CA2413526A1 - Micro-organisms active in the digestive environment - Google Patents

Abstract

The invention concerns the development of genetically modified micro-organisms, in particular genetically modified yeast, capable of surviving in a digestive environment and efficiently express therein one or several genes of interest. The invention also concerns the galenic form and the delivery mode of said micro-organisms. Moreover, the invention concerns the use of artificial digestive systems simulating the human gastrointestinal functions for studying, controlling and selecting said micro-organisms. Furthermore, the invention concerns direct uses of secretion and/or bioconversion in situ, in particular for developing preventive or curative treatments for pathologies, in particular diseases related to the metabolism of xenobiotics.

Description

MICROORGANISMS ACTIVE IN THE DIGESTIVE ENVIRONMENT
The present invention relates to the development of microorganisms genetically modified, including genetically modified yeast, which can survive in a digestive environment and effectively express one or more genes there of interest. The dosage form and mode of administration of these microorganisms are also proposed. In addition, the invention relates to the use of systems artificial digestives simulating the gastrointestinal functions of humans for studying, control and the selection of the aforementioned microorganisms. The invention also relates to direct in situ bioconversion applications, in particular in the development point of preventive or curative treatments for pathologies, especially related diseases at metabolism of xenobiotics.
Living things are continuously in contact with xenobiotics, substances said to be foreign to the “normal” metabolic pathways of the cell (drugs, pesticides, pollutants, food additives, "natural" molecules of plants ...). These compounds, often too hydrophobic to be eliminated directly by kidneys or degraded by bile, accumulate in lipids of (organism. Enzymes of xenobiotics metabolism (or EM ~ are responsible for facilitating their elimination in making them more hydrophilic.
These enzymes are classified into three categories - phase I enzymes (functionalization phase): making the xenobiotic plus hydrophilic by the introduction of polar groups (most often by reactions oxidation). These are mainly cytochrome P450 monooxygenases, hemoproteins with a maximum characteristic absorption around of 450 nm, when reduced and linked to carbon monoxide (Omura, 1964, 1993). In mammals, the P450s are mainly at the level of the smooth endoplasmic reticulum membrane, mainly in the liver, but also in the lungs and (intestine, organs playing a role in the control of the entry of many xenobiotics into the body.

2 Each P450 is part of a multienzymatic complex where it is associated with of them flavoprotein electron transport systems, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase.
The P450s operate on a catalytic cycle during which the iron passes through different degrees of oxidation, which leads to the incorporation of an atom oxygen in the substrate R according to the following reaction (Guengerich, 1993) RH + 02 + 2e- + 2H + ~ ROH + H20 The P450s have original catalytic properties since, unlike the majority of enzymes, a given P450 can act on many substrates presenting very different structures and, conversely, the same substrate can be recognized by different P450s (Coon and aL, 1992). Endogenous factors (species, strain...), physiological (age, sex ...), but also exogenous factors can modulate P450 activity (Guengerich, 1993), by acting on different levels:
expression of gene, protein stabilization, activity modulation.
- phase II enzymes (conjugation phase): making metabolites from the even more hydrophilic phase I (if they are not directly excreted), by conjugation with various endogenous molecules (glutathione, glucuronide ...).
These enzymes are found in almost every cell in the body, main being glutathione S-transferase (GST), Ia N-acetyl transferase (NAT), Ia quinone reductase (QR), aldehyde dehydrogenase (ALDH), uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase, epoxide hydrolase and O-acetylase.
- phase III enzymes (evacuation phase): responsible for eliminating Ies cells metabolites resulting from detoxification reactions (ABC transport pumps, in especially MDR multidrug resistant pumps).
Overall, the metabolism of xenobiotics is considered to be a pathway detoxification. However, phase I enzymes can give rise to more reactive metabolites than the original molecule, capable of binding with proteins (causing necrosis or autoimmune diseases), nucleic acids (responsible for mutagenesis or carcinogenesis) or lipids (peroxidation

3 lipid). This is particularly the case when phase II enzymes are absent or too low activity.
EMX activity is genetically determined and highly variable depending on the individuals (genetic polymorphism). Molecular analysis of correlations existing between major genes involved in the detoxification and biotransformation, and pathologies is currently the subject of numerous works of research. Indeed, anomalies in the detoxification and biotransformation of exogenous compounds have been clearly identified in of patients with so-called "multifactorial" diseases (diseases combining genetic predisposition and role of environmental factors), very widespread at this day in the world, such as different cancers, various pathologies digestive, endometriosis, chronic bronchitis ...
For example, the risk of oil-induced lung cancer polycyclic aromatics is increased about 40 times when the individual East GSTM1 deficient (GSTMl 0/0 phenotype) and has a strong P450 lAl activity enzymatic (Raunio et al., 1995). In addition, according to epidemiological studies, the populations who are at the greatest risk of developing colon cancer are those who have the “fast metabolizer” phenotype for NAT2 and P4501A2 and that are exposed to high levels of heterocyclic amines in their food (Lang et al., 1994). Regarding breast cancer, correlations may have been also be established. Women with a highly inducible form of P450 lAl see their risk of having breast cancer increased. Likewise, the risk of Cancer breast enlarged (x4) in postmenopausal women, moderately smoking (presence of aromatic amines) and having a slow phenotype for NAT2. The risk group for bladder cancer is represented by individuals deficient in GSTMl and NAT2 (Brockrnoller et al., 1996). A positive correlation has been established between endometriosis and women deficient in GSTM1, as well as with the phenotype slow metabolizer for NAT2 (Baranova et al., 1999).
Food is one of the main sources of xenobiotics involved in the

4 multifactorial diseases, hence the importance of ensuring the detoxification of these xenobiotics before they have negative effects on the target organs.
Thus, the present invention relates to the development of a "drug living ", genetically modified microorganisms, and more particularly yeasts, expressing in the digestive environment one or more genes) of interest allowing to correct dysfunctions of the metabolism of xenobiotics, such than those mentioned above. This or these genes of interest will be chosen after determination of correlations between genetic heritage and presence of pathology and after evaluation of the prevalence of this pathology.
Besides the main detoxification application, two other objectives of the present invention are described below, by way of examples.
The first is to express in the digestive environment by said microphone-organisms a gene of interest coding for an enzyme allowing activation in situ of pre-substance to active substance. The administration of such a "drug living "
allowing to control the bioconversion of pre-substance into active substance is all particularly interesting in the case where the active substance has effects unwanted side effects and / or is toxic from a certain dose. Among these applications include the transformation of provitamins into vitamins.
The second consists in making produce in the digestive environment by these microphone-organisms an active peptide or polypeptide, in particular having properties antigenic. The production of such peptides, at different points in the tract digestive, results in the development of "live vaccines" allowing to circumvent a certain number of difficulties encountered in conventional methods administration belonging to the state of the art, such as degradation problems.
Fahl et al (W0 99! 27953) constructed E. coli strains expressing enzymes mammals (P450! Al, NADPH-cytochrome P450 reductase and GST) whose genes were inserted into a plasmid. These model constructions are made in order to to develop “chemoprotective bacterial strains”, in particular of Lactobacilli, expressing mammalian enzymes involved in the detoxification of procarcinogens and administered in the digestive environment in order to supplement the gastrointestinal cell detoxification system.
Fahl et al. do not specify the survival time of Lactobacilli genetically

5 modified in a digestive environment and if they can express it effectively a gene of interest.
To answer these questions, we have put in place digestive systems artificial which allow to study, adapt, control and select microorganisms which survive at least for a minimum time and which can effectively express in situ (i.e. in the digestive environment) a or several heterologous genes of interest. Of course, the survival time depends on microorganisms in question and the digestive environment but we can define in the scope of the invention this parameter as being greater than 6 hours in the stomach small intestine and 48 hours in the colon, i.e. the average duration of transit in these digestive compartments in humans.
Among the preferred microorganisms of the invention, there may be mentioned yeasts.
Indeed, in the same way as the bacteria targeted by the patent application previously cited, yeasts are organisms present in animal feed and / or human and well-known hosts in the digestive environment. Some of them are GRAS or “generally recognized as safe” bodies and have been used since a few years as probiotics.
Their ease of production explains their large-scale use in industry food, for example in the beer and beer manufacturing processes bread or as an additive in animal and human food. S cerevisiae is the most known and the most commercially exploited. Today, S cerevisiae and S
boular ° dü are also used in the pharmaceutical industry as medicines (carbolevure ~ and ultralevure ~ respectively), mainly intended for treatment disorders secondary to antibiotic therapy such as diarrhea, colitis or candidiasis (Ligny G, I975). Numerous studies carried out in animals and the man emphasize both the safety and the positive impact of these yeasts on the environment digestive (antagonism towards Candidas for example).

6 Compared to bacteria, the use of yeasts for medication living has considerable advantages - Unlike bacteria, yeasts are eukaryotic organisms.
Genomic integration of heterologous fragments is easily achievable (highly efficient homologous recombination) and results in a construction more stable than that obtained by plasmid insertion, which in particular limits the potential transfers of the gene of interest to the endogenous human flora.
In addition, the yeasts have a subcellular organization very close to that of ZO higher eukaryotes and have transport mechanisms intracellular and post-translational processing essential for functional expression and compartmentalized by many heterologous genes. For example, it was shown that they have the necessary criteria for the synthesis of human P450 functional since a protein addressing system ("Signal Recognition Particle" or SRP) allows the translocation of the synthesized P450 to the endoplasmic reticulum, this who results in correct intracellular localization and folding of the P450 (Urban and al., 1994a; Pompon et al., 1997). Yeasts also have a environment membrane allowing the stability of P450 and proteins transfer of electrons (NADPH- cytochrome P450 reductase and cytochrome b5) in the membrane of the endoplasmic reticulum, necessary for (enzymatic activity of P450.
- Yeasts, well characterized at the molecular level (especially S
cerevisiae, fully sequenced since 1996-Goffeau et al.), constitute a tool for basis for (genetic engineering and i ~ vitro bioconversions under conditions of not only laboratory but above all large-scale fermentation (production industrial).
- Certain yeasts such as S ce ~ evisiae have the capacity to live in aerobic and anaerobically, which makes it possible to envisage the use of such yeasts at any level of digestive tract.
- Other advantages concerning the construction of recombinant yeasts are also worth highlighting. Indeed, the yeasts are selected by auxotrophy and not based on antibiotic resistance, which avoids being confronted to the

7 problems with the administration of genetically modified organisms to humans possessing an antibiotic resistance gene. In addition, the insertion genomics of gene of interest is facilitated by the recombination property that have certain yeasts, including S cerevisiae (Bellamine, 1996).
S cerevisiae strains have been developed allowing the expression of three main types of genes involved in the different phases of metabolism of xenobiotics: genes from human P450 cytochromes (phase I), genes "Phase I environment" enzymes (NADPH-cytochrome P450 reductase and cytochrome b5 for example) and phase II genes. Such strains have been described by Pompon et al. (US 5,635,369 and EP 595,948).
In the context of the present invention, strains of S cerevisiae have been used as study models. On the one hand, they express the human P450 lAl and the NADPH-cytochrome P450 reductase from yeast or man (respectively strains W (R) pHlAl and W (hR) pHlAl), on the other hand the plant P450 73A1 and NADPH-yeast cytochrome P450 reductase (strain W (R) pP73AI).
P450 lAl exists in the majority of animal species, including (man.
The human form has been isolated by Jaiswal et al. (1985). It intervenes in the metabolism of polycyclic aromatic hydrocarbons, especially in that of benzo (a) pyrene, a compound found particularly in cigarette smoke and barbecue products (Sims et al., 1974; Gautier et al., 1996a). It is hardly detectable in the liver without faction of an inducer but exists in many fabrics extrahepatic to (constitutive state. It is highly inducible by compounds polycyclic aromatics such as 3-methylcholanthrene (3-MC), by compounds halogenated aromatics such as 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD) or by flavonoids such as (3-naphtoflavone.
P450 73A1 (cinnamate 4-hydroxylase or CA4H activity) first catalyzes step of the phenylpropanoid pathway in higher plants by transforming acid trans-cinnamic to p-coumaric acid. This path constitutes the beginning of the synthesis oh flavonoids (antioxidants).
The survival of "model" yeasts, the maintenance of their activity after passage in artificial digestive systems and their in situ activity have been demonstrated in part of the present invention. Such capacities for survival and enzymatic activity are reveal be very important if you want to use living microorganisms like drug.
As such, the method for assessing the fate of microorganisms medication in the human digestive environment is also an object of this invention.
As mentioned earlier, one of the main problems encountered in the stake of a "living" drug lies in its selection, in the evaluation of his survival, its activity and its control, within an environment digestive. For To overcome these difficulties, the present invention proposes the use of systems artificial digestives reproducing in the most reliable and most dynamic possible the human digestive environment. Technically advantageous, these systems allow the control of the main parameters of digestion and ensure the reproducibility of experimental conditions, factors that cannot be or very difficult to control in laboratory animals. In addition, these systems allow samples to be taken at any level of the digestive tract and of get rid of all the ethical constraints linked to the experiments animal or human (potentially toxic molecules). Such systems give also the possibility of assessing the impact of genetically modified micro-organisms on the human endogenous flora (implanted in the artificial digestive system).
Thus, the artificial digestive systems make it possible to make a selection micro-genetically modified organisms, including genetically engineered yeast modified, which have not only adapted to the digestive environment but which canservant a good ability to express a heterologous gene of interest.
Any artificial digestive system with essential characteristics of the system human digestive system can be used in the context of the invention, Various in vitro systems very simple (individual digestive compartments) have been described. Their use is however limited by the fragmentation of the digestive compartments and (absence of dynamism. It is therefore advantageous to use the artificial digestive system full described in WO 94/09895.
Another advantage of the present invention comes from the formulation specific of living drug to protect microorganisms and target specifically an action in this or that part of the digestive system.
Microorganisms, and more particularly yeasts, which must be active at the level of the gastrointestinal tract, the route of administration is the oral route or rectal. The recombinant microorganisms should be administered in a dosage form allowing both to transport them to their place of action, to keep in a active state and protect them from the outside environment. Fireworks or processes galenics used to achieve these goals are different depending on the sensitivity of microorganisms to the digestive environment and depending on where they will have to achieve their activity (level of the gastrointestinal tract). Among the processes galenical used, we can cite: lyophilization of microorganisms and their nutrient medium, encapsulation of microorganisms or coating of the dosage system containing using polymers sensitive to pH or having properties muco-adhesives, the mixture with polymers exhibiting muco-adhesives, the compression of microorganisms in the presence of excipients with properties muco-adhesives, their incorporation in hydrophilic or lipophilic gels.
The resulting galenical systems are monolithic forms of the type monolayer or multilayer tablets, gelatin capsule type (hard or soft) or multiparticulate forms of microgranule or microcapsule type. These shapes allow normal or delayed release of microorganisms within the tract digestive.

DESCRIPTION
Micro or ~ isms Thus, in a first aspect, the present invention relates to micro-organisms comprising at least one heterologous DNA fragment comprising one or more several genes of interest capable of being expressed in the environment digestive.
Advantageously, said microorganisms are also characterized in that that they can survive in the digestive environment for at least 6h, 10h or 12h in 10 stomach and small intestine and / or at least 12h, 24h, 60h or 72h in the colon.
Indeed, in the artificial digestive systems described below, we obtain micro-organisms with survival rates greater than 25%, 50%, 75%, 80%, preference 90% or 95% at the outlet of the ileum after 6 h of digestion and is greater than 2 %, 4%, of preferably 8% after 12 h of fermentation in the colon.
These measurements can be easily made by counting from samples easily taken from artificial digestive systems, asset time of digestion or fermentation.
Preferably, the above-mentioned microorganisms are capable to perform either a bioconversion reaction, or the secretion of a peptide of interest or a protein in all digestive compartments.
In the context of the invention, yeasts are chosen, preferably yeast selected from Saccharomyces cerevisiae, S boulardü, S uvarwna, Schizosaccharomyces pombe, Kluyveromyces lattis and Yarr ~ owia lipolitica. We can also choose bacteria such as lactobacillus described in Fahl et al (W0 99/27953).
Genes of interest By genes of interest is meant genes coding for peptides of interest vaccine, peptides of hormonal interest (such as insulin), peptides of interest therapeutic (like anti-proteases, coagulation factors), peptides with an effect antibiotic, peptides capable of trapping or complexing molecules, of enzymes or all peptides having bioconversion activities, in particular of detoxification or secreted enzymes in the digestive environment.
In the sense of the invention, the terms polypeptides, peptides or proteins will be able be used interchangeably and do not in any way constitute a limitation to a given size.
The genes of interest will preferably be chosen from the enzymes of the metabolism of xenobiotics, namely phase I enzymes (P450 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 73A1), phase environment enzymes I
(NADPH-cytochrome P450 reductase and cytochrome b5), phase II enzymes such as glutathione S-transferase (GST), N-acetyl transferase (NAT), quinone reductase (QR), aldehyde dehydrogenase (ALDH), uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase, epoxide hydrolase and O-acetylase and phase III enzymes (ABC transport pumps, in particular pumps multidrug resistant MDR).
Glutathione S-transferase is a gene superfamily made up of 4 main classes: alpha (GSTZ), mu (GSTM), pi (GSTP) and teta (GSTT).
These enzymes play an essential role in cell resistance against lipid peroxidation, DNA damage and protein alkylation caused by electrophilic chemical species. They catalyze the binding of numerous xenobiotics electrophilic to glutathione and are involved in the detoxification oxygen radicals. The different classes of GST are present in large quantities in many tissues, with a certain specificity of tissues depending on the class considered.
The genes coding for these different enzymes have been located on the chromosomes humans. Different polymorphisms have been highlighted, in particular concerning the gene coding for GSTM1. Indeed, three different alleles of GSTM1 have summer identified: the GSTM1A and GSTM1B alleles code for activity enzymes similar while the GSTM10 allele codes for a non-functional enzyme.
In the most populations studied worldwide, 35% to 50% of subjects are homozygous for the GSTM10 allele and are therefore devoid of activity enzymatic corresponding. Because of this failure in their detoxification system, the individuals of genotype GSTM10 / 0 are considered to be individuals at risk when exposed to high levels of carcinogens and products chemical toxic. Indeed, GSTM1 deficiency is associated with an increase of risk of cancer (mainly lung and bladder cancer) and some chronic diseases (endometriosis, chronic bronchitis ...).
Two N-acetyltransferases (NAT1 and NAT2) have been identified, catalyzing N-acetylation reactions of arylamines, as well as other reactions of detoxification.
They are also involved in the metabolism of drugs such as the sulphonamides and in drug interactions. Variations existing in NAT activity corresponds to their ability to transfer acetate from acetyl coenzyme A to the substrate.
Only the NAT2 gene has a polymorphism allowing differentiation Between slow acetylator (SA) and fast acetylator (RA). Available studies suggest that slow acetylation is more of a risk factor than rapid acetylation.
Indeed, the SA phenotype is considered a risk factor for cancer of the bladder. Of more, postmenopausal women with the SA phenotype are 4 times more likely to develop breast cancer than women with the RA phenotype.
Construction of micro-gold. ~ Recombined anisms: for example yeasts Strains of S. cerevisiae have been developed allowing (expression of three main types of genes involved in the different phases of metabolism of xenobiotics. In this context, a series of expression cassettes for genes of mammals (humans, mice, rabbits) or plants (Jerusalem artichokes, for example) a summer produced, cassettes carried by autonomous mufti-copy vectors or integrated into chromosomal DNA from yeast. For example, the human P450s expressed in the yeasts are the isoenzymes lAl, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 19A.
At the same time, phase I gene coexpressions have been developed, of "phase I environment" (NADPH-cytochrome P450 reductase and cytochrome b5 for example) and phase II. The efficiency of these constructions has been demonstrated. For example, the three stages of biotransformation of benzo (a) pyrene involving the IA1, NADPH-cytochrome P450 reductase and epoxide hydrolase were reproduced in yeast S ce ~ evisiae: analysis of the incubation products of benzo (a) pyrene with these recombinant yeasts shows the formation of all of the metabolites expected (Gautier et al., 1996b).
Such systems can be used as a model for studying certain aspects of human metabolism, as an aid in the analysis of the toxicity of pollutants and mostly as a bioconversion tool.
It is possible, after design of selection and control markers of viability completely original types, to demonstrate and control survival, and when needed death and elimination of functional recombinant yeasts in the environment human digestive system.
It is possible to identify and create by molecular engineering structures of control of heterologous genes (promoter sequences in particular), allowing a adapted and modular activity in the human digestive environment.
For expression of the gene of interest, promoters can be used constitutive or inducible, strong promoters, specifically active promoters in one digestive tract segment under aerobic, semi anaerobic or anaerobic, promoters regulable by a molecule introduced into the medium intestinal. It is possible to develop a genetic engineering technology respecting criteria essential for in vivo use in humans, in particular the absence of mobilizable vectors, the absence of antibiotic resistance markers, a conditional genetic stability of heterologous functions allowing their destruction by an external chemical factor if necessary (interruption of the treatment for example).
The heterologous DNA fragment may also have a genetic marker allowing selection or counter selection or a genetic bar code.

Study and selection of recombinant microorganisms by using systems artificial digestives In a second aspect, the invention relates to a method for studying, control, for adaptation and / or selection of the recombinant microorganisms described above above at by means of an artificial digestive system.
By artificial digestive system is meant any device incorporating the characteristics essentials of the human digestive system.
We can define the artificial digestion system as a device described reaction in WO 94/09895.
This system is the most complete model in the world today.
In Indeed, it has all the compartments of the digestive system (stomach, duodenum, jejunum, ileum and colon, Figures 1 and 2 below) and reproduces the more faithfully possible (human or animal digestive environment monogastric. It takes into account the essential parameters of digestion such as peristaltic movements, variations in pH at the gastric level and intestinal, the gastric emptying and intestinal transit time, secretions gastric, biliary and pancreatic, absorption of digestion and fermentation products, absorption of water, faecal microflora (Minekus et al., 1995 and 1999). Steering computer science system allows continuous parameter control. This system allows mimic different situations, both physiological and pathological. he present number advantages in terms of reproducibility, control of conditions experimental (difficult to control in laboratory animals), standardization, reliability, speed, possibility of collecting samples at any level of the tract digestive, everything by limiting a large number of technical constraints and problems acceptability (impossibility of carrying out experiments with potentially molecules toxic in humans).
Concretely, each compartment is made up of glass units containing walls flexible internals. A water passage between the flexible walls and those of glass allows maintain a constant temperature of 37 ° C and mimic the movements peristaltics (thanks to variations in water pressure). The different compartments are connected together by peristaltic valves, of which the opening is computer-controlled (sending or not sending nitrogen).
S The passage of chyme from one compartment to another is controlled by the number of food present in each compartment (presence of level sensors).
An exponential formula is used to model the gastric emptying and ileal (Minekus et al., 1995): f = 1-2-t ~ tli2 ~~;
where f represents the fraction of meal delivered, t1 / 2 the half-time of meal release, t The meal release time and a parameter describing (pace of the curve).
The volume of the digestive contents is continuously recorded by a sensor pressure and kept constant by absorption of water through fibers of dialysis thanks to a pump.
1 SL 'stomach (TIM 1 We choose the amount of food to introduce into the artificial stomach and the duration of digestion (Minekus et al., 1995). Pepsin is delivered continuously through to one pump. The pH is continuously readjusted with fHCI or Skin, so that to follow a predetermined curve, established according to in vivo data.
The intestine ._yël ~ IM11 We program (artificial small intestine to simulate a transit time closer possible from that observed ih vivo. The pH is maintained at the most physiological possible with NaHC03 (6, S in the duodenum, 6.8 in level 2S of the jejumum and 7.2 at the level of the ileum). At the duodenum level, pumps provide pancreatic enzymes (mixture of pre-enzymes which will be activated by trypsin) and bile acids. The absorption of water and digestion is performed through a dialysis system working with fiber units hollow, connected at the level of the jejunum and (ileum.
The colon ~ TIM2) In its basic form, the artificial colon consists of a loop where circulates cyclically a faecal flora previously installed. It has been verified that the composition and fermentation activity of this fecal microflora, derived from stool voluntary donors and implanted in the digestive system, were well representative those of the human colonic flora and that this flora remained stable and functional over time (Minekus et al., 1999).
A flow of nitrogen keeps the system anaerobic, a condition controlled by an indicator (resazurin solution). The nutritional medium is stored in a unit glass maintained at 4 ° C where an influx of nitrogen allows its mixing permanent in anaerobic. A set of dialysis fibers crossing the entire colon allows mimic (absorption of water and nutrients. The pH is kept constant and adjusted to the value 6.5 with NaOH solution (instead of bicarbonate ih vivo). The total production gas (such as H2, C02 and CH4) can be determined by measuring the amount of water displaced in a bottle of Mariotte. The different gases, as well as acids volatile fats produced (acetate, butyrate and propionate mainly), can be qualitatively and quantitatively analyzed by phase chromatography carbonated.
In a more elaborate form, the artificial colon is made up of three sub-units simulating the three parts, proximal, transverse and distal where sit activities different fermentation (associated with different pH).
Of course, all or part of the aforementioned artificial digestive systems can to be put in work in the context of the invention.
Thus, the invention relates to a method for studying and controlling micro-organizations including the steps of a) add the microorganisms according to the invention in a nutritive medium liquid, b) introducing said medium at the entry of a system as defined above, c) incubate and count the recombinant microorganisms and / or at a test of the activity of the gene (s) of interest after passage through the systems digestive or in situ. For example, this step may include a PCR test. The test activity may include a secretion test and / or bioconversion.
In another embodiment, the method can include the steps consists in a) add the microorganisms according to the invention in a nutritive medium liquid, b) introducing said medium at the entry of a system as defined above, c) incubate and recover the surviving microorganisms after passage through the TIMl and / or TIM2;
- if necessary repeat steps a) to c) until a population homogeneous of micro-organisms adapted to the digestive environment and capable IO to effectively express the gene (s) of interest.
The invention also relates to microorganisms capable of being obtained by the process mentioned above. These microorganisms can be characterized in this that their survival rate is greater than 25%, 50%, 75%, 80%, preferably 90%
or 95% at the outlet of the ileum after 6 h of digestion and is greater than 2%, 4%, of preferably 8% after 12 h of fermentation in the colon. Preferably, it it's about yeasts.
The survival of microorganisms can be assessed by counting. The presence of or genes of interest can be identified by molecular techniques (for example Polymerase Chain Reaction). Expression of the gene (s) of interest may be estimated by biochemical techniques such as mono electrophoresis or two-dimensional or Western Blot. The activity of expression products of or some genes of interest is appreciated by specific techniques (for example dosage enzymatic activity).
In a particular embodiment, the invention relates to micro-organizations capable of carrying out a bioconversion reaction or secreting a peptide or a protein of interest in all digestive compartments.

Method of administration of microorganisms Know-how in the field of galenics and biopharmacy allowing to optimize the administration of the active ingredients by oral route developed.
The effectiveness of a drug is conditioned by its bioavailability;
say the proportion of active ingredient arriving at the level of general circulation, and according to what kinetics. One of the aims of the galenical formulation is therefore to make a optimized form best suited for the treatment of a disease or syndrome determined, i.e. a form presenting an easy administration for the patient and good availability.
For conventional active ingredients (origin and chemical nature defined), the oral constitutes one of the preferred channels of supply, by its convenient administration and his low cost. The active ingredients thus administered can be absorbed while iong of the gastrointestinal tract. They will eventually suffer at this level different biodegradation under the action of enzymes, digestive juices and pH variations.
The fraction absorbed from the digestive tract can reach the site action after passage through the liver and possible metabolism.
In contrast, in the case of microorganisms, these will not absorbed by the intestinal mucosa but will stay in the tract for a while or less long in order to exert their action at the level of the defined target. The form galenic containing the microorganisms is a form for bringing them intact at target level and maintain their integrity at that level for a time defined more or less long.
Under these conditions, the techniques used with the active ingredients classics for ensure sustained release apply to microorganisms after adaptation of technology and operating parameters.
All additional details regarding these dosage forms are available in EP 904021995 (dosage forms improving the bioavailability), EP0542824 (muco-adhesive forms) and FR 9806958 (oily gel and bigels).

These different techniques or processes, as well as any other process of the state of the coating and encapsulation technique, can be applied to micro-organizations of the invention.
Lice substances sensitive to atmospheric humidity or those sensitive to oxidation by ambient air oxygen, protection is necessary for the preparation of dosage forms (capsules, tablets) to guarantee their conservation and maintain their therapeutic activity. The coating allows by deposit direct protective agent (resin, polymer) to protect the active ingredient In the case microorganisms, in particular yeasts, according to the invention, the coating has for purpose of isolating them from the hostile external environment in order to protect them from assaults enzymes, pH and juices present in the digestive system. The result of the coating is obtaining a "sphere" or microsphere whose diameter is directly proportional to the quantity of coating product used and to the size of the produced at originally coat. Operating parameters, technical equipment and adjuvants used to achieve this coating are important factors for obtaining microspheres with defined characteristics. The coating is either a polymer sensitive to pH and dissolving at a given pH of the gastrointestinal tract, either a polymer including the nutrients necessary for the survival of micro-organizations, either a mucoadhesive product facilitating the mucoadhesion of microorganisms to level of the gastrointestinal tract, a combination of these different compounds. The result obtained consists of microspheres which are then conditioned in hard or soft capsules.
Thus, another aspect of the invention relates to a pharmaceutical composition comprising the microorganisms defined above advantageously found under a dosage form suitable for oral or rectal administration so as to the route intact to the target site of the digestive tract and maintain their integrity at this level for a given time.
In this pharmaceutical composition, microorganisms, in particular the yeasts, can be protected by an associated composition or covered by a coating and / or a resin type membrane and / or polymer type forming spheres, microspheres, granules or microgranules.
Preferably, the composition is in the form of capsules, tablets or from 5 sugared almonds. The composition can also be in the form of a suspension cellular.
The invention also relates to a combination product comprising the micro-organisms described above and an active substance or a precursor of a substance active for simultaneous, separate or spread over time use.
10 This product is preferably characterized in that the said microorganisms interact with the active substance or the precursor. The active substance is intended for favor the action of recornbined microorganisms (for example, it may be a substance promoting the survival, growth or activity of microorganisms) or, conversely, microorganisms act on the active substance or on the precursor.
Conclusion Another aspect of the invention relates to the use of micro-organizations according the invention for the preparation of a medicament such as detoxification, metabolic correction agents, agents capable of modifying the flora intestinal agents capable of increasing the bioavailability of a active substance or its precursor and for the preparation of a medicament capable of providing in situ active peptides, peptides of vaccine interest, peptides of interest hormonal, peptides of therapeutic interest, peptides with antibiotic effect or peptides capable of trapping or complexing molecules.
Legends Figures 1 and 2: schematic representations of the digestive systems artificial.
Figure 1 (artificial stomach and small intestine or TIM 1) Figure 2 (artificial colon or TIM 2) Figure 3: genomic and plasmid constructs for gene expression of P450 lAl human and NADPH-cytochrome P450 reductase (A) or yeast (B) and for the expression of the Jerusalem artichoke P450 73A1 gene and NADPH-cytochrome P450 yeast reductase (C).
A- The strain obtained is called W (hR) pHlAl.
B- The strain obtained is called W (R) pHlAl.
C- The strain obtained is called W (R) pP73A1.
URA 3: wild gene for orotate decarboxylase; ADE 2: wild gene for the amino-imidazole ribonucleotide carboxylase; ura 3: mutated orotate gene decaxboxylase;
Y red: yeast P450 reductase; H red: human P450 reductase; PGK
phosphoglycerate kinase; Y red ORF: P450 reductase coding sequence for yeast; H red ORF: coding sequence for human P450 reductase; H 1A1 ORF
coding sequence for human P450 Al; P 73A1 ORF: coding sequence of Jerusalem artichoke P450 73A1; pro: promoter; ter: terminator.
FIG. 4: release kinetics of the recombinant yeasts W (hR) pHlA1 and W (R) pHlA1 at the ileum outlet.
The results obtained during three digests in Ie TIM1 are presented digestion by digestion (A) and averages (B).
The results are expressed in cumulative rate of surviving yeasts found output ileum relative to the number of yeasts ingested. Or, for each withdrawal (t =
120, t = 240 and t = 360 min) corresponding to a time interval of collecting cumulative outings (0-120 min, 120-240 min and 240-360 min), the number total of yeast is listed and expressed as a percentage of the total number of yeasts ingested.
These percentages are then added up over time.
The dilution factor due to the volume of digestive secretions is taken into account.
account in calculations.
Figure 5: monitoring of the recombinant yeasts W (hR) pHlAl and W (R) pHlA1 in the artificial colon.

Different samples were taken in order to be able to carry out a follow-up yeasts in TIM 2 - at t = 0, the number of cells per ml of yeast suspension is listed recombinant, before introduction into the colon, - at t = Ofec: the number of endogenous yeast cells per ml of content colic, before seeding of the colon, - at t = 1, t = 12, t = 24, t = 36, t = 48 and t = 60h: the number of cells of yeasts (recombinant and endogenous) per ml of colonic content taken from the colon.
The results obtained are expressed in number of cells per ml (scale logarithmic) taking into account the dilution factor due to the volume of the content colic.
Figure 6 PCR results carried out on W (R) pHlA1 and W (hR) pHlA1 before passage through the digestive systems (A), at the outlet of small intestine (B) and colon (C) A: before passage through the digestive systems (t = Omin) B: at the exit of the small intestine (t = 120 min) C: at the exit of the colon (t = 12h) Primers (see Figure 6 + 7'3 + 4 1 + ~ 1 + 8 5 + 2'3 + 4 1 + 2 1 + 8 3 + 4 3 + 4 3) Fragment size 593 650 351 459 461 650 338 459 0 650 (bp) Strain W (R) pHlAl W (hR) pHlAl T <0 T> 0 T = Witness Figure 7: monitoring of W (R) pP73A1 yeasts in the different compartments of the TIM 1 (7A: stomach, 7B: duodenum, 7C: jejunum, 7D: ileum) W (R) pP73A1 yeasts were monitored within each digestive compartment at TIM1 digestion course (n = 3). To do this, direct debits of 1 ml are made in (food before its introduction into (stomach (t = 0), then at different levels of the digestive tract: in the stomach after 15, 30, 45, 60 and 90 min from digestion, in the duodenum after 15, 30, 45, 60, 75, 90, 120 and 150 min of digestion, in the jejunum after 30, 45, 60, 90, 120, 150, 180 and 240 min of digestion, in (ileum after 30, 45, 60, 90, 120, 150, 180 and 240 min of digestion.
The number of yeasts present in each sample is expressed in percentage of the total number of yeasts ingested. Yeast tracking curves recombined in each compartment were compared with those of a transit marker no absorbable.
Figure 8: release kinetics of the recombinant yeasts W (R) pP73A1 in out of ileum The results are expressed in cumulative rate of surviving yeasts found output ileum relative to the number of yeasts ingested (n = 3). Or, for each sample (t = 30, 45, 60, 90, 120, I80 and 240 min) corresponding to a collection time of cumulative outings (0-30 min, 30-45 min, 45-60 min, 60-90min, 90-120 min, 120-180 min and 180-240 min), the total number of yeasts is listed and expressed in percentage of the total number of yeasts ingested. These percentages are then accumulated over time.
The dilution factor due to the volume of digestive secretions is taken into account.
account.
Figure 9: monitoring of W (R) pP73A1 recombinant yeasts in the artificial colon Different samples were taken from TIM2 in order to be able to make a monitoring of yeasts in the colonic environment - at t = 0, the number of cells per ml of yeast suspension is listed, before their introduction into the colon, - every hour until 12 noon, the number of yeast cells is listed through ml of colonic content taken from the colon.
The results obtained are expressed as a percentage of the number of yeasts introduced in the colon, taking into account the dilution factor due to the volume of the content colic.

FIGURE 10: transformation kinetics of (trans-cinnamic acid to p- acid coumaric with yeasts W (R) pP73A1, in (TIM 1 (A) together and in the different digestive compartments of TIMl (B) In order to be able to follow the conversion reaction of trans-cinnamic acid in acid p-coumarique, samples are taken throughout the digestion in the different digestive compartments of TIM 1 (n = 3). Direct debits are so performed in the stomach after 15, 30, 45, 60 and 90 min of digestion, in the duodenum after 15, 30, 45, 60, 75, 90, 120 and 150 min of digestion, in Ie jejunum after 30, 45, 60, 90, 120, 150, 180 and 240 min of digestion, in (ileum after 30, 45, 60, 90, 120, 150, 180 and 240 min of digestion and in the exits cumulative ileal over 0-30 min, 30-45 min, 45-60 min, 60-90 min, 90-120 min, min and 180-240 min at 30, 45, 60, 90, 120, 180 and 240 min.
FIG. 10A presents a summary of the bioconversion reaction in the whole TIM 1 (n = 3). The production of p-coumaric acid is expressed as% of the acid trans cinnamic introduced into the stomach.
Figure l OB represents the production of p-coumaric acid (expressed as a% of acid very cinnamic years introduced into the stomach) in the different TIMI compartments (stomach, duodenum, jejunum and ileum), during digestion (n = 3).
Figure 11: Constructs of plasmids allowing secretion by yeasts S.
cerevisiae, either of a "tagged" peptide (A), or of a "tagged" protein (B) A: The plasmid obtained is called ppV5H6.
B: The plasmid obtained is called ppGSTV5H6_ PGAL1: promoter highly inducible by galactose; CYC1 TT: terminator of uncomfortable CYCI; pUC ori: origin of pUC replication allowing maintenance and replication of the plasmid in bacteria; 2 ~, origin: origin of replication 2 ~
allowing maintenance and replication of the plasmid in yeast;
Ampicillin resistance gene (ampicillin serving as a selection marker in the bacterium;
URA 3: auxotrophy marker allowing the selection of yeasts transformed;
BamH1: cleavage site of (restriction enzyme BamHI; EcoRl: site of break restriction enzyme EcoRl; Pre: pre sequence, allowing addressing of peptide a to the endoplasmic reticulum; Pro: pro sequence allowing a efficient secretion of peptide a; KR: protease cleavage site I ~ ex2 (acids amines lysine-arginine); GST: Glutathione-S-transferase; VS: epitoge V5; H6 : string 5 polypeptide of 6 histidines; Stop: stop codon.
Fi ur ~ e 12: detection of the secretion products of the WppV5H6 (A) and WpGSTVSHg (B) in Erlen-Meyer The Western-Blots obtained with the WppV5H6 and WpGSTV5H6 strains are represented 10 in FIG. 12A and 12B respectively. Samples were taken from Culture growth and in the induction culture, 0, 2, 4 and 6 hours after addition of galactose. The samples are noted "expo" when the induction culture has been seeded low cell density and "stat" when it was seeded at high density. The protein of the supernatant are precipitated, separated by gel electrophoresis polyacrylamide 15 and analyzed by Western-Blot (anti-VS body). Lice each sample, we deposit the proteins of the supernatant corresponding to a culture volume containing 5 *
10 ~
yeasts (except (sample B6 which corresponds to 4 * 108 cells).
A1: peptide-VSH6, growth culture control; A2: peptide-VSH6, t0 culture induction; A3: peptide-VSH6, expo t2h; A4: peptide-VSH6, stat t2h; AS:
peptide-20 VSH6, expo t4h; A6: peptide-VSH6, stat t4h; A7: peptide-VSH6, expo t6h ; AT 8 peptide-VSH6, stat t6h.
B1: GST-VSH6, growth control witness; B2: GST-VSH6, t0 culture induction;
B3: GST-VsH6, expo t2h; B4: GST-VSH6, stat t2h; BS: GST-VSH6, expo t4h;

GST-VSH6, stat t4h (4 * 108 cells); B7: GST-VSH6, stat t4h (5 * 10 ' cells); B8 25 GST-VSH6, expo t6h; B9: GST-VSH6, stat t6h.
a) peptide-VSH6 (calculated PM: 5.7 kDa), b) precursor of peptide-VSH6 (PM
calculated 16 kDa), c) glycosylated forms of the peptide-VSH6 or its precursor, d) forms hyperglycosylated peptide-VSH6 or its precursor, e) GST-VSH6 (calculated PM
29.7 kDa), f) precursor of GST-VSH6 (calculated PM: 40 kDa), g) forms glycosylated or hyperglycosylés of GST-VSH6 or its precursor.

Figure 13: Detection, in the various TIM1 compartments, of the peptide "tagged" secreted by the WppV5H6 strain FIG. 13 represents the Western-Blots obtained after analysis of the samples taken from the various compartments of TIM 1. A positive control has also was carried out in Erlen-Meyer (samples marked "witness"). All of proteins contained in the samples supernatant has been deposited (corresponding to a volume of 4 mI in the stomach and duodenum and 2 mI in the food, jejunum and the ileum).
1: Food; 2: Witness t90 min; 3: Stomach t90 min; 4: Duo t90 min; 5:
Jej t90 min; 6: Island t90 min; 7: Esto t120 min; 8: Empty; 9: Witness t160 min; 10 : Duo t160 min; 11: Jej t160 min; 12: Island t160 min; 13: Duo t210 min; 14: Jej t 210 min; 15 Island t210 min; 16: Island t270 min; 17: Witness t270 min.
Figure 14: Detection, in the various TIMl compartments, of the protein "Tagged" GST secreted by the WppGSTV5H6 strain FIG. 14 represents the Western-Blots obtained after analysis of the samples taken from the various compartments of TIM 1. A positive control has also was carried out in Erlen-Meyer (samples marked "control"). All of proteins contained in the samples supernatant has been deposited (corresponding to a volume of 4 ml in (stomach and duodenum and 2 ml in food, jejunum and (ileum).
1: Witness t90 min; 2: Stomach t90 min; 3: Duo t90 min; 4: Jej t90 min;
5: Island t90 min; 6: Empty; 7: Duo t160 min; 8: Jej t160 min; 9: Island t160 min; 10:
Witness growing culture; 11: Witness t0 min; 12: Food; 13: Witness 160 min; 14 Duo t210 min; 15: Jej t210 min; 16: Island t 210 min; 17: Island t270 min; 18 : Witness t270 min.
Figure 15: Quantification of "tagged" GST, secreted in TIM 1 by the strain WppGSTV5H6 In order to approximate the amount of GST secreted in the digestive environment, we compared (intensity of the detection of a standard range with that of samples from TIM 1. 20 ng, 10 ng and 1 ng of GST
85% pure were thus deposited on the same gel as the proteins samples from TIM 1 samples.
1: Growth culture witness; 2: Food; 3: Esto 120 min; 4: Duo t90 min; 5 Jej t90 min; 6: Island t90 min; 7: GST 20 ng; 8: GST 10 ng; 9: GST 1 ng;
10: GST
20 ng; 1I: GST 10 ng; I2: GST I ng; 13: Jej 160 min; I4: Island 160 min;
15: Duo t160 min; 16: Duo t210 min; 17: Jej t210 min; 18: Ile 210 min.
Example 1: The yeast Saeclaaromyces cerevisiae as a system for expressing P450 humans.
S. ce ~ evisiae was the first system used for (expression of a P450 of rat (Oeda et al., 1985). Since then, it has been widely used as a system expression heterologous in many laboratories. In particular, the team of Dr Denis Pompon fa chosen as study model for the expression of human P450 (isoenzymes 1A1, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 19A).
Use yeast as host for heterologous expression of P450 present of many advantages. Indeed, yeasts have the necessary criteria to the synthesis of functional human P450s (Urban et al., 1994a; Pompon et al., 1997).
They present the intracellular architecture and the majority of post changes of eukaryotic cells, as well as a system for addressing protein ("Signal Recognition Particle" or SRP) allowing the translocation of the P450 synthesized towards the endoplasmic reticulum, which results in localization intracellular and folding of the P450 correct. Yeasts have also a lipid membrane environment allowing the stability of P450 and protein electron transfer (NADPH- cytochrome P450 reductase and cytochrome b5) in the endoplasmic reticulum membrane, necessary for enzyme activity of P450.
The introduction into yeasts of expression cassettes can be carried out of them ways: on plasmid or by genomic integration.
In addition, yeasts allow the stable coexpression of several DNAs complementary (cDNA) and the techniques used are low cost and risk free.
The yeast S. cerevisiae allows the characterization of monooxygenase activities human (Urban et al., 1994a; Gautier, 1994), viz.
- the study of the specificity of substrate in an activity-free context contaminating monooxygenase (S ce ~ evisiae contains endogenous P450 weakly expressed of which (activity does not risk interfering with that of human P450), - the study of genetic diversity and enzymatic polymorphism (possibility to express scant forms of P450), - the study of the structure / function relationship by site-directed mutagenesis and by manufacture of fusion proteins (search for amino acids involved in an aspect particular of the function of human P450).
Despite the advantages previously listed, the use of yeasts as heterologous expression system (Urban et al., 1994a; Pompon et al., 1996) present some limits - the subcellular fractions are di ~ ciles to prepare because of the wall very resistant yeasts, - the level of endogenous NADPH-cytochrome P450 reductase is low, which limit (P450 activity expressed, - the enzymes NADPH-cytochrome P450 reductases from yeast and from humans low sequence similarity (33%), which reduces the effectiveness of coupling between human P450 and yeast NADPH-cytochrome P450 reductase, - the production of human P450 (between 50 and 500 pmol of P450 per mg of protein microsomal) is weaker than in E soli bacteria (around nmol / mg) but this drawback is more than made up for by the fact that, unlike the bacteria yeasts produce functional P450s.
In order to best overcome some of these limitations (Urban et al., 1994a;
Pompon et al., 1995) different expression systems have been developed - First generation systems: optimization of (expression of P450 human.
An expression cassette, inserted into a plasmid, allows the production of a human. To obtain a sufficient level of expression, the 5 'and 3' sequences no coders of the eDNA were deleted before insertion into the yeasts. Moreover, the strong promoter GAL10-CYC1 (totally suppressed by glucose but strongly galactose inducible) was introduced to separate the phase growth exponential, required to constitute the biomass, of the expression phase.
- Second generation systems: optimization of (specific activity human.
Several types of genomic changes have been made to increase the activity specific for P450 from 10 to 100 times that obtained with first generation - the construction of a fusion protein between human P450 and NADPH-yeast cytochrome P450 reductase, overexpression of yeast NADPH-cytochrome P450 reductase, under the control of the promoter GAL 10-CYC 1, the coexpression of human cytochrome b5 and human P450.
Coexpression of phase II enzymes (such as epoxide hydrolase) and P450 human also allowed to simulate metabolic couplings between enzymes phase I and phase II (Gautier et al., 1993).
- Third generation systems: "humanization" of (environment redox.
At the genome level, the yeast NADPH-cytochrome P450 reductase has been replaced by a human reductase, which caused a sharp increase of the specific activity of human P450 x10 to 500 compared to the first generation.
Example 2: Obtaining strains of recombinant yeasts W (hR) pHlAl, W (R) pHlA1 and W (R) pP73A1 The S cerevisiae W (hR) pHlAl strain (FIG. 3A) is derived from the W (hR) strain.
The two strains W (R) pHlA1 (Figure 3B) and W (R) pP73A1 (Figure 3C) are derived from strain W (R).
The construction of strains W (R) and W (hR) have been described respectively by Truan et al. (1993) and Urban et al. (I993).

Genomic modifications have thus enabled the construction of W (R) strains and W (hR) from laboratory strain W3031B (sex sign MAT a), which possesses mutations in certain genes (Ieu 2, his 3, trp 1, ura 3, ade 2), thus allowing its selection by auxotrophy.
10 W (R) strain: the open reading phase of NADPH-cytochrome P450 reductase yeast (2072 bp) was overexpressed by insertion of the strong promoter GAL10-(480 bp) in place of the NADPH-cytochrome P450 reductase promoter yeast.
The terminator for the yeast NADPH-cytochrome P450 reductase gene has been preserved.
15 W strain (hR): the open reading phase of NADPH-cytochrome P450 reductase human (2034 bp) has been inserted into the yeast genome in place of that of the NADPH-cytochrome P450 reductase from yeast and placed under the control of promoter strong GAL10-CYC1. The terminator of the NADPH-cytochrome P450 reductase gene yeast was kept.
The two strains W (hR) pHlAl and W (R) pHlAl result respectively from Ia transformation of W (hR) and W (R) with the plasmid plAl.
Plasmid plA1 (See Figure 3) results from insertion into plasmid pV60 of the sequence coding for human P450 lAl (1539 bp), placed between the promoter strong GAL10-CYCZ and the terminator of the phosphoglycerate kinase (PGK) gene. I1 contains the gene encoding 13-lactamase which confers resistance to ampicillin at E coli.
This plasmid can be maintained in yeasts thanks to the markers auxotrophy URA 3 and ADE 2 which complement the corresponding deficient genes in the yeast genome. The addition of the ADE 2 auxotrophy marker allows a better vector stability when transformed yeasts are cultivated in one culture medium becoming spontaneously limiting in high density adenine cellular.
The plasmid can multiply in bacteria thanks to an origin of replication of E
coli and in yeasts thanks to a fragment of (origin of replication of plasmid 2 ~ ,.
Transformation of the two strains W (hR) and W (R) with the plasmid plA1 The yeast transformation protocol, adapted from Gietz et al. (1992), need first, to make the yeasts competent. To this end, the strains W (R) and W (hR) are cultured in YPGA medium (glucose 20 g / 1, yeast extxact 10 g / 1, bactopeptone 10 g / 1 and adenine 20 mg / 1) until a D06oonm IO between 0.5 and 2. After centrifugation (6000 g, 3 min, 20 ° C), the pellet cell is washed twice with 25 ml of sterile distilled water, then once with 2 ml of Tris-HCl buffer 100 mM, EDTA 1 mM, lithium acetate O, 1M. The cell pellet is resumed in 1 ml of this same buffer. Then, in order to proceed with the transformation of yeasts are mixed faliquot of the transforming vector plAl, 50 ~ g of denatured sperm DNA
of salmon, 100 ~ 1 competent yeasts and 300 ~, l of a solution of PEG-4000 to 40%
in 100 mM Tris-HCl buffer, lmM EDTA, 0.1 M lithium acetate. The mixture is incubated for 30 min at 28 ° C then 15 min at 42 ° C. After centrifugation (11000 g, 3 min, 20 ° C), the cell pellet is taken up in 100 μl of Ringer's liquid.
The 100 ~ .1 are spread on SGI medium (glucose 20 g / 1, yeast nitrogen base 7g / 1, bactocasamino acids 1g / 1, tryptophan 20 mg / I and agar 20g / 1). The dishes are incubated 2 to 3 days at 30 ° C.
Similarly, the strain W (R) pP73A1 results from the transformation of the strain W (R) with the plasmid pV60 in which the cinnamate 4-hydroxylase gene of Jerusalem artichoke or P450 73A1 (plasmid called p73A1- see Figure 3) has been inserted (Urban et al., 1994b).
Example 3: Example of a digestion protocol in the TIM1 digestive systems and TIM2 (modified protocol from WO 94/09895) A Digestion in the artificial stomach and small intestine or TIM 1 (see hard The variable digestion time is programmed between 240 and 360 minutes.

Solutions used during digestion Different solutions simulating stomach and intestinal secretions are prepared and stored in ice for those containing enzymes. All the solutions are prepared with demineralized water.
- solutions simulating stomach secretions * Gastric juice: NaCI 53 mM, KCI 15 mM, CaCIa I mM and NaHC03 7mM, pH 4, to which pepsin is added at a rate of 588 U / ml, * lipase: NaCl 53 mM, KCl 15 mM, CaCl2 1 mM and NaHC03 7 mM, pH 4, at which lipase is added at a rate of 37.5 U / ml, * 1M HCl so that the pH in the stomach follows a predetermined curve.
- solutions simulating secretions from the small intestine * 7% pancreatin solution, * 4% bile solution (for the first 30 minutes of digestion) and at 2% (for the rest of the digestion), * 1X electrolyte solution: 86 mM NaCl, 8 mM KCl and 2 mM CaCl2, * 1M NaHC03 or 1X electrolyte solution to keep the pH at 6.5 to level of the duodenum, to 6.8 at the level of the jejunum and to 7.2 at the level of (ileum.
Digestion Prior to (food, are introduced into the different compartments of the system digestive residues simulating what remains on average in an fasting individual : the residue gastric (gastric juice adjusted to pH 2 and preincubated 15 min at 37 ° C), Ie duodenal residue (bile 2%, pancreatin 1.75%, electrolyte solution 0.25X of the small intestine and trypsin 7773 U / ml, preincubated 10 min at 37 ° C) and jejunal and ileal residues (solution 1X of electrolytes from the small intestine).
Dialysis fluids for jejunum (1.55% bile, 8 mM KCl, 86 mM NaCl) and the ileum (1X electrolytes) are maintained at 37 ° C for the time of digestion and circulate to a flow rate of 10 ml / min (dialysis fibers: LOBE, HG-400, threshold = 8000 Da).
The yeast strain studied, resuspended in 300 ml of half milk skim or YPL culture medium (yeast extract 10g / 1, bactopeptone 10g / 1, galactose 20g / 1), is introduced into the stomach. The yeasts are prepared as described in the examples 4and5.
Kinetic The solutions simulating the different secretions are delivered according to a kinetic preset according to in vivo parameters * in the stomach: gastric juice and lipase are delivered to a 0.25 flow ml / min, fHCI is introduced at a flow rate of 0.25 ml / min if necessary.
* at the duodenum level: the 7% pancreatin solutions are delivered to a flow of 0.25 ml / min, the bile solution (4 or 2%) at a flow rate of 0.5 ml / min, the NaHC03 1M
and the 1X electrolyte solution at a total flow rate of 0.25 ml / min.
* at the level of the jejunum and the ileum: NaHC03 1M is introduced at a rate 0.25 ml / min, if necessary.
The gastric and ileal emptying curves are modeled by the formula f =
1-2 '~~ tli2> a where t1 / 2 is the half-drain time and 13 is a defining parameter (pace of the curve.
B Fermentation in the artificial colon or TIM 2 (see Figure 2) After a 2-day flora stabilization phase, fermentation can to be maintained for up to a week after seeding of the colon.
Colon seeding All the steps of (seeding are carried out under nitrogen flow.
200 g of fresh faeces (from healthy volunteers) are mixed in 200 ml of buffer 0.1 M phosphate at pH 6.5, previously autoclaved and placed under nitrogen flow while at least 2 h. The mixture obtained is filtered through sterile gauze. The filtrate is centrifugal (25000g, 30 min, 4 ° C). The bacterial pellet and mucus are taken up in from the middle of base (Ileum delivery medium) itself diluted 10 times in phosphate buffer 0.1 M.
About 10g of filtration retentate are added to this solution.
bacterial in order to enrich it with fiber. The mixture is mixed again and then introduced into the colon using of a syringe.

Solutions used during fermentation Different solutions entering into the constitution of the nutritive medium of the faecal flora and of colon dialysis solution are prepared, autoclaved 15 min at 120 ° C then stored at 4 ° C until use (except the vitamin solution which is sterilized by filtration and stored at -20 ° C). All solutions are prepared with water demineralized.
- a 10 X electrolyte solution: heme 0.1 g / 1, bile salts 0.5 g / 1, cysteine 4 g / 1, FeS04 0.05 g / 1, CaCl2 4.5 g / 1, MgS04 5 g / 1, NaCl 4.5 gll and K2HP04 25 g / 1, - a 20 X carbohydrate solution: pectin 12 g / 1, xylan 12 g / 1, arabinogalactan 12 g / 1, amylopectin 12 g / 1 and starch 100 g / 1, - a solution containing lipids and proteins (TBC 40X): tween 80 80 m1 / 1, bacteropeptone 120 g / 1 and casein 120 g / 1, - a solution of vitamins 10 X: menadione 10 rng / l, biotin 20 mg / l, vitamin B 12 5 mg / l, pantothenate 100 mg / 1, nicotinamide 50 mg / 1, p-aminobenzoic acid 50 mg / 1 and thiamine 40 mg / l.
Stabilization of flora and fermentation Dialysis solution (1 X colon electrolyte solution and vitamins 0.01 X, pH 6.5) is bubbled under nitrogen flow overnight.
She is delivered at a flow rate of 1 ml / min during the whole stabilization period and of the fermentation (dialysis fibers: JFM MSID X Flow).
"Ileum delivery medium" (1 X colon electrolyte solutions, solution hydrates carbon 10 X, TBC 10 X, solution of vitamins 0.01 X and mucus 20 g / 1, pH
6.5), simulating the product of digestion from filëon, is introduced into the colon due to 3 ml per hour.
The studied yeast strain, resuspended in demineralized water or YPL culture medium (10g / 1 yeast extract, 10g / 1 bactopeptone, galactose 20g / 1), is introduced into the colon, after stabilization of the flora. The yeasts are prepared as described in examples 4 and 5.
Example 4: Monitoring of the recombinant yeasts W (R) pHlA1 and W (hR) pHlA1 in artificial digestive systems Preparation of yeasts The W (R) pHlAl and W (hR) pHlA1 strains are cultured in medium minimum SGI (glucose 20 g / 1, yeast nitrogen base 7g / 1, bactocasamino acids 1g / 1 and 5 tryptophan 20 mg / 1) up to a concentration of 106-10 'cells / ml.
The culture is then centrifuged in sterile plastic tubes (3000 g, 10 min, 4 ° C). The supernatant is discarded and the cells are washed in 15 ml physiological water (NaCl 9 g / 1).
After centrifugation (3000 g, 10 min, 4 ° C), the supernatant is discarded and the cell pellet 10 is resuspended in the food.
The yeasts are introduced into TIM 1 resuspended in 300 ml of milk semi-skimmed or are introduced into the TIM2 resuspended in 5 ml of water demineralized.
15 Enumeration of yeasts - Collection of samples in TIM 1: 1 ml of the starting food (containing the two strains of yeast) is taken before introduction into the stomach (t = 0), then 1 ml is taken at times 120, 240 and 360 min in the collected ileal effluents on the time intervals 0-120 min, 120-240 min, 240-360 min, 20 - Sampling in TIM 2: 1 ml of the colonic content is taken before inoculation of the colon (t = Ofec) as well as 1 ml of the yeast suspension before introduction into the colon (t = 0), then 1 ml of the colonic content is removed all the 12 hours in the colon (t = 1, 12, 24, 36, 48 and 60 h), - Yeast culture: the samples are diluted in water physiological and 25 spread on SGI medium. Ampicillin (100 ~ g / ml) is added to the middle afm eliminate contaminants from the digestive system and / or flora fecal as well than ethanol (3% v / v) to promote the development of yeasts.
- Enumeration: the enumeration of the boxes is carried out after 2 to 3 days incubation at 30 ° C.
Results: The experiments carried out have demonstrated viability in the artificial digestive systems of recombinant strains of .S ce ~ evisiae W (hR) pHlA1 and W (R) pHlAl, introduced simultaneously into the stomach or the colon.
50% ~ 18.9 (n = 3) of the recombinant yeasts introduced into the stomach are found at the outlet of the ileum after 6 h of digestion (Figure 4).
The survival rate of yeasts in the colon appears lower since we don't finds, after 12 h of fermentation, that 4% of the yeasts introduced (n = 1) (figure 5).
The causes of this sharp decrease in the survival rate in the colon remain indeterminate, all the more that after one hour of fermentation, all yeasts introduced was still present in the colon. Two hypotheses have been advanced involving the competition exerted by the faecal flora and the conditions almost anaerobic prevailing in the artificial colon.
Identification of yeasts by Polymerase Chair Reaction (PCR) The PCR technique had to be optimized for monitoring recombinant yeasts in the digestive environment PCR can be carried out from colonies isolated on a Petri dish or from yeasts returned to liquid culture. In the first case, we take a head pin of a colony which is resuspended in 10 ~ 1 of distilled water before do it boil 10 min. In the second case, the culture must be in phase exponential of growth (D06oonm between 0.5 and 2). After centrifugation (10000g, 10 min, 4 ° C), the cell pellet is taken up in 50 ~, 1 of distilled water and put to boil 10 min.
In liquid culture, the best results (in terms of intensity of amplifiers and reproducibility of the experiments) are obtained when the yeasts are in phase exponential growth (D06oonm from 0.5 to 2), while on solid medium the stage of yeast growth does not seem to influence the results.
The primers used are either internal to the genes (P450 lAl and NADPH-cytochrome P450 human or yeast reductases), either located on both the gene and his promoter GAL10-CYC1 in order to verify its stability (Figure 3).
They are chosen so that they hybridize as little as possible on themselves, with themselves and between them using the program on the Internet at the following address http: // www. Williamstone. com / primers / calculator / calculator. cgi PCR samples contain 1 X PCR buffer, 1.5 mM MgCl2, all 4 dXTP
0.2 mM, primers S 'and 3' 0.5 p. M, Taq polymerase 25 U / mI and the matrix DNA
0.06%.
The PCR program used is as follows (30 cycles): deanification 5 min 95 ° C, denaturation 30s 9S ° C, priming 30s 51 ° C, DNA synthesis 2min 72 ° C and extension final 10 min 72 ° C.
After PCR, the samples are mixed with a stop solution (blue Bromophenol 0.25%, Ficoll 10% and SDS 1%) and deposited on a 1.5% Agarose gel containing of BET 0.5 ~, g / ml. Migration is carried out in an electrophoresis tank containing 40 mM Tris-base buffer, 20 mM glacial acetic acid, 1 mM EDTA, for 15 minutes at 100V. After migration, DNA is visualized under UV.
Results: The yeast strains were identified by PCR both in Release the small intestine up to 6 h after their introduction into the stomach and colon exit up to 48 hours after their introduction into it (Figure 6).
Example 5: Monitoring of W (R) pP73A1 Recombinant Yeasts in Systems artificial digestives Preparation of yeasts The yeasts are precultured for 12 hours at 28 ° C., with stirring, in 30 ml SGI
(glucose 20 g / I, yeast nitrogen base 7g / I, bactocasamino acids 1 g / I and tryptophan 20 mg / 1). Then, a culture of 48 h at 28 ° C, with stirring, is carried out in 250 ml YPGE (glucose 20 g / 1, yeast extract 10 g / I, bactopeptone 10 g / I and ethanol 3%).
During the last 12 hours, induction of CYP73A1 is caused by adding galactose (25 ml of a 200 g / l solution) until reaching a density of 3 at 4 x 10 ' yeasts / ml.
The culture is then centrifuged in autoclaved plastic tubes at 3000 g, 10 min, 4 ° C. The supernatant is discarded and the cells are washed in 15 ml of water physiological (NaCI 9 g / I).

After another centrifugation (3000 g, 10 min, 4 ° C), the yeasts are introduced in TIM 1 resuspended in 300 ml of YPL culture medium (galactose 20 g / I, yeast extract 10 g / 1, bactopeptone 10 g / 1) and are introduced into the resuspended in 10 ml of YPL medium.
Enumeration of yeasts - Sampling in TIM I: 1 ml of (starting food (containing the yeasts) is taken before introduction into the stomach (t ~ 0), then 1 ml is taken from different levels of the digestive tract: in the stomach after 15, 30, 45, 60 and 90 min of digestion, in the duodenum after 15, 30, 45, 60, 75, 90, 120 and 150 mins digestion, in the jejunum after 30, 45, 60, 90, 120, 150, 180 and 240 min from digestion, in the ileum after 30, 45, 60, 90, 120, 150, 180 and 240 min of digestion and in cumulative outings of 0-30 min, 30-45 min, 45-60 min, 60-90 min, 90-120 min, 120-180 min and I80-240 min at 30, 45, 60, 90, 120, 180 and 240 min.
- Sampling in TIM 2: 1 ml of colonic content is taken before introduction of yeast into the colon (t = Ofec) as well as I ml of the suspension of yeast before introduction into the colon (t = 0), then 1 ml of colonic content East taken every hour for 12 hours, then 24 hours after introduction of the yeasts, - Yeast culture: the samples are diluted in water physiological and spread on SGI medium. (Ampicillin (100 ~, g / mI) is added in Ie middle so eliminate contaminants from the digestive system and / or flora fecal.
- Enumeration: the enumeration of the boxes is carried out after 2 to 3 days incubation at 30 ° C.
Results: The experiments carried out made it possible to demonstrate the viability in the artificial digestive systems of yeasts W (R) pP73Al.
These yeasts show great resistance to gastric secretions and intestinal since no significant difference is observed in each compartment digestive of TIM1 between the curve obtained for yeasts and that of a marker of nonabsorbable transit (Figure 7).
79% ~ 13 (n = 3) of the recombinant yeasts introduced into the stomach are found in exit from ileum after 4 h of digestion (Figure 8).
The survival rate of yeasts in the colon is lower since can't find, after 12 h of fermentation, only 1% of the yeasts introduced (n = 3) (Figure 9).
Two hypotheses have been put forward calling into question the competition exerted by the flora fecal and near anaerobic conditions prevailing in the colon artificial.
Example 6: Measurement of the ethoxyroresorufin-O-deethylase (EROD) activity of the yeast W (hR) pHlA1 after passage through TIM 1 The W (hR) plilAl strain is used to demonstrate the maintenance of activity of Recombinant yeasts after passage through TIM1.
P450 lAl has the ethoxyrësorufine-O-deethylase (EROD) activity and catalyzes it transformation of 7-ethoxyroresorufin into resorufin.
Yeast culture The yeasts are precultured for 12 hours at 28 ° C., with stirring, in 30 ml SGI
(glucose 20 g / 1, yeast nitrogen base 7g / 1, bactocasamino acids 1 g / 1 and tryptophan 20 mg / 1). Then, a culture of 48 h at 28 ° C, with stirring, is carried out in 250 ml YPGE (glucose 20 g / 1, yeast extract 10 g / 1, bactopeptone 10 g / I and ethanol 3%).
During the last 12 hours, the induction of P450 lAl is caused by adding galactose (25 ml of a 200 g / I solution). The yeast culture is then be used as it is for the assay of EROD on cells, or undergoes different treatments so to prepare microsomes (Pompon et al., 1996).
P450 lAl content The content of P450 lAl was measured on microsomes of the strain W (hR) pHlAl after passage through the digestive systems.
For this, we use the method of Omura and Sato (I964), based on the property that the hemoprotein to have a maximum absorption around 450 nm when it is reduced and linked to carbon monoxide. The suspension of microsomes East taken up in 50 mM Tris-HCl buffer, 1 mM EDTA, pH 7.4 in order to obtain a D06oonm of about 0.8, then reduced by a tip of a dithionite spatula sodium.
The absorption spectrum is recorded before and after bubbling CO in the suspension. P450 IA1 concentration (only detectable P450) is calculated according to Beer-Lambert's law (x448 nm = 91 mM-ICrri 1).
5 The microsomal proteins are dosed with the Protein Assay ESL kit (Boehringer Mannheim).
Determination of EROD activity The enzymatic activity of ethoxyroresorfin-O-deethylase (EROD) is characteristic of 10 cytochrome P450 1A1. It is measured on the W (hR) pHlAl strain, after passage in artificial digestive systems. This activity is dosed according to Ia method fluorimetric described by Burke et al. (1985) which measures the increase in fluorescence linked to the formation of resorufin, a dalkylation product of the ethoxyresorufin.
The EROD activity is measured on microsomes or on cells at 24 ° C.
In the first case, I m1 of 50 mM Tris-HCI buffer, 1 mM EDTA, pH 7.4, 5 ~, l of NADPH (10 mg / ml) and 2 ~ .1 of a solution of 7-ethoxyroresorufin saturated in ethanol are mixed. After adding the microsomes, the increase in fluorescence (~, ex = 530 nm; ~, em = 586 nm) is saved. In the second case, after centrifugation (11000 g, 20 30 s, 4 ° C) from 1 ml of culture to a D06oonm of 3 minimum, the pellet cell is resumed in 1 ml of 50 mM Tris-HCl buffer, I mM EDTA, pH 7.4. We record (increase in fluorescence (~, ex = 530 nm; 7 ~ em = 586 nm) consecutive to (addition of 2 ~ .1 of a solution of 7-ethoxyroresorfine saturated in ethanol.
25 Results: The W (hR) pHlA1 strain always has EROD activity, feature P450 1A1, at the outlet of (small intestine and in the colon, whether it is dosed on microsomes (approximately 20 pmol of resorufin per ml of microsomal suspension and through minute) or on cells (approximately 5 pmol of resonxfine per ml of culture and per minute).
Example 7: Monitoring of the Cinnamate-4-Hydroxylase Activity of the Yeasts W (R) pP73A1 in the various compartments of TIM 1 and in TIM 2 The yeast strain used to demonstrate activity in situ is the strain W (R) pP73Al.
P450 73A1 has cinnamate 4-hydroxylase activity and catalyzes the transformation from tr ahs-cinnamic acid to p-coumaric acid.
Preparation of yeasts The yeasts are precultured for 12 hours at 28 ° C., with stirring, in 30 ml SGI
(glucose 20 g / 1, yeast nitrogen base 7g / 1, bactocasamino acids I g / 1 and tryptophan 20 mg / 1). Then, a culture of 48 h at 28 ° C, with stirring, is carried out in 250 ml YPGE (glucose 20 g / 1, yeast extract 10 g / 1, bactopeptone 10 g / 1 and ethanol 3%).
During the last 12 hours, induction of CYP73A1 is caused by adding galactose (25 ml of a 200 g / l solution) until reaching a density of 3 at 4 x 10 ' yeasts / ml.
The culture is then centrifuged in autoclaved plastic tubes at 3000 g, 10 min, 4 ° C. The supernatant is discarded and the cells are washed in 15 mI
of water physiological (NaCI 9 g / 1).
After centrifugation (3000 g, 10 min, 4 ° C), the supernatant is discarded and the cell pellet is resuspended in the food (300 ml of YPL medium for monitoring the bioconversion reaction in TIM 1 and 10 ml of YPL medium for monitoring of the reaction in TIM 2).
Tim 1 digestion process The substrate (tracs-cinnamic acid) is introduced immediately into stomach to the concentration of 670 ~ uM (concentration in the food). Digestion is performed on 240 minutes.
Samples (1 ml) are taken at different levels of the digestive tract : in (food (t0), in the stomach after 15, 30, 45, 60 and 90 min of digestion, in the duodenum after 15, 30, 45, 60, 75, 90, 120 and 150 min of digestion, in the jejunum after 30, 45, 60, 90, 120, 150, I80 and 240 min of digestion, in the ileum after 30, 45, 60, 90, 120, 180 and 240 min of digestion and in the ileal outings accumulated over 0-30 min, 30-45 min, 45-60 min, 60-90 min, 90-120 min, 120-180 min and 180-240 min at 30, 45, 60, 90, 120, 180 and 240 min.
TIM 2 fermentation process The substrate (trans-cinnamic acid) is introduced into the colon at the concentration of 1.7 mM (initial concentration in the colon). Samples (1 ml) are made at t0 then 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300 and 360 min after introduction simultaneous yeast and substrate in the colon. The samples are diluted to I / 2 before being processed as described below.
Sample processing 100 μl of trifluoroacetic acid (25% in water w / v) are added to 1 ml sample to stop the enzyme reaction. After microcentrifugation (3 min, 10,000 rpm), the supernatant is collected and filtered (0.45 μm). Samples are kept at -20 ° C
before analysis.
HPLC analysis After filtration, 10 p1 of the samples are injected into the Merck column Lichrospher 100 RP-18 (5 p, m, 12.5 cm * 4 mm). A gradient between 2 phases A and B
East performed over 34 min with a flow rate of 1 ml per minute, as described below Phase A: Water 95, Methanol 5 and acetic acid 0.1 (v / v), Phase B: Acetonitrile 95, Methanol 5 and acetic acid 0.1 (v / v), 0-16 min: 90% A, 10% B, 16-18 min: linear gradient up to 80% A, 20% B, 18-32 min: 80% A, 20% B, 32-34 min: linear gradient up to 90% A, 10% B.
Detection is carried out by spectrophotometry at 314 nm between 0 and 16 min, then at 280 nm between 16 to 34 min.
Compound retention time is 6 ~ 0.5 min for p-coumaric acid and of 22 ~ 0.5 min for tans-cinnamic acid.

Results Cinnamate 4-hydroxylase enzyme activity was detected in all digestive compartments of TIM 1 (stomach, duodenum, jejunum and ileum). Of control digestions ensured the stability of the substrate and the produced in TIM 1, as well as the specificity of the bioconversion reaction.
Figure 10A shows a summary of the acid bioconversion reaction trans-cinnamic to p-coumaric acid (n = 3), over time, in (set of TIM 1 compartments. Thus 41% ~ 6 (n = 3) of trans-cinnamic acid introduced in the stomach is converted to p-coumaric acid in TIM 1 under the action of W (R) yeast pP73Al.
Figure lOB represents p-coumaric acid produced in the different compartments of TIM 1 over time (n = 3), in% of (acid t ~~ years-cinnamic introduced into the stomach at the start of digestion. It is in the duodenum that see you large amount of p-coumaric acid is produced by yeasts.
Enzyme activity, although very low, has also been detected in the colon artificial (results not shown). Witnesses ensured that the specificity of the reaction. The low survival rate of yeast in the colon, as well as existence secondary metabolic pathways (metabolism of (trans-cinnamic acid and of (p-coumaric acid by colon bacteria) could explain that the activity cinnamate 4-hydroxylase is very weak in the colon.
EXAMPLE 8 Genetic Constructions of Yeast Strains Secreting a peptide "tagged" model (strain WppV5H6) and a "tagged" model protein (strain WppGSTV5H6) Choice of secretion signal A signal sequence originating from peptide a, peptide naturally secreted by the yeast of the sexual type a, was chosen as a secretion signal. The sequence signal of peptide a is divided into three domains, located directly upstream of the cc peptide and necessary for its maturation and its secretion 1- the sequence of addressing the peptide to the endoplasmic reticulum:
sequence Pre, 2- a sequence whose glycosylation allows efficient secretion of the peptide Pro sequence, S 3- the cut-off region of the proteases Kex2, Stel3 and Kexl.
A signal sequence consisting of the Pre and Pro sequences and the sequence coding for the two amino acids lysine-arginine (KR), the Kex2 cleavage site, was detention (noted PreProKR). Indeed, in this case, the function of the protein Stel3 is not required (activity becoming limiting in the event of protein overexpression heterologists) and the protease Kex2 is sufficient to obtain a mature peptide.
Construction of the plasmids ppV5H6 and ppGST VSH6 The bacteria / yeast shuttle vector pYES2-CT (commercial plasmid, Invitrogen) at served as the basis for the construction of the plasmids ppV5H6 and ppGSTV5H6 (Figure 11A
and 11B). pYES2-CT carries an expression cassette comprising the strong promoter (inducible by galactose) and the terminator of the CYCl gene. This vector presented by elsewhere, under the control of GAL 1, a sequence coding for a so-called epitoge VS and for a polypeptide chain of 6 histidines. These two sequences form a tag (multi tag VSH6) which can be recognized through its epitoge VS (recognized by of specific antibodies), in particular during a Western Blot analysis.
Construction of the plasmid ppV5H6 The plasmid ppV5H6 was constructed by insertion of the sequence coding for the PreProKR upstream of the V5 epitope coding sequence. To do this, sites of BamHI and EcoRI restriction were added, by PCR amplification, respectively at the S 'and 3' ends of the sequence coding for PreProKR. The PCR product at then was subcloned into the vector pYes2 / CT, opened by double digestion BamHl-EcoRl.
Construction of the plasmid ppGSTV5H6 The construction was carried out from the plasmid ppV5H6 in which the sequence of the coding phase of GST was inserted, upstream of the VSH6 sequence. This insertion was carried out by homologous recombination directly in the host yeast.

For this, homologous sequences were added, by PCR amplification, to the ends of the coding phase of GST (GST from S'chistosoma japonicum).
Obtaining the yeast strains WppV5H6 and WppGSTV5H6 Plasmids ppV5H6 and ppGSTV5H6 were introduced by transformation into the yeast strain W303 of sexual sign MAT a (leu 2, his 3, trp 1, ura 3 and ade 2), giving respectively the strains WppV5H6 and WppGSTV5H6.
The "tagged" model peptide (denoted peptide-VSH6) secreted by the strain WppV5H6 at a size of 42 amino acids (calculated PM: 5.7 kDa).
The "tagged" model protein (noted GST-VSH6) secreted by the strain WppGSTV5H6 at a size of 29.7 kDa (calculated PM).
Example 9. Monitoring of the secretion products of WppV5H6 yeasts and WppGSTV5H6 in Erlen-Meyer Preparation of yeasts The WppV5H6 or WppGSTV5H6 strains are precultured (24h, 30 ° C, 210 rpm) in minimum SRAI medium (bactocasamino acids 1g / 1, yeast nitrogen based 7g / 1, adenine 2 mg / ml and raffinose 20 g / 1), until it is in phase stationary. Then, a growth culture (12 h, 37 ° C, 210 rpm) is carried out in full middle YPRA (yeast extract 10 g / 1, bactopeptone 10 g / 1, adenine 2 mg / ml and raffinose 20 g / 1), until being in stationary phase. The culture is centrifuged (2000 g, 6 min, room temperature), the supernatant is removed, then the induction medium YPLRA
(yeast extract 10 g / 1, bactopeptone 10 g / 1, adenine 2 mg / ml, galactose 20 g / 1 and raffinose 10 g / 1) is seeded either at high cell density (D06oonm of 4), either at low cell density (DO6oonm 0.6). The induction culture is performed 6 h, at 37 ° C, with stirring (210 rpm).
Samples are taken from the growth culture and from the culture induction at t0, t2h, t4h and t6h, under the two concentration conditions cellular.

Sample processing Protein precipitation After centrifugation (3000 g, 10 min, 4 ° C) to remove the cells, the proteins contained in the supernatant are precipitated by adding trichloro acetic acid (TCA) (final concentration 10%). The samples are incubated at 4 ° C for 1h then centrifuged (10,000 g, 10 min, 4 ° C). The protein pellets are then rinsed 2 times with 100% acetone. After drying, the pellets are taken up in buffer of charge and heated for 5 min at 95 ° C.
Protein electrophoresis For each sample, the proteins of the corresponding supernatant are deposited.
has a culture volume containing 5 * 10 ~ yeasts. Proteins are separated by 12% polyacrylamide gel electrophoresis (GST-VSH6) or 16.5% (peptide-VSH6), in denaturing conditions (addition of SDS). A migration buffer containing the tricin, more suitable for the separation of low molecular weight proteins, has been used in the case of the peptide-VSH6 (Shâgger H. and Von Jagow G., 197). The settings of the migration are as follows: 30 mA, 150 V, 1 hour (GST-VSH6) or 3 hours (peptide-VSH6).
Western-Blot After electrophoresis, the proteins are transferred to the membrane polyvinyldifluorene (PVDF). The transfer parameters are as follows: 350 mA, 100 V, 1 h (GST
VSH6) or 20 min (peptide-VSH6).
The peptide-VSH6 and the GST-VSH6 are revealed after successive incubations of the membranes with specific anti-VS antibody and a secondary antibody coupled with alkaline phosphatase (Western-Breeze Kit, Invitrogen, WB7103). The addition of substratum (BCIP / NBT) causes the formation of a purple precipitate.
Results FIG. 12 represents the Western-Blots obtained with the strain WppV5H6 (12A) and with the strain WppGSTV5H6 (12B).
In both cases, the basal level of secretion obtained on raffinose is too Weak To be visible on Western-Blot. The secretion products are detectable as soon as 2h after induction of the GAL1 promoter pax galactose. In view of the Western Blots, the secretion seems more important when the induction culture is carried out at low density cell, either for peptide-V5H6 or for GST-VSH6. In both cases, the secretion products are also detectable as precursors (with the Pre-Pro-KR sequence) and in glycosylated or hyperglycosylated form.
Example 10. Monitoring of the secretion products of WppV5H6 yeasts and WppGSTV5H6 in the various compartments of TIM 1 Preparation of yeasts The WppV5H6 or WppGSTV5H6 strains are precultured (24h, 30 ° C, 210 rpm) in minimum SRAI medium (bactocasamino acids 1 g / 1, yeast nitrogen based 7g / 1, adenine 2 mg / ml and raffinose 20 g / 1), until it is in phase stationary. Then, a growth culture (12 h, 37 ° C, 210 rpm) is carried out in full middle YPRA (yeast extract 10 g / 1, bactopeptone 10 g / 1, adenine 2 ml and raffinose 20 g / 1), until being in stationary phase. The culture is centrifuged (3000 g, 10 min, room temperature) and the cell pellet is resuspended in 300 ml of YPLRA induction medium (yeast extract 10 g / 1, bactopeptone 10 g / 1, adenine 2 mg / ml, galactose 40 g / 1 and raffinose 20 g / 1), thus constituting (food which will be introduced in the TIM 1.
Tim 1 digestion process Digestion is carried out according to a conventional digestion protocol (cf.
example 3) but without digestive proteases (to avoid degradation of the products of secretion).
Samples are taken from different levels of the digestive tract: in the food before introduction into the system (t0), in the stomach after 90 and 120 min from digestion, in the duodenum after 90, 160 and 210 min of digestion, in the jejunum after 90, 160 and 210 min of digestion and in the ilëon after 90, 160, 210 and 270 min of digestion.
Counts are carried out in the food and in the outlets accumulated on 0-90, 90-180 and 180-270 min, at 90, 180 and 270 min, by enumeration on medium Sabouraud containing (ampicillin (100 ~, g / ml).

A positive Erlen-Meyer control is performed in parallel for both strains.
Sample processing Protein precipitation The precipitation protocol is that described in Example 9. The samples from of the stomach are neutralized beforehand with sodium bicarbonate 1 M.
Protein electrophoresis The electrophoresis protocol is that described in Example 9. The whole proteins contained in the supernatant of the samples is deposited (corresponding to a volume of 2 ml in TIM 1, except for the stomach and duodenum where the volume of samples is 4 ml, and 600 ~ l in the Erlen Meyer).
Western-Blot The Western-Blot protocol is that described in Example 9. The antibody anti-GST a been used in place of (anti-VS antibody in order to achieve a semi quantification of GST-VSH6 secreted.
GST-VSH6 semi-quantification In order to approximate the amount of GST secreted in the digestive environment we compared the intensity of the signals of detection of a standard range with that of samples from TIM 1. 20 ng, 10 ng and 1 ng of GST
85% pure were deposited on the same gel as the sample proteins from TIM 1 samples. After transfer to PVDF membrane, the presence of GST
has been demonstrated with a primary anti-GST antibody and the detection WesternBreeze (Invitrogen, WB7103).
Results Figures 13 and 14 represent the Western-Blots obtained during the digestion in the TIM 1 in the presence of the WppV5H6 and WppGSTV5H6 strains respectively. The VSH6 peptide has been identified in all TIM 1 compartments from 90 minutes after the start of digestion. The amount of peptides detected increases at during digestion and along the digestive tract (from (stomach to ileum) for a time given digestion. GST-VSH6 has been detected in all compartments except the stomach. The amount of protein secreted also increases during the digestion.
FIG. 15 represents a serification of the GST-VSH6 produced during of a digestion in TIM 1 in the presence of the strain WppGSTV5H6. Concentration of GST-VSH6 in digestive samples can be assessed at - <1 ng / ml for duo, jej and ile 90 min samples, - 10 10 ng / ml for the jej and ile samples at time 160 and 210 min.

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Claims (22)

1. Microorganisms containing at least one fragment of heterologous DNA
comprising a gene of interest capable of being expressed in the environment digestive, characterized in that its tracking rate is greater than 25%, 50%, 75%, 80%, of preferably 90% or 95% leaving the ileum after 6 hours of digestion and is superior at 2%, 4%, preferably 8% after 12 hours of fermentation in the colon. 2. Microorganisms according to claim 1 characterized in that the fragment of heterologous DNA consists of an expression cassette comprising at less a gene of interest, a promoter and a terminator. 3. Microorganisms according to one of the preceding claims, characterized in this that they are yeasts selected in particular from Saccharomyces cerevisiae, S
boulardii, S uvarum, Hansenula anomala, Schizosaccharomyces pombe, Kluyveromyces lattis and Yarrowia lipolitica. 4. Microorganisms according to one of the preceding claims, characterized in this that the gene of interest encodes a product selected from the peptides to interest vaccine, peptides with hormonal interest, peptides with therapy, the peptides with an antibiotic effect, peptides capable of trapping or complexing of the molecules, enzymes or any peptide possessing activities of bioconversion, including detoxification enzymes or secreted into the environment digestive. 5. Microorganisms according to one of the preceding claims, characterized in this that the gene of interest encodes a xenobiotic metabolism enzyme selected from phase I enzymes (P450 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 73A1), the environmental enzymes of phase I (cytochrome P450 reductase and cytochrome b5) and phase enzymes II
such as glutathione S-transferase (GST), N-acetyl transferase (NAT), the quinone reductase (QR), aldehyde dehydrogenase (ALDH) and uridine diphosphoglucuronosyl transferase (UGT). 6. Microorganisms according to one of the preceding claims, characterized in this that the gene of interest is placed under the control of a promoter chosen from them constitutive or inducible promoters, strong promoters, promoters assets specifically in a segment of the digestive tract under aerobic conditions, semi-anaerobic or anaerobic, and promoters regulatable by a molecule introduced into the intestinal environment. 7. Microorganisms according to one of the preceding claims, characterized in this that they are capable of carrying out a bioconversion reaction or of secreting a peptide or protein of interest in all digestive compartments. 8. Process for the study, control and/or selection of microorganisms according to moon of claims 1 to 7 characterized in that it implements a system digestive artificial. 9. Study and/or control method according to claim 8 comprising the steps consists in:
a) adding a microorganism according to one of claims 1 to 7 in a environment liquid nutrient, b) introducing said medium at the inlet of an artificial digestive system including at least one of the compartments, c) incubate and enumerate and/or test for gene activity of interest after passage through the digestive systems or in situ, the test of activity which may include a secretion and/or bioconversion test. 10. Selection method according to claim 7 comprising the steps consists in:
a) adding microorganisms according to one of claims 1 to 7 in a environment liquid nutrient, b) introducing said medium at the inlet of an artificial digestive system including at least one of the compartments, c) incubating and recovering the surviving microorganisms after passing through said system ;

d) optionally repeating steps a) to c) until a population homogeneous micro-organisms adapted to the digestive environment and capable to effectively express the gene of interest. 11. Microorganism obtained from the method according to one of the claims 8 to 10. 12. Microorganism according to claim 11 characterized in that its rate survival is greater than 25%, 50%, 75%%, 80%, preferably 90% or 95% at the output of ileum after 6 hours of digestion and is greater than 2%, 4%, preferably 8%
after 12 hours of fermentation in the colon. 13. Microorganism according to one of claims 11 and 12 characterized in that that he it is a yeast. 14. Combination product comprising a microorganism according to one of the claims 1 to 7 and 11 to 13 and an active substance or a precursor of a active substance for simultaneous, separate or spread use in the time. 15. Product according to claim 14 characterized in that the microorganism acts on the active substance or vice versa. 16. Pharmaceutical composition comprising microorganisms according to one of claims 1 to 7 and 11 to 13 being in a pharmaceutical form suitable for a administration orally or rectally so as to transport them intact to the level of the target site of the digestive tract and to maintain their integrity at this level for a given time. 17. A pharmaceutical composition according to claim 16 wherein the micro-organisms, in particular yeasts, are protected by a coating and/or a membrane of the resin type and/or of the polymer type forming spheres, microspheres, granules or microgranules. 18. Pharmaceutical composition according to claim 17, characterized in that what is in the form of capsules, tablets, dragees or suspension cellular. 19. Use of microorganisms according to one of claims 1 to 7 and 11 at 13 for the preparation of a medicament. 20. Use according to claim 19 for the preparation of agents of detoxification, metabolic correcting agents or agents capable of modifying the flora intestinal. 21. Use according to claim 19 for the preparation of a medicament intended for decrease the toxicity and/or increase the bioavailability of a substance active or of its precursor. 22. Use according to claim 19 for the preparation of a medicament able to provide in situ active peptides, peptides of vaccine interest, peptides to hormonal interest, peptides with therapeutic interest, peptides with antibiotic or peptides capable of trapping or complexing molecules.