α. The WATl l α, WAT21 α, WCA41α strains are therefore completely isogenic (with the exception of the sexual locus) to the WATl 1, WAT21 and WCA41 strains.

The diploid test of the WAT1 x WCA41α and WAT21 x WCA41α crosses is carried out according to the conventional methods of yeast genetic engineering well known to those skilled in the art. As internal controls during bioconversion measurements implementing the CA4H activity are also included the crossings WAT11 x WAT11 (11/11), WAT21 x WAT21 (21/21) and WCA x W100 (integration of the empty expression cassette , see figure 15). No CA4H activity should be detectable in these strains. The diploid strains thus obtained, named C11 (WAT1 1 x CWA41) and C21 (WAT21 x WCA41) completely isogenic on the alleles except for the yeast NADPH cytochrome P450 reductase loci which respectively carry a copy of the cytochrome NADPH gene P450 Arabidopsis reductase and a copy of the Jerusalem artichoke CA4H gene. V) CHARACTERIZATION OF PLANT CA4H ACTIVITY IN DIFFERENT YEAST STRAINS IN THE PRESENCE OF ENDOGENOUS VEGETABLE REDUCASES NADPH CYTOCHROME P450.

CA4H, cytochrome P450 ubiquitous in the plant kingdom, catalyzes the hydroxylation of cinnamate in position 4 to give coumarate. With a view to applying recombinant yeast strains as biocatalysts in order to carry out precise chemical reactions there, by means of biotechnology, it is essential to prove, firstly, that the substrate as well as the product formed are stable in the reaction medium. Advantageously, the substrate enters without any apparent restriction in the biocatalyst and the product of the reaction to Cytochrome P450 does not accumulate in the cell but is exported to the medium where it can be easily recovered. Advantageously again, it is ensured that neither substrate nor product is toxic to the cell at concentrations compatible with a bioconversion process. To this end, cultures of C11 and C21 are carried out in YPGAL, WRCA, WAT11 / CA4H, WAT21 / CA4H, WCA41 / ATR1 and WCA41 / ATR2 medium in minimum galactose medium up to OD = 3 (end of exponential growth phase ); 100 μM tr-cinnamate are added, then incubated for 30 minutes at 30 ° C. with moderate agitation.

The extraction process and the separation by HPLC are described in detail in paragraph 6 (4) below. Table 1 shows the bioconversion measures.

The +++ assessment indicates that the bioconversion has been complete, no more traces of cinnamate can be detected. All the spectrograms shown in FIG. 7 clearly show that the cinnamate -> coumarate conversion is due to the presence of CA4H, since the control strains 11/11, 21/21 and Cl 00 show no appearance of peak corresponding to the coumarate . Also, it is clear that in the absence of CA4H activity, cinnamate is stable in the reaction medium.

A more detailed characterization of the plant CA4H expressed in yeast is given with the example of the WRCA strain. VI) Characterization of the enzymatic activity of CA4H in S. cer.

6.1) Description of the genetic environment of the WRCA strain In Figure 3 is schematic the yeast strain

WRCA which has been constructed so as to overexpress simultaneously in a gala ctosed medium the exogenous CA4H and the endogenous P450 Reductase. The WRCA strain is obtained by transformation of the PES 1-3U strain with the plasmid pCA4H / YeDPβO described above. The PES 1-3U strain, which carries as known mutations Ura3, Ade2, His1, and Leu2, was transformed according to a standard method with lithium chloride (Pompon, 1988, Eur. J. Biochem., 177, pp 285 -293), followed by a selection for the restoration of the Ade + phenotype which is brought in the presence of the plasmid. The PES 1-3U strain, the detailed description of which is the subject of a patent application in France No. 91 08884, was deposited with the National Collection of Cultures and Microorganisms on March 17, 1992, under deposit number 1 -1187. This haploid strain (mating type alpha) is completely isogenic with the laboratory strain W303.1B, except the promoter region preceding the coding phase of the endogenous P450 Reductase, which is modified by homologous recombination so as to replace the natural promoter of P450 Reductase by the inducible promoter GAL10-CYC1. 6.2 Determination of the Cytochrome concentration

P450 in the microsomal fraction of the WRCA strain A preculture (150 ml) of the WRCA strain is carried out in SW5 medium at 28 ° C. with moderate stirring (100 rpm) up to a cell density of 3 OD, then 1 vol is added. / theft of YPGAL medium (induction of CA4H and P450 Reductase) and the culture is continued up to a cell density of 10 OD. The culture is stopped by centrifuging the cells and then the microsomes are prepared according to a standard process involving mechanical breakage of cells (Cullin and Pompon, 1988, Gene, 65, pp 203-217). A homogeneous microsome solution of 2.8 ml is thus obtained, stored at -80 ° C. in the form of aliquots of 100 microliters, and which serves as a stock solution for all the experiments described below. To measure the total protein concentration in this stock solution of microsomes we use the Pierce method with serum albumin as standard. The overall concentration of Cytochrome P450 contained in these microsomes is measured by differential spectrophotometry according to the method described by Omura and Sato (1964, J. Biol. Chem. 239, pp 2379-2387) which highlights the formation of the characteristic absorption peak around 450 nm of reduced Cytochrome P450 after fixing a carbon monoxide molecule on the iron atom of the chromophore, compared to the reduced form of cytochrome P450 in the absence of carbon monoxide. More precisely, two spectrophotometric cuvettes are distributed in equal amounts of the stock solution of microsomes (approximately 2 mg of protein proteins) diluted 10 times in a buffer composed of 50 mM Tris-Hcl pH 7.4 and 1 mM EDTA and to which a few seeds of sodium dithionite were added beforehand in order to completely reduce the P450 cyto¬ chromes present in the microsomes and to consume any free oxygen dissolved in the solution. The two cuvettes containing equal amounts of reduced microsomes are used to make the baseline between 400 and 500 nm. A dozen bubbles of carbon monoxide are then bubbled into the test cuvette, which leads to the formation of the absorption peak around 450 nm due to the fixation of carbon monoxide to the iron ion of the heme group present. in cytochrome P450, compared to reduced microsomes in the control tank. By using the absorption coefficient which is 91 mM -1.cm-1 for the cytochromes P450 one can directly deduce from the amplitude of the peak the total concentration in cytochromes P450 contained in microsomes. In Figure 4 is shown the difference spectrum in the presence of carbon monoxide obtained with a solution of microsomes 10 times diluted from the stock solution. From the spectrum which shows the characteristic peak at 453.5 nm, we have determined the overall concentration in the microsomes after induction is 1.2 micromolar. Since the control culture (PES 1-3U) does not show, under the same experimental conditions and the same sensitivity conditions, no detectable trace of cytochromes P450, we can conclude that the peak observed with the microsomes of the culture induced from the WRCA strain must come from the expression of the CA4H cDNA on the plasmid. 6.3 Obtaining a type I perturbation spectrum following the addition of tr-cinnamic acid to the micro¬ somes obtained from the induced culture of the WRCA strain

One of the most interesting properties of P450 cyto-chromes is the possibility of being able to monitor the interaction of different molecules with the active site. Indeed, the strength of the ligand field determines typical and well-coded spectra (type I, II or I in¬ versed). The type I spectra, generally characterizing the interaction of a P450 with its specific substrate, are defined by the formation of a negative peak around 420 nm and a positive peak around 390 nm following the fixation of the substrate cytochrome P450. In order to provide additional proof that the peak at 453.5 nm observed in FIG. 4 is indeed due to the presence of CA4H from topinam¬ bour in the yeast microsomes and that the protein is there in an active form capable of fixing its clean substrate, it has been attempted to obtain such a spectrum by the addition of tr-cinnamate to the microsomes. More precisely, equal amounts of the stock solution of microsomes (induced culture of the WRCA strain) are distributed 10 times diluted in a buffer containing 50 mM Tris-Hcl pH 7.4 and 1 mM EDTA in two spectrophotometric cuvettes. In the presence of oxygen dissolved in the medium can be rai _¬ ably assume that cytochromes P450 in microsomes are in their oxidized form, having attached oxygen on the heme group. A baseline is made between 370 and 490 nm with the two identical cuvettes. Then tr-cinnamate is added to the test tank at a concentration of 100 micromolar, and a differential absorption spectrum is produced (oxidized cytochrome P450 versus oxidized cytochrome P450 plus specific substrate) between 370 and 490 nm. In FIG. 5 is shown the obtaining of a type I disturbance spectrum characteristic of a P450 after fixing of its substrate, using the same stock solution of microsomes as in 6.2 which clearly shows the formation of an important negative peak at 418 nm and a positive peak at 390 nm after the addition of tr-cinnamate. This same type of differential spectrum was observed after the addition of tr-cinnamate to micro¬ somes of Jerusalem artichoke (Benveniste and Durst, 1974, CR Acad. Se. Paris 278D, pp 1487-1490). Thus, the polypeptide derived from the Jerusalem artichoke CA4H cDNA all has the spectral characteristics of a cytochrome P450 in yeast microsomes and is capable of specifically fixing tr-cinnamate as a substrate. 6.4 Demonstration of the conversion of tr-cinnamate to tr-coumarate by a culture of WRCA in a lactose medium

It is essential to prove that the spectrum of disturbance observed following the addition of tr-cinnamate to microsomes containing Jerusalem artichoke CA4H is correlated with a catalytic activity of CA4H which results in the formation of tr-coumarate. It is also very important to prove that tr-cinnamate does not meet a diffusion barrier during a culture of whole cells and that the coumarate formed will be accumulated in the culture medium, for application of bioconver- whole cells on an industrial scale. To this end, the following experiment was carried out: 0.5 ml of a WRCA preculture in minimum medium was added to 2 ml of fresh YPGAL medium containing 10 microM trinnamate, then incubated at 27 ° C. moderate agitation. After 30 minutes, the cells are centrifuged and 1 ml of the supernatant is removed, to which 1 ml of a solution saturated with NaCl and 1 ml of ethyl acetate are added. After having rigorously mixed the two phases on a vortex, the organic and aqueous phases are separated by centrifugation, and an aliquot of the organic phase (ethyl acetate) is removed. Evaporated in air and taken up in a 10% methanol solution. 20 microliters of the sample are separated by HPLC on column C18 using a linear gradient.

COMPOSITION OF THE HPLC GRADIENT

TABLE 1

COMPOSITION FLOW TIME (min) (ml / min) A (%) B (%)

0.00 1.00 100

1.00 1.00 90 10

15.00 1.00 70 30

15.10 1.00 0 60

17.00 1.00 100

A = 0.01% TFA B = Acetonitrile A preliminary calibration with tr-cinnamate and pure commercial tr-coumarate had demonstrated that under the chromatography conditions described the two products are well separated. In fact, the coumarate comes out after about 8 minutes, while the tr-cinnamate comes out after 11.5 min. In Figure 6 is shown the chromatogram of the sample from the experiment described above. The chosen detection wavelength of 292 nm is a compromise where the two substances absorb well. It is clearly visible that there are only negligible and barely detectable quantities left, whereas a single major peak appears at the position of tr-coumarate. Thus it has been demonstrated that the polypeptide derived from the cloned plant cDNA is indeed a cytochrome P450 enzyme on the basis of its spectral properties and that its catalytic activity is the hydroxylation of tr-cinnamate in position four to form the tr -coumarate, without appearance of secondary products. Tr-cinnamate is apparently accessible to intracellular cytochrome P450, while the transformation product is found in the supernatant. Neither the substrate nor the product of the conversion are apparently toxic to yeast. 6.5 Determination of the CA4H affinity constant for tr-cinnamate in yeast microsomes The total amplitude observed between the positive maximum at 370 nm and the negative peak at 418 nm in a type I disturbance spectrum , varies as a function of the specific substrate added, when the substrate is in limiting concentration with respect to the cytochromes P450 capable of fixing this substrate present in the microsomes. If the inverse values of the absorption amplitudes measured in a type I perturbation spectrum are plotted as a function of the inverse value of the increasing concentrations of added substrate (representation of Line eaver-Burk), one obtains a right and the affinity constant of the cytochrome P450 considered for its specific substrate can be calculated directly from the point of intersection of the straight line with the X axis. In FIG. 7 is shown a representation of Lineweaver-Burk by plotting the variation of the amplitude of absorption in a type I spectrum as a function of the tr-cinnamate added in increasing amounts to oxidized microsomes originating from an induced culture of the WRCA strain. The experimental conditions being identical to those described in 3.3 with the exception of the tr-cinnamate concentrations used ranging from 0.2, 1.0, 3.0, 10.0 to 30.0 icroM, they will not be repeated here . The affinity constant of topiambour CA4H in yeast microsomes in the presence of endogenous yeast P450 Reductase thus measured is 2.2 microM, which is in excellent agreement with the values obtained with Jerusalem artichoke microsomes which vary between 1 and 10 microM depending on the preparations (F. Durst, personal communication). It is therefore evident that the yeast S. cer. provides the plant-like environment for efficient expression of CA4H. 6.6 Determination of the coupling efficiency between the yeast P450 Reductase and the Jerusalem artichoke CA4H in the microsomes of the WRCA strain

Cytochromes P450 are not self-sufficient to perform the hydroxylation of their substrates. The active enzyme complex consists of two components, cytochrome P450 and P450 Reductase, which provides the electron transport chain necessary for the hydroxylation of NADPH to Cytochrome P450. In the event of perfect coupling (illustrated in FIG. 8), the consumption of one molecule of NADPH per molecule of product formed will therefore be observed. Since no plant cytochrome P450 has ever been expressed in yeast, it is essential to ensure that the yeast provides the reducing environment suitable for the enzymatic activity of Jerusalem artichoke CA4H, For this purpose the following experiment was carried out: a reaction mixture consisting of 6 nM CA4H (dilution 1 to 200 of the stock solution of microsomes) in a 50 mM phosphate buffer pH 7.4 and reduced NADPH 0.1 mg / ml is distributed in equal parts in two spectrophotometric cuvettes. The quantity of reduced NADPH in the two tanks being identical, the initial differential absorption measured at 350 nm is zero. Then tr-cinnamate is added to the test dish at the final concentration of 10 microM. In the event of perfect coupling, a reduction in absorption corresponding to the consumption of an equivalent quantity of NADPH must be recorded at 350 nm. In Figure 8 is shown the recording of this experience. The addition of 10 nmoles of tr-cinnamate results in a reduction of the initial absorption in the es¬ sai tank of 0.06 OD.

Using the reduced NADPH molar coefficient (6200 M -1 cm-1), it is easy to convert the absorption variation into moles of oxidized NADPH. Indeed, after the addition of 10 nmoles of tr-cinnamate 9.7 nmoles of NADPH were oxidized, which results in a coupling factor of 1.03. It is therefore obvious that the yeast P450 Reductase is capable of perfectly ensuring the electron transport chain towards the Jerusalem artichoke CA4H in order to make it work. 6.7 Determination of the maximum rate of conversion of CA4H in yeast microsomes as well as the Km

The maximum speed of conversion of plant cytochrome P450 measured in yeast microsomes is a very important value to assess whether S. Cer is an interesting industrial tool for carrying out bio¬ conversions using cytochromes P450, the speed of enzymatic reaction being one of the main criteria. This is all the more so since the mammalian cytochrome P450 are known to be so-called "slow" enzymes. In the same experiment, the Michaelis constant will also be determined, which gives the substrate concentration necessary for the CA4H to operate at 50% of its maximum speed. In 3.6 it was shown that the coupling efficiency between the yeast P450 Reductase and the CA4H expressed by the relation of NADPH consumed for converted substrate is very close to 1. Therefore it is possible to follow the activity of the CA4H as a function of the concentration of tr-cinnamate added by the rate of consumption of NADPH. Indeed, the reduced NADPH absorbs strongly at a wavelength of 340 nm (molar absorption coefficient = 6200) and the slope of the variation in absorption over time is directly proportional to the speed of the transformation. tr-cinnamate to coumarate by CA4H. However, in order to avoid interference with the coumarate formed which absorbs weakly at 340 nm, the measurements will be made at 350 nm, a wavelength where only the reduced NADPH shows strong absorption. More specifically, a reaction mixture, consisting of a 200-fold dilution (final concentration of CA4H is 6 nM), is carried out of the stock solution of microsomes in a 50 mM phosphate buffer at pH 7.4 and NADPH reduced to excess to 0.1 mg / ml. This mixture is distributed in equal parts in two spectrophoto etric cuvettes, increasing concentrations of tr-cinnamate are added at time zero, then the decrease in initial absorption at 350 nm over time is followed by differential etrop spectrophoto in the test tank compared to the control tank. The recordings of this experiment were made for tr-cinnamate concentrations of 0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.6 and 25 microM. From the slopes obtained, it is therefore possible to convert them directly into CA4H activity and to perform a representation according to Linevreaver-Burk by reporting the inverse of CA4H activities measured as a function of the inverse of the concentration of tr-cinnamate added to the reaction medium. The point of intersection of the line thus obtained (Figure 9 with the Y axis gives the value of the maximum conversion speed of the CA4H, while the point of intersection with the X axis gives the value of Km The maximum in vitro speed of 400 +/- 50 min is the highest obtained to date for a eukaryotic P450, which clearly shows that in a genetic context where it is possible to simultaneously overexpress the yeast P450 Reductase and the CA4H, S. cer provides the best environment currently known in order to operate a plant cytochrome P450 very efficiently. On the basis of activities measured in vitro, the WRCA strain is an excellent candidate for carrying out the bioconversion of tr-cinnamate in couma¬ rate by CA4H on an industrial scale. In this sense the very low value of Km of 1.2 microM acquires its importance, since low intra¬ cellular concentrations of tr-cinnamate of the order micromolar is enough to run the CA4H at full speed.

5.8 Direct measurement of the maximum CA4H conversion speed on WRCA microsomes

Measuring the consumption of NADPH is certainly very appropriate at the experimental level, but is only indirect proof of the enzymatic activity of CA4H. It therefore remains very important to validate the results thus obtained under 3.7 by real activity measurements, that is to say the comparable values for the maximum CA4H conversion speed must be found when monitoring the activity. enzymatic by measuring the amount of tr-coumarate formed. To this end, the following experiment was carried out: a reaction mixture of 1 ml containing 50 mM phosphate buffer pH 7.4, 50 microM tr-cinnamate, 0.1 mg / ml NADPH and 12 poles of CA4H (dilution 1 for 1000 of the solution stock of microsomes) is prepared on ice. The micro¬ somes are added last, then the reaction is started by placing the complete reaction medium at 37 ° C. The reaction is stopped at the times indicated in Table 2, by taking a sample of 50 microliters to which 2.5 microliters of trifluoroacetic acid is added, then separation by HPLC under the conditions described above. Figure 10 shows the kinetics of coumarate formation using the values from the table. In fact, in an initial 6 minutes when the substrate is in excess, the formation of tr-coumarate is linear over time and rapidly declines to reach the plateau after only 15 minutes. This results in a rapid decline in maximum speed when the substrate begins to become limiting. It is very important to note that the value of the maximum conversion speed of the

-1 CA4H is 350 min when the substrate is in excess, a value which is in perfect agreement with the values found by indirect test, which reinforces the comments made under 3.7.

V _U ) Bioconversion process using a yeast strain overexpressing endogenous P450 Reductase and a plant cytochrome P450. Exemplification by the production of tr-coumarate from tr-cinnamate on whole cells containing the Jerusalem artichoke CA4H.

A 200 ml culture in a 1 L Erlenmeyer flask of the WRCA strain is incubated at 27 ° C. with vigorous stirring (200 rpm) in YPGAL medium up to 5 DO (stationary saturation phase). At this time, cinnamate is added to a final concentration of 100 microM, then the incubation is continued. Samples of 100 microliters are taken at the time of the addition of the tr-cinnamate and then 1, 10, 30, and 100 minutes after the addition. Then the extraction is carried out according to the methods described in 3.3, and the samples are analyzed by HPLC chromatography (see 3.3). The analysis wavelength is voluntarily chosen at 314 nm, a wavelength where the absorption of tr-coumarate is almost maximum while tr-cinnamate absorbs very little. The amplitude of the absorption peaks is therefore not identical for the same concentration of tr-cinnamate and tr-cou¬ marate. The aim of this experiment being to quantify the evolution of the amount of tr-coumarate found in the culture medium as a function of time, a scale of known concentrations of tr-coumarate was used for the calibration, making it possible to translate the amplitude of the coumarate peak in concentration or in number of molecules of tr-coumarate formed. The superimposition of 4 chromatograms was carried out on the 4 samples taken at the times indicated above. It is obvious that very quickly after the addition of tr-cinnamate, large quantities can be detected in the culture medium without the appearance of secondary products. Given the value of the affinity constant of CA4H for tr-cinnamate which is micromolar, it is clear that at least 95% of the substrate initially present in the culture will be transformed into tr-coumarate before the concentration becomes limiting. The fact of finding large quantities of tr-coumarate quickly in the culture medium suggests that the tr-cinnamate diffuses sufficiently quickly in the cell so that the CA4H can operate at full speed, and also that the product formed n is not retained or metabolized in yeast but excreted in the medium. This makes it possible to recover and then purify the product during bioconversion on an industrial scale, but also explains the apparent inocuity of coumarate for the cell. It is indeed unlikely to obtain a large quantity of a product by bio-conversion if it is accumulated inside the cell. On the basis of the peaks observed in FIG. 11, the calculated productivity is 10 micromoles of coumarate formed / minute. liter of culture. In FIG. 11 is shown a kinetics which reports the number of tr-coumarate formed per yeast cell (calculated from the chromatograms) as a function of time. It is clear that this bioconversion process is completely stable over time. This is an essential criterion for the application of the WRCA strain for an industrial process for obtaining bioconversion tr-coumarate. By extrapolating this striking stability of the Jerusalem artichoke CA4H in yeast compared to other mammalian cytochrome P450, the productivity cited above will be approximately 10 grams of coumarate formed per day per liter of culture under conditions laboratory. These values can reasonably be improved by a factor of 5 during an industrial fermentation process.

Claims

1) Yeast strain allowing the co-expression of a plant cytochrome P450 mono-oxygenase activity and an endogenous or heterologous NADPH- cytochrome P450-reductase, characterized in that one of the genes of NADPH-cytochrome P450-reductase or cytochrome P450 is integrated into the chromosome of said strain, and, the strain is transformed by a vector carrying an expression cassette for the other gene.

2) A strain according to claim 1, characterized in that the microsomal cytochrome P450 gene of plant origin is integrated into the chromosome of said yeast strain.

3) Yeast strain according to claim 1 or 2 allowing the co-expression of a heterologous cytochrome P450 monooxygenase activity and an endogenous or heterologous NADPH-cytochrome P450-reductase, characterized in that the NADPH gene -cytochrome P450- reductase is integrated into the chromosomal genome of said strain and the strain is transformed by a vector carrying an expression cassette for the plant cytochrome P450 gene.

4) Strain according to one of claims 1 or 2, characterized in that said vector is an integration vector or a replicative vector.

5) A strain according to claim 3, characterized in that the cytochrome P450 and NADPH-cytochrome -P450-reductase genes are integrated into the chromosome of said strain.

6) Strain according to one of claims 1 to 5, characterized in that the cytochrome P450 is the CA4H protein of Helianthus tuberosus.

7) Strain according to one of claims 1 to 6, characterized in that said cytochrome P450 is the CA4H protein of Arabidopsis thaliana.

8) Strain according to one of claims 1 to 7, characterized in that said gene of said cytochrome P450 is a hybrid gene. 9) Strain according to one of claims 1 to 8, characterized in that the NADPH-cytochrome P450-reductase is the endogenous yeast NADPH-cytochrome P450-reductase.

10) Strain according to one of claims 1 to 9, characterized in that said gene of NADPH-cytochrome P450-reductase consists in the cDNA sequence coding for heterologous NADPH-cytochrome P450-reductase. 11) Strain according to one of claims 1 to 10, characterized in that said gene of NADPH-cytochrome P450-reductase is that of a NADPH-cytochrome P450-reductase of plant origin.

12) Strain according to claim 11, characterized in that the NADPH-cytochrome P450-reductase is a NADPH-cytochrome P450-reductase from Arabidopsis thaliana ..

13) A strain according to claim 11, characterized in that the NADPH-cytochrome P450-reductase is a NADPH-cytochrome P450-reductase from Helianthus tuberosus. 14) Strain according to one of claims 1 to 13, characterized in that the NADPH-cytochrome P450-reductase and cytochrome P450 genes are integrated into the locus of the endogenous NADPH-cytochrome P450-reductase on different alleles.

15) Yeast strain according to one of claims 1 to 14, characterized in that the yeast is chosen from the genera

Saccharomvces. Hansenula. Kluvveromvces. Pichia. and Yarrowia.

16) Strain according to claim 15, characterized in that the yeast is of the genus Saccharomvces cerevisiae.

17) Strain one of claims 1 to 16, characterized in that in said expression vector the cytochrome P450 gene is placed under the control of an inducible promoter.

18) Strain according to claim 17, characterized in that said vector is the vector pYEDP60.

19) Strain according to one of claims 1 to 18, characterized in that the NADPH-cytochrome P450-reductase gene is placed under the control of an inducible heterologous promoter.

20) Strain according to one of claims 1 to 19, characterized in that the NADPH-cytochrome P450-reductase gene and the cytochrome P450 gene are placed under the control of the same inducible promoter.

21) Strain according to one of claims 17 to 20, characterized in that said inducible promoter is the GAL10 / CYC1 promoter.

22) Strain according to one of claims 1 to 21, characterized in that said strain is obtained from the strain PES 1-3-U deposited in the National Collection of Cultures of Microorganisms (CNCM) under the n 'I- 1187. 23) Strain according to one of claims 1 to 22, characterized in that said strain is the WRCA strain obtained by transformation of the PES 1-3-U strain with the vector pCA4H / YeDP60.

24) Yeast strain according to one of claims 1 to 23, characterized in that it allows the co-expression of NADPH-cytochrome P450-reductase and cytochrome P450 in a NADPH-cytochrome P450-reductase / cytochrome molar ratio P450 optimum which is between 1/3 and 1/10.

25) A method of co-expression in a yeast of the mono-oxygenase activity of microsomal cytochrome P450 of plant and of endogenous or heterologous NADPH-cytochrome P450, characterized in that a strain according to l is cultivated in an appropriate culture medium 'One of claims 1 to 24.

26) Use for bioconversion purposes of a yeast strain according to one of claims 1 to 24.

27) Bioconversion process intended for the specific monoxidation of a substrate compound of a cytochrome P450, characterized in that a medium containing said compound or a precursor is converted with one of the strains according to one of claims 1 to 24, expressing the monoxygenase activity of said cytochrome P450 and in that said monoxygenated substrate compound is recovered.

28) Method according to claim 27, characterized in that said medium is a culture medium.

29) Bioconversion process according to one of claims 27 or 28, characterized in that the monoxygenation is a stereospecific hydroxylation.

30) Method according to claim 29, characterized in that the cytochrome P450 is CA4H, the substrate compound is trans-cinnamate, and the trans-coumarate is recovered. 31) cDNA sequence coding for Helianthus tuberosus cytochrome P450 CA4H.

32) cDNA sequence coding for the cytochrome P450 CA4H of Arabidopsis thaliana.

33) cDNA sequence according to claim 31 as shown in Figure 1.

34) cDNA sequence according to claim 32 as shown in Figure 12. 35) Sequence of cDNA hybridizing under weakly stringent conditions at 50 ° C and / or having at least 60% homology with one of the sequences according to one of claims 31 to 34.