ARTICLE IN PRESS

Metabolic Engineering 11 (2009) 148–154

Contents lists available at ScienceDirect

Metabolic Engineering

1096-71

doi:10.1

� Corr

Ingenie

Faculte

Clermon

E-m

journal homepage: www.elsevier.com/locate/ymben

Genetically engineered yeasts as a new delivery vehicle of active compoundsto the digestive tract: In vivo validation of the concept in the rat

G. Garrait a,�, J.F. Jarrige a, S. Blanquet-Diot a, M. Alric a

aUniversite Clermont1, UFR Pharmacie, Centre de Recherche en Nutrition Humaine (CRNH), Equipe de Recherche Technologique ‘‘Conception, Ingenierie et

Developpement de l’Aliment et du Medicament’’ (ERT CIDAM), Clermont-Ferrand F-63001, France

a r t i c l e i n f o

Article history:

Received 1 August 2008

Received in revised form

17 December 2008

Accepted 15 January 2009Available online 21 January 2009

Keywords:

Biodrug

Recombinant S. cerevisiae

Digestive environment

Rat

Secretion

76/$ - see front matter Crown Copyright & 20

016/j.ymben.2009.01.001

esponding author at: Equipe de Recherche

rie et Developpement de l’Aliment et du Medi

de Pharmacie, Universite d’Auvergne, 28,

t-Ferrand Cedex, France. Fax: +33 4 73 17 83 9

ail address: ghislain.garrait@u-clermont1.fr (G

a b s t r a c t

An innovative ‘‘biodrug’’ concept based on oral administration of living recombinant microorganisms as

a vehicle to deliver active compounds directly into the digestive tract has recently been developed. To

validate this concept, we studied a recombinant Saccharomyces cerevisiae strain in order to investigate

its viability and its ability to produce a protein (glutathione-S-transferase (GST)-V5H6) in the rat.

Following oral administration, the recombinant yeast showed a survival rate of around 40% in the upper

parts of the digestive tract, but was more sensitive to the conditions in the large intestinal, where

viability dropped to 1%. Western blot analysis was able to detect the model protein throughout the

digestive tract, including stomach, duodenum, jejunum (proximal, median and distal), ileum, cecum and

colon. The gastrointestinal sac technique was employed to quantify GST-V5H6 in all the digestive

compartments. These results suggest that S. cerevisiae may represent a useful host for producing

compounds of interest directly in the digestive tract.

Crown Copyright & 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction

An innovative ‘‘biodrug’’ concept based on oral administrationof living recombinant microorganisms as a vehicle to deliverpharmacologically active compounds directly into the digestivetract has recently been developed (Alric et al., 2000; Blanquetet al., 2001). This new kind of vector offers several advantagesover classical dosage forms, making it possible to administerdrugs sensitive to digestive conditions, target specific sitesthroughout the digestive tract and/or control drug release throughthe regulation of gene expression. It has been suggested that acid-or protease-mediated degradation of the active compound has tobe avoided upstream from its absorption or reaction sites, andthat similar therapeutic effects could potentially be obtained atlower doses (Steidler et al., 2000). Genetically modified micro-organisms can be exploited to carry out bioconversion reactions(e.g. in situ biodetoxification) or produce compounds of interest(such as hormones, enzymes, interleukins or antigens) directlyinside the digestive tract (Braat et al., 2006; Chen et al., 2000;Corthesy et al., 2005; Drouault et al., 2002; Prakash and Chang,2000a).

09 Published by Elsevier Inc. All r

Technologique ‘‘Conception,

cament’’ (ERT CIDAM), CBRV,

Place Henri-Dunant, 63001

2.

. Garrait).

Even if lactic acid bacteria have been as the main candidatehosts for this new ‘‘biodrug’’ concept (Steidler et al., 2003),yeasts may prove advantageous (Blanquet et al., 2001), especiallywhen an eukaryotic environment is required for the functionalexpression of heterologous genes. Moreover, this would makeit possible to ensure the absence of bacterial sequencesliable to promote gene transfer to host bacteria by deployingthe efficient site-targeted homologous recombination machineryof the yeast to introduce the heterologous gene into theyeast genome. Yeasts offer the added advantage of not beingsensitive to antibacterial agents, thus allowing concomitantadministration antibiotics with recombinant microorganisms.We opted to use common baker’s yeast Saccharomyces cerevisiae

(S. cerevisiae) as host, due to its ‘‘generally recognized as safe’’GRAS status, easy culture, and high level of resistance to gastricand small intestine secretions (Blanquet et al., 2003; Blanquetet al., 2004).

The scientific feasibility of the ‘‘biodrug’’ concept has beendemonstrated in vitro using a gastric and small intestine model(TIM) and recombinant S. cerevisiae expressing either thecytochrome P450 73A1 model (WRP45073A1 strain; Blanquetet al., 2003) or the model of glutathione-S-transferase (GST) infusion with V5 epitope (V5) and a polyhistidine (H6) tag(WppGSTV5H6 strain; Blanquet et al., 2004). Garrait et al. (2007)recently demonstrated in vivo that the recombinant yeastWRP45073A1 survived gastric and intestinal conditions and wasable to carry out a bioconversion reaction directly in the ratdigestive tract. The next step in biodrug development is therefore

ights reserved.

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to determine whether recombinant yeast is able to initiatesynthesis and secrete heterologous protein in vivo.

For this purpose, we evaluated the viability of the recombinantyeast strain WppGSTV5H6 and its ability to produce the modelprotein GST-V5H6 in the rat. All the experiments were conductedin the digestive tract organs as well as in ex vivo gastrointestinalsacs.

2. Materials and methods

2.1. Chemicals

Sodium chloride, sodium phosphate dodecahydrate, potassiumphosphate monobasic, gelatin, trisaminomethane base, raffinoseand galactose were purchased from Acros Organics (Morris Plain,NJ). Krebs Henseleit modified buffer, Tween 20, glycine, acryla-mide/bis-acrylamide, bromochloroindolyl phosphate (BCIP)/nitroblue tetrazolium (NBT), adenine and tryptophan were supplied bySigma Chemical Co. (St Louis, MO). Sodium dodecyl sulfate (SDS)was provided by Fluka Chemical Corp. (Ronkonkoma, NY). All theproducts for yeast culture media were sourced from Difco (Le Pontde Claix, France).

2.2. Yeast strains

The S. cerevisiae strain was derived from the haploidstrain W303-1B (MATa; ade2-1; his3-11,-15; leu2-3,-112; ura3-1;CanR; cyr+). The strain was genetically engineered as previ-ously described by Blanquet et al. (2004). The recombinantyeast strain WppGSTV5H6 expresses the model protein GSTfrom Schistosoma japonicum in fusion with the V5 epitope and aH6 tag. The synthesis of GST-V5H6 (MW: 31.5 kDa, 246amino acids) was induced by adding galactose to the culturemedium. Control experiments used another yeast strain namedWppV5H6, which is deprived of the sequence encoding S.

japonicum GST.

2.3. Yeast culture conditions

The S. cerevisiae strain was cultured as described in Blanquet etal. (2004). Briefly, the recombinant yeasts were precultured tostationary growth phase at 28 1C in 30 mL SRAI broth. Preculturewas then carried out in 150 mL YPRA, and cells were grown in ashaking incubator (28 1C, 220 rpm, 12 h) until cell density reached108 cells/mL. The cellular suspension (10 mL) was then harvested(4 1C, 3 min, 5000g) and yeast cells were resuspended in 2 mL ofPBS (0.1 mol/L, pH 7.4) containing 20 g/L of galactose to induceheterologous gene expression. The cell suspension was orallyadministered to rats or introduced on the mucosal side of thegastrointestinal sacs.

2.4. Animals

The experiment used adult male Wistar rats (Elevage Depre, St-Doulchard, France) weighing 300720 g. The rats were housed foran acclimatization period with free access to food (A04, lot 50803,UAR, Epinay-sur-Orge, France) and tap water. Before the experi-ments, they were individually housed in metabolic cages for 3days, and food was withheld on the fourth day. The animals weremaintained at constant room temperature (2272 1C) and exposedto natural light. All the animal care and handling procedures wereapproved by the Institutional Authority for Laboratory AnimalCare.

2.5. Sacrificed rat experiments

Rats (n ¼ 36) were fasted for 24 h before being administered asingle gavage dose of 109 cells (WppGSTV5H6 or WppV5H6)suspended in 2 ml of PBS containing 20 g/L galactose. For eachyeast strain, three rats were decapitated at 0.5, 2, 4, 8 and 24 hafter the oral administration. In addition, three rats weresacrificed immediately after oral administration of 2 mL of PBS(0.1 M, pH 7.4) containing 20 g/L of galactose to determine thenumber of endogenous living yeasts (control experiments).Different sections of the digestive tract, i.e. the stomach,duodenum (between the pylorus and the ligament of Treitz),jejunum (divided in three equal-length parts: proximal, medianand distal), ileum, cecum and colon were all promptly removed.The mucosal fluid of each organ was collected on ice by gentlyscraping the luminal surface with a glass slide, and then diluted in5 mL of PBS/g of organ content. Aliquots of 100mL were thenplated immediately onto SGAI solid medium (see yeast countbelow) to evaluate yeast survival rates. All samples were stored at�80 1C until Western blotting analysis and enzyme-linkedimmunosorbent assay (ELISA).

2.6. Ex vivo experiments

After a 24-h fasting period, rats (n ¼ 15) were anesthetizedbefore sacrifice, and the different parts of the digestive tract werequickly removed as previously described (Garrait et al., 2006).Two milliliters of PBS with 20 g/L of galactose containing 109

recombinant yeasts (WppGSTV5H6 or WppV5H6, n ¼ 5 for eachyeast strain) or not (control group, n ¼ 5) were introduced insidethe mucosal side using an 18-gauge catheter (BD, Rutherford, NJ,USA). Gastrointestinal sacs were immediately transferred to atissue chamber containing 10 mL of warmed (37 1C), oxygenated(95% O2/5% CO2) Krebs Henseleit modified buffer, as previouslydescribed by Garrait et al. (2007). The chamber was closed with arubber stopper to prevent evaporation. Gastrointestinal prepara-tions were incubated for 180 min, and the 18-gauge catheter wasused to collect 300-mL samples from the mucosal side at 0, 60, 90,120 and 180 min. At the end of the experiment, the gastrointest-inal sacs were dried and weighed, and the mucosal fluid wascollected onto ice. The survival rate of the recombinant S.

cerevisiae was immediately evaluated by plating the samples ontoSGAI solid medium (see the yeast counts section below). Themucosal fluids collected were stored at �80 1C until Westernblotting analysis and ELISA.

2.7. Yeast counts

The mucosal fluid samples (from sacrificed rats and ex vivo

experiments) were diluted in 0.9% NaCl and plated onto SGAI solidmedium (7 g/L of yeast nitrogen base without amino acids, 1 g/L ofbacto casamino acids, 20 mg/L of tryptophan, 20 mg/L of adenine,20 g/L of glucose and 20 g/L of agar) supplemented with ampicillin(100mg/mL). The results were expressed as either number of cellsor percentage of the initial number of cells.

2.8. Western blot analysis

GST-V5H6 synthesis was examined by Western blotting inmucosal samples (sacrificed rats and gastrointestinal sacs). All thesamples were centrifuged (4 1C, 3 min, 5000g) to remove yeastcells. The supernatant containing the proteins was then boiled inLaemmli buffer for 5 min and subjected to sodium dodecylsulfate–12% polyacrylamide gel electrophoresis (PAGE). SDS-PAGEwas performed at 30 mA for 45 min. Proteins resolved by

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SDS-PAGE were then transferred onto a polyvinylidene fluoridemembrane (Bio-Rad Laboratories, Marnes-la-Coquette, France)with a Bio-Rad electroblotter (100 V, 350 mA, 35 min). Non-specific sites were blocked using 5% (w/v) nonfat dry milk inPBS containing 0.1% Tween-20 (v/v) under gentle shaking (1 h,room temperature). After rinsing, 2 mL of alkaline phosphatase(AP)-conjugated anti-V5 mouse monoclonal antibodies (1:2000,R962-25, Invitrogen, Carlsbad, CA) were added, incubated undergentle shaking (2 h, room temperature), and washed. Themembranes were then incubated with 5 mL of BCIP/NBT substrate(B-3679) until the appearance of the blots.

2.9. ELISA

ELISA was used to quantify the amounts of GST-V5H6 producedin sacrificed rats and gastrointestinal sacs; 96-well microtiterplates (Immulon, Thermo Labsystems, Issy Les Moulineaux,France) were coated with 40mg/mL of anti-GST rabbit polyclonalantibody (1:250, G7781, Sigma) in PBS and incubated for 1 h at37 1C, then overnight at 4 1C. Non-specific sites were blocked with0.5% (w/v) gelatin in PBS for 1 h at room temperature. All thesamples were centrifuged (4 1C, 3 min, 5000g) to remove yeastcells, and 200mL of supernatant was added to the wells. Afterincubation for 2 h at room temperature, 20mg/mL of rabbitperoxidase-conjugated anti-GST antibody (1:500, A7340, Sigma)was added. The color reaction was developed with 0.4 mg/mL oforthophenylene diamine dihydrochloride in 0.05 M phosphate–citrate buffer containing 0.04 mg/mL of urea hydrogen peroxide.Absorbance was recorded on a microplate reader (Multiskan

Fig. 1. Survival rate of WppGSTV5H6 yeast strain orally administered to rats. Values are

cells with a logarithmic scale or (B) mean percentages7SEM (n ¼ 3) relative to the in

galactose.

Spectrum, Thermo Fisher Scientific, Waltham, MA) operating at awavelength of 450 nm. GST-V5H6 synthesis was quantified usingstandard curves plotted with commercial GST from S. japonicum

(G5663, Sigma).

2.10. Statistical analysis

Values are presented as means 7 SEM. Between-groupcomparisons were performed using the Student’s t-test. Allstatistical evaluations were processed using SAS system software(Software Version 8.1. SAS Institute Inc., Cary, NC). The level ofstatistical significance was set at Po0.05.

3. Results

3.1. Yeast survival rate

3.1.1. Sacrificed rat experiments

Following gavage with 0.1 M PBS (control experiment), no yeastwas detected in the various organs studied (Fig. 1A). After oraladministration of 109 WppGSTV5H6 cells, the yeasts colonized allthe digestive organs. At 30 min after gavage, yeast cells weredetected in each organ of the upper digestive tract (from stomachto ileum), where 4373% of total ingested yeasts were recoveredalive (Fig. 1B). Two hours after gavage, the large intestine (cecumand colon) was colonized by the recombinant cells, giving asurvival rate of 2672%. At the end of the experiment (24 h post-administration), WppGSTV5H6 yeast cells were found only in the

expressed as (A) mean counts7SEM (n ¼ 3 for each sacrifice timepoint) of viable

itial amount of yeasts introduced. Control rats received PBS containing 20 g/L of

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feces (around 1% of the yeast content initially administered).Similar distribution patterns and survival rates were recorded inrats given an oral administration of 109 WppV5H6 yeast cells (datanot shown).

3.1.2. Ex vivo experiments

At the end of the experiment (180 min), the survival rate ofWppGSTV5H6 cells ranged from 3676% in the ileum to 103715%in the stomach (Table 1). In each gastrointestinal sac, the survivalrate of WppV5H6 yeast cells was not significantly different(P40.05) from that observed with WppGSTV5H6 strain (Table 1).

3.2. Detection and quantification of GST-V5H6

3.2.1. Sacrificed rat experiments

Control experiments with PBS alone or with the WppV5H6

strain showed no signal detectable by Western blot analysis. Ratsgiven an oral administration of WppGSTV5H6 yeast cells revealeddifferent forms of GST-V5H6 in all the various digestive organs(Fig. 2). The expected protein (calculated MW: 31.5 kDa) appearedas a doublet around 31 kDa (Fig. 2, a signal). The recombinantWppGSTV5H6 strain also secreted a large amount of immatureforms—the precursor that Blanquet et al. (2004) named prepro-KR-GST-V5H6 (calculated MW: 41.8 kDa, Fig. 2, b signal) togetherwith signals of higher MWs that may have corresponded toglycosylated and hyperglycosylated forms of this expected protein(Fig. 2, g signal). A band below 18 kDa was also detected, whichcorresponded to the degradation products of the protein (Fig. 2).In all rats, 30 min after gavage, GST-V5H6 was detected from the

Table 1Survival rate of WppV5H6 and WppGSTV5H6 yeast strains at 180 min after their

introduction into gastrointestinal sacs.

Survival rate of recombinant yeasts strain (% of yeast

initial number)

WppV5H6 WppGSTV5H6

Stomach 70719 103715

Duodenum 52710 65720

Jejunum 61720 3878

Ileum 2774 3676

Cecum 49711 80726

Colon 4274 47720

Values are expressed as mean percentages7SEM (n ¼ 5 for each sac) relative to the

initial amount of yeasts introduced.

Fig. 2. Western blot analysis of GST-V5H6 produced by WppGSTV5H6 cells in the various

109 recombinant cells. Signal a (mature GST-V5H6), signal b (prepro-KR-GST-V5H6) and s

different forms of GST-V5H6. Stom ¼ stomach; Duo ¼ duodenum; Prox jej ¼ proxim

Ce ¼ cecum; Col ¼ colon.

stomach to ileum as a signal at around 31 kDa and signals ofhigher MWs (Fig. 2A). At 2 h post-administration, the modelprotein was detectable in all digestive samples (Fig. 2B). At 4 hpost-administration, GST-V5H6 was detected only in the largeintestine (cecum and colon) of a single rat (data not shown). Nosignal was observed in the rats sacrificed at 8 h and 24 h post-gavage (data not shown).

Using ELISA, GST-V5H6 could be quantified only in the stomachof sacrificed rats at 30 min after introduction of the yeasts (notshown). The concentration of model protein recovered in thestomach was around 10 ng/mL. The secreted quantities, over30 min, represented 13 ng of GST-V5H6.

3.2.2. Ex vivo experiments

No trace of GST-V5H6 was found in the gastrointestinal sacsafter introduction of either PBS or WppV5H6 yeast cells (notshown). Following the introduction of 109 WppGSTV5H6 cells,Western blotting detected the different forms of GST-V5H6 in allthe gastrointestinal sacs (data not shown). At 60–180 min post-administration, GST-V5H6 concentrations ranged between 1771and 6277 ng/mL (Table 2). After a 180-min incubation period, thehighest concentrations were measured in the duodenum and thelowest in the stomach. This is consistent with the results obtainedby Western blot analysis. The amounts of GST-V5H6 produced inthe various gastrointestinal sacs are presented in Fig. 3. After a 60-min incubation period, the quantity of GST-V5H6 ranged from4176 (ileum) to 68713 ng (colon). The amount of model proteinthat remained was stable until the end of the experiment.

digestive organs of rats sacrificed 30 min (A) or 2 h (B) after oral administration of

ignal g (glycosylated or hyperglycosylated prepro-KR-GST-V5H6) correspond to the

al jejunum; Med jej ¼ median jejunum; Dist jej ¼ distal jejunum; Ile ¼ ileum;

Table 2Concentration of GST-V5H6 after introduction of WppGSTV5H6 cells into gastro-

intestinal sacs.

Time after introduction

(min)

Concentration of GST-V5H6 (ng/mL)

Stomach Duodenum Jejunum Ileum Cecum Colon

0 070 070 070 070 070 070

60 2073 2672 2672 2273 1771 2773

90 2173 3073 2872 2673 1971 3075

120 2473 3673 3273 3172 2272 3977

180 3074 6277 4374 4875 3675 5579

Values are expressed as means7SEM (n ¼ 5) in ng/mL of GST-V5H6 in the different

sacs.

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Fig. 3. Production of GST-V5H6 by WppGSTV5H6 strain in gastrointestinal sacs. The

values indicate mean amounts7SEM (n ¼ 5) in ng of GST-V5H6 produced by the

yeast in the mucosal side of each organ.

G. Garrait et al. / Metabolic Engineering 11 (2009) 148–154152

4. Discussion

In order to validate the biodrug concept in terms of secretion,this study used a recombinant strain (named WppGSTV5H6)secreting the model protein GST-V5H6. The survival rate ofWppGSTV5H6 strain and its ability to secrete the model polypep-tide have been previously demonstrated in vitro in a gastro-intestinal tract model (Blanquet et al., 2004).

The next step in the development of our new drug deliverysystem consists in in vivo validation in the rodent. This study wasdesigned to determine the viability and biosynthesis/secretionactivities of recombinant WppGSTV5H6 yeast in the rat digestiveenvironment.

4.1. Viability of WppGSTV5H6 yeast strain

4.1.1. In sacrificed rat

At 30 min after gavage, 43% of the ingested yeasts wererecovered alive in the upper part of the digestive tract. TheWppGSTV5H6 yeast cells were resistant to gastric conditions, asalready demonstrated for Saccharomyces boulardii (Blehaut et al.,1989). They also resisted against bile salts and proteolyticactivities of pancreatic secretions, as their survival rate was notmodified between the duodenum and the ileum. Conversely, therecombinant strain was strongly affected by the colonic condi-tions, as its survival rate dropped to around 1% in the colon(Fig. 1B). Our results are similar to those recently obtained undersame experimental conditions for a S. cerevisiae strain expressingthe cytochrome P450 73A1 (Garrait et al., 2006). Moreover, theviability of WppGSTV5H6 in the upper digestive tract of rats isconsistent with previously reported in vitro results for the gastric/small intestinal system TIM1 (Blanquet et al., 2004).

Few studies have focused on evaluating the viability ofSaccharomyces spp. throughout the length of the rat gastrointest-inal tract. Single or multiple oral administrations of S. boulardii

(Albert et al., 1977; Blehaut et al., 1989) resulted in a similardistribution pattern to that reported here. In fact, yeast viabilityhas been mainly evaluated in feces. In the present study, thesurvival rate observed in feces (around 1%) was consistent withthat obtained by Boddy et al. (1991), who reported a fecal recoveryrate of 0.8% in rates after gavage with S. boulardii. Klein et al.(1993) also found a similar yeast survival rate (0.1%) in fecesof healthy humans after a single oral administration of 1010

S. boulardii. Moreover, according to Pecquet et al. (1991), when

healthy volunteers were given a daily dose of 3�108 S. cerevisiae

for 5 days, 2% of living yeasts were excreted in stools throughoutthe administration period.

The significant mortality of yeasts in the large intestine can beexplained by several biochemical processes. According to Duclu-zeau and Bensaada (1982), the yeast cell-wall polysaccharidesglucans and mannans can be hydrolyzed by degrading enzymesnative to the large intestine. Indeed, the production of b 1–3glucanases by Bacteroides, which is the most prevalent genus ofintestinal bacteria in humans, has already been demonstrated(Salyers et al., 1978). As previously shown by Ducluzeau andBensaada (1982) in mouse, endogenous colonic microflora actingas a ‘‘barrier’’ towards orally administered S. boulardii may alsoexplain the extensive elimination of yeasts in the digestive tract.

4.1.2. In gastrointestinal sacs

We also evaluated the survival rate of the recombinant strainin each digestive compartment of the rat using the ex vivo

gastrointestinal sac technique. After a 180-min incubation period,the absence of yeast mortality in the stomach indicated that therecombinant S. cerevisiae strain was highly resistant to gastric pH.A similar finding was previously obtained in vitro with S. boulardii,

incubated in artificial gastric juice for several hours (Gedek andHagenhoff, 1989). Taken together, the data on each of the organsstudied in the present work show that on average, about 60% ofthe introduced yeasts were recovered alive at the end of theexperiment. This result is consistent with the data from ratssacrificed 4 and 8 h after the oral administration of WppGSTV5H6

yeast cells.

4.2. Biosynthesis/secretion activity

4.2.1. In sacrificed rats

In order to profile the heterologous activity of the yeast insacrificed rats, recombinant WppGSTV5H6 yeasts and galactose –the gene inducer – were simultaneously orally administered to theanimals. This is the first demonstration of the ability ofrecombinant yeasts to produce a model protein in the rat digestiveenvironment. Indeed, 30 min and 2 h after gavage, the expectedGST-V5H6 was detected by Western blot analysis in all the organsstudied. The presence of uncleaved precursors of GST-V5H6 wasalso revealed. These immature forms had previously beenreported when S. cerevisiae was used as host for protein secretion(Guisez et al., 1991; Kjeldsen, 2000; Zsebo et al., 1986). Immatureforms may result from saturation of proteolytic enzymes (such asKex2) or from their inefficiency during the protein-processingpathway (Guisez et al., 1991; Kjeldsen, 2000). Blanquet et al.(2004) recently found similar secretion products in all compart-ments of the TIM1 artificial digestive system.

Although living WppGSTV5H6 cells were present in the variousareas of the rat gastrointestinal tract and in feces, GST-V5H6 wasweakly or not detectable in the lower digestive compartments at4, 8 and 24 h post-gavage. These results may reflect thedegradation of the heterologous protein in the upper part of thedigestive tract. Moreover, in the large intestine, the lack of GST-V5H6 could also be the consequence of the gastrointestinalabsorption of galactose, the gene inducer. This monosaccharideis actively transported across the brush border membrane of thesmall intestine (Wright et al., 2007), and consequently the livingWppGSTV5H6 yeasts present in the large intestine were deprivedof galactose, probably explaining their lack of heterologousactivity in the cecum and colon.

Although Western blotting revealed the presence of GST-V5H6

in the digestive organs at 30 min and 2 h after administration ofrecombinant yeasts, the model protein could be quantified only in

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the 30-min gastric sample. Despite the relative high sensitivity ofour immunoenzymatic method, GST-V5H6 could not be quantifiedin the other parts of the rat digestive tract.

Only a few studies have focused on the production ofheterologous protein by microorganisms directly in the digestivetract. Steidler et al. (2000) investigated the in vivo synthesis ofhuman cytokine interleukin-10 (IL10) by recombinant Lactococcus

lactis (L. lactis) throughout the mouse gastrointestinal tract. Theydetected recombinant IL10 in the colon but not in other digestiveorgans. Oozeer et al. (2004) also showed that recombinantLactobacillus casei (Lb. casei) in mice harboring human microbiotawas able to initiate the synthesis of luciferase during transit withthe diet. In most of the studies, the in vivo bacterial production ofheterologous protein was assessed by monitoring the biologicaleffect of the secreted protein. A number of studies in animals andhumans (Cong et al., 2005; Corthesy et al., 2005; Garmory et al.,2003; Lee and Faubert, 2006) have shown that geneticallyengineered bacteria can induce an immune response character-ized by antibody production after the synthesis of heterologousantigen directly in the gastrointestinal tract. However, only onestudy (Chen et al., 2000) has described the efficiency ofheterologous protein secreted by recombinant yeasts. The authorsshowed improved growth in chickens when recombinant Pichia

pastoris cells secreting growth hormone were added to the diet.

4.2.2. In gastrointestinal sacs

The second step in this study was to quantify the production ofthe heterologous protein in the separate digestive organs. Usingthe gastrointestinal sac technique, we showed for the first timethat the recombinant yeasts were able to produce GST-V5H6 in allthe gastrointestinal compartments of the rat. Indeed, substantialamounts of the heterologous protein were recovered from 60 to180 min after the introduction of WppGSTV5H6 yeast cells. Theamounts of GST-V5H6 produced from 60 min to the end of theexperiment were constant, whatever the organ studied. Thesedata led to the conclusion that WppGSTV5H6 yeast activityoccurred very quickly and took place only during a shortincubation period. In each organ studied, the galactose and thegenetically engineered cells were introduced on the mucosal sidesimultaneously. The monosaccharide was certainly rapidly ab-sorbed across the digestive epithelium and passed into the serosalmedium. Consequently, the WppGSTV5H6 cells were quicklydeprived of the inducer, which probably explains the observedabsence of activity after the first 60-min incubation period. Thedegradation of the model protein seemed to be more limited inour ex vivo experiment than in sacrificed rats. The reason could bethat, in sacrificed rats, the GST-V5H6 was rapidly degraded byenzymes of the pancreatic juice, whereas most of the endogenousrat proteases were probably removed from the mucosal side of thegastrointestinal sacs by rinsing during the preparation step. TheGST-V5H6 concentrations in ex vivo experiments were close tothose measured in batch cultures (Blanquet et al., 2004), i.e. from17 to 62 ng/mL in gastrointestinal sacs vs. 10–25 ng/mL in batchcultures. This demonstrated that the yeast’s ability to produce theheterologous protein was not significantly affected by thedigestive conditions. However, the levels of GST-V5H6 obtainedin both batch cultures and the gastrointestinal sacs remained verylow. As previously reported, heterologous activity may depend onthe size of the protein (Zsebo et al., 1986) and/or the geneticconstruction (Blanquet et al., 2004). Protein secretion may beimproved with another heterologous protein or a differentexpression vector (Kjeldsen, 2000).

Few studies have tested the production of heterologous proteinusing a recombinant strain in the various parts of the digestivetract. A recent study assessing genetically modified L. lactis

secreting human IL10 in the intestine of conscious pigs (Steidleret al., 2003) showed that recombinant lactic acid bacteria wereable to produce IL10 in the ileal loop contents after a 4-hincubation period.

In conclusion, our results indicate that the WppGSTV5H6

recombinant yeast was able to resist the stringent conditionsfound in the upper part of the rat digestive tract. However, yeastsurvival rate decreased strongly after the cells reached the largeintestine. For the first time, we showed that genetically en-gineered yeasts were able to produce a model protein directly inthe rat digestive tract. Using the gastrointestinal sac technique,substantial amounts of GST-V5H6 were further quantified fromstomach to colon. These in vivo results support the possibility ofusing recombinant S. cerevisiae as a potential host for thedevelopment of ‘‘biodrugs’’, particularly as a means to deliveractive compounds directly to the human digestive tract. Thenumerous potential medical applications include the develop-ment of oral vaccines, the production of various biologicalmediators (e.g. insulin, cytokines or growth factors) and thecorrection of metabolism disorders resulting from gastric orintestinal enzyme deficiencies or organ failure.

Acknowledgments

This work was supported by the Delegation Generale pourl’Armement (D.G.A.). The authors thank Angelique Gardes and EveArmente for their skilled technical assistance.

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