AU754020B2 - Chemoprotective bacterial strains - Google Patents

Description

WO 99/27953 PCT/US98/25315 CHEMOPROTECTIVE BACTERIAL STRAINS Pursuant to 35 U.S.C. §202(c), it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health, Grant No. CA22484.

This application claims priority to U. S.

Provisional Application No. 60/066,969, filed November 28, 1997, the entirety of which is incorporated by reference herein.

FIELD OF THE INVENTION The present invention relates to the field of genetically engineered bacteria for therapeutic and prophylactic use. In particular, the invention provides recombinant probiotic bacteria expressing enzymes that facilitate detoxification of carcinogens, and methods for their use in prevention and treatment of certain forms of cancer.

BACKGROUND OF THE INVENTION Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.

Each year, nearly a million people in the world die from colorectal cancer. About 15% of all colorectal cancers have been attributed to the presence of a gene carried within certain families that significantly increases the likelihood of developing the cancer. It has been proposed to begin screening of general populations to identify those "at risk" populations, so that protective measures could be applied to the reduce WO 99/27953 PCT/US98/253 the risk for colorectal cancer in these populations.

It is widely recognized that genetic and environmental factors both contribute to the onset and development of all types of cancer. In colorectal cancer, development of tumors is generally attributed to the food content of the normal Western World diet, such that even persons not genetically at risk are at risk due to dietary intake of certain kinds of food. Much of the food consumed in the Western World diet contains a variety of procarcinogens (promutagens) arising from mold by-products during storage, consumption of mold byproducts by cattle and carryover into our food, chemicals endogenous to the foods, and chemicals produced by pyrolysis during the cooking and preparation of the food.

Dietary intake of such procarcinogens not only contributes to colorectal and other types of gastrointestinal (GI) cancers, but also can increase the risk of all kinds of cancer, once the procarcinogen is absorbed into the body's circulation from the GI tract.

A significant additional source of GI procarcinogen consumption is from tobacco byproducts.

These can include procarcinogens generated during roasting of tobacco leaves and then solubilized in saliva and swallowed during the normal use of chewing or smokeless tobacco. These can also include the procarcinogens present in tobacco smoke that are solubilized in saliva and swallowed during smoking of tobacco products.

Consumption of procarcinogens is also of concern in livestock, where acute forms of toxicity, including death, can result from oral consumption and GI absorption of naturally-occurring plant toxins. Another area in which consumption of specific procarcinogens is of concern is populated environments that are heavily contaminated with specific environmental contaminants whose identities are known.

Detoxification systems in cells of the GI tract WO 99/27953 PCT/US98/25315 -3are the first line of defense against procarcinogens and other ingested xenobiotic compounds. In general, the enzymes of detoxification convert reactive compounds to less reactive species, and/or to species that can be more easily excreted from the cell. Enzymes of this system are often polymorphic: one isozyme may have a higher specificity toward a particular xenobiotic chemical than another. The enzymes of detoxification have been classified into two groups: Phase I and Phase II. Phase I enzymes function to render the xenobiotic compound reactive, whereafter it can be acted on by the Phase II enzymes.

Chief among the Phase I enzymes are different isozymes of cytochrome P450 (Cyt P450). Cyt P450 contains a heme group which acts as the cofactor in the catalysis of oxygenation of the procarcinogens, thereby providing a reactive chemical group for the Phase II family to conjugate to a second molecule, which increases the hydrophilicity of the resulting compound.

A prominent member of the Phase II family is glutathione S-transferase (GST). It catalyzes the conjugation of glutathione, the tripeptide y-glutamylcysteinylglycine, to a variety of toxic electrophilic compounds. The reaction renders many of these toxic electrophiles less reactive against cellular components, such as genomic DNA, and often sends them to subsequent metabolic and excretion pathways. GST has been suggested to be the most important cellular factor against these electrophiles.

Even with such detoxification mechanisms in place, clearly the cells of the GI tract are not sufficiently equipped to defend against the onslaught of all procarcinogens and other toxic agents introduced into the body in the foods we eat. A need exists, therefore, to enhance or supplement the body's own natural defenses against such agents, preferably at the site where they are often first introduced the gastrointestinal system.

WO 99/27953 PCTUS98/25315 Such supplementation of the GI detoxification system would serve to reduce the overall bioavailability of carcinogens, and by so doing, reduce the incidence of cancer throughout the body.

SUMMARY OF THE INVENTION This invention provides compositions and methods for supplementing the detoxification systems of GI cells to protect the body from ingested procarcinogens and other toxic substances.

The compositions of the invention comprise probiotic microorganisms which have been genetically modified to express mammalian enzymes involved in Phase I and Phase II detoxification. In preferred embodiments of the invention, the probiotic microorganisms are GI tractcolonizing or -associated bacteria, such as various strains of Lactobacillus.

The invention is practiced by feeding the engineered probiotic organisms to an animal or human.

The organisms colonize or otherwise become associated with the GI tract of the individual, whereupon they grow and multiply, continuing to produce the detoxifying enzymes. As "friendly" residents of the GI tract, these bacteria scavenge and detoxify food procarcinogens, releasing the biologically innocuous metabolites into the bowel contents for excretion. The overall bioavailability of carcinogens is thereby reduced, which reduces the risk and incidence of all types of cancers, and GI cancers in particular.

In a preferred composition of the invention, the probiotic microorganism is selected from the group consisting of Lactobacillus, Lactococcus, Bifidobacteria, Eubacteria and non-pathogenic strains of Escherichia coli. The enzymes are selected from the group consisting of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione-S-transferase, gamma-glutamylcysteine synthetase, N-acetyltransferase, aldehyde dehydrogenase, WO 99/27953 PCT/US98/25315 and aldehyde reductase. The procarcinogenic or toxic substances are selected from the group consisting of polycyclic aromatic hydrocarbons, mycotoxins, arylamines, heterocyclic amines, nitrosamines and benzene.

In a preferred embodiment of the invention, genes encoding three classes of recombinant drugmetabolizing enzymes are used to transform probiotic bacteria. These genes encode one or more of the commonly available forms of Cyt P450 for Phase I metabolism, a common form of the enzyme NADPH-P450 reductase that associates with, and contributes electrons to, Cyt P450 to enable efficient Phase I metabolism; and either common or unique forms of glutathione

S-

transferase (GST) for Phase II metabolism to produce nonreactive conjugates. In a particularly preferred embodiment a plurality of different probiotic microorganisms are used, which express a plurality of different enzymes.

In another preferred embodiment, the abovedescribed metabolically fortified bacteria are used in products like yogurt to populate the human GI tract, and by so-doing, put in place a system that steadfastly acts to capture and detoxify procarcinogen molecules as they are presented to the GI mucosal surface where the bacteria reside.

Other features and advantages of the present invention will be understood from the description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram showing the specific detoxification and elimination of the procarcinogen, benzo(a)pyrene by recombinant probiotic bacteria expressing cytochrome P450, NADPH P450 reductase and glutathione S-transferase. Other major procarcinogen contaminants of human food and their detoxification steps are also depicted.

WO 99/27953 PCT/US98/25315 -6- Figure 2. Schematic diagram showing two examples, Construct 1 and Construct 2, which contain functional expression cassettes for each of three recombinant drug-metabolizing enzymes in a preferred embodiment of the invention. Construct 1 is 9742 nucleotides, with components as follows: Cyt P450 1A1 cDNA at bases 1-1625; NADPH Cyt P450 reductase at bases 1637-4099; M13 origin of replication at bases 4267-4665; P-lactamase CDS at bases 4082-5732; ColEl origin at bases 5792-6491; Human GST pi cassette at bases 6786-7678; Laq I' at bases 8136-9402; Taq promoter at bases 9577-9629; Taq promoter at bases 9652-9736. Construct 2 is 9641 nucleotides, with components as follows: Cyt P450 1A2 cDNA at bases 1-1523; NADPH Cyt P450 reductase at bases 1535-3997; M13 origin of replication at bases 4165-4563; P-lactamase CDS at bases 4700-5630; ColEl origin at bases 5690-6389; Human GST pi cassette at bases 6684-7576; Laq

I

q at bases 8034-9300; Taq promoter at bases 9455-9527; Taq promoter at bases 9550-9634. Additional variants of these are also used, in which additional cytochrome P450 and/or GST cDNAs are exchanged at the indicated sites.

Figure 3. Western immunoblot showing rec cytochrome P450 1Al production in E. coli DH5a cells transformed with the Construct 1 plasmid. Cultures were induced overnight with IPTG. Bacterial membranes were prepared according to Parikh et al. (1997). Proteins g of bacterial membrane protein per lane) were separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using an ECL kit according to the manufacturer's protocol (Pierce Biochemicals, Rockford). A polyclonal rabbit anti-human CYP450 1A antibody (RDI Research Diagnostics) was used as the primary antibody (1:1000 dilution). CYP450 1Al (57 kDa) was detected using a polyclonal goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000 dilution) and chemiluminescent substrate. Lane 1 is Control (pIC20r); Lane 2 is pCW lAl/reductase; Lane 3 WO 99/27953 PCT/US98/25315 -7is pCW lAl/reductase/GST pi (construct Lane 4 is a blank lane; Lane 5 is molecular weight markers (kDa).

Figure 4. Western immunoblot showing rec cytochrome P450 1A2 production in E. coli DH5a cells transformed with the Construct 2 plasmid. Cultures were induced overnight with IPTG. Bacterial membranes were prepared according to Parikh et al. (1997). Proteins (100 pg of bacterial membrane protein per lane) were separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. CYP450 1A2 was visualized using a goat anti-human CYP 1A2 polyclonal antibody (1:2000 dilution; RDI Research Diagnostics, Inc.) and a swine anti-goat secondary antibody conjugated to horseradish peroxidase (1:5000, Boehringer Mannheim) with chemiluminescent substrate (Pierce Biochemicals, Rockford). Lane 1 is molecular weight markers (kDa); Lane 2 is purified CYP 450 1A2 (0.2 gg, Panvera Biochemicals); Lane 3 is purified CYP 450 1A2 (0.4 /g, Panvera Biochemicals); Lane 4 is control (pIC20r); Lanes 5 and 6 are blank; Lane 7 is pCW 1A2/reductase; Lane 8 is pCW 1A2/reductase/GST pi clone #3 (construct 2).

Figure 5. Western immunoblot showing rec NADPH-P450 reductase production in E. coli DH5a cells transformed with Construct 1 or Construct 2 plasmids.

Cultures were induced overnight with IPTG. Bacterial membranes were prepared according to Parikh et al.

(1997). Proteins (100 pg of bacterial membrane protein per lane) were separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using an ECL kit according to the manufacturer's protocol (Pierce Biochemicals, Rockford).

A polyclonal rabbit anti-human CYP450 1A1 antibody (RDI Research Diagnostics) was used as the primary antibody (1:1000 dilution). P450 reductase (77 kDa) was detected using a polyclonal goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000 dilution) and chemiluminescent substrate. The gel was overloaded, WO 99/27953 PCT/US98/25315 -8leading to smearing of the 77 kDa P450 reductase band.

Lane 1 is rat liver extract showing NADPH-P450 reductase; Lane 2 is blank; Lane 3 is the control (pIC20r); Lane 4 is pCW lAl/reductase; Lane 5 is pCW 1A1 reductase/GST pi (Construct Lane 6 is pCW 1A2/reductase/GST pi (Construct Lane 7 is blank; Lane 8 is molecular weight markers (kDa).

Figure 6. Western immunoblot showing rec GST pi production in E. coli DH5a cells transformed with Construct 1 or Construct 2 plasmids. Cultures were induced overnight with IPTG. Bacterial membranes were prepared according to Parikh et al. (1997). Proteins g9 of crude bacterial supernate per lane) were separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using an ECL kit according to the manufacturer's protocol (Pierce Biochemicals, Rockford). A polyclonal rabbit anti-GST pi antibody (RDI Research Diagnostics) was used as the primary antibody (1:1000 dilution). GST pi was detected using a polyclonal goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000 dilution) and chemiluminescent substrate. Lane 1 is molecular weight markers (kDa); Lane 2 is purified human GST pi (0.1 gg); Lane 3 is the control (pIC20r); Lane 4 is pCW 1A1/OR; Lane 5 is pCW 1A1/OR-GST; Lane 6 is pCW 1A2/OR; Lane 7 is pCW 1A2/OR-GST pi.

Figure 7. Reduced carbon monoxide difference spectrum of rec cytochrome P450 1A2 in membranes of bacterial cells transformed with Construct 2 plasmid.

Intact difference spectrum illustrates that the cytochrome P450 1A2 apoprotein synthesized in the bacterial cells is properly binding the heme prosthetic group produced by the bacterial cells to yield functional holoenzyme. The rec cytochrome P450 content of the bacterial membranes was determined by measuring the absorbance difference at 450 nm between the reduced hemoprotein (Fe+2) versus the reduced hemoprotein bound WO 99/27953 PCT/US98/25315 -9to carbon monoxide (Fe+2-CO).

Figure 8. Results of Ames mutagenesis assay showing linear, dose-dependent induction of mutations in TA98 tester strain using benzo(a)pyrene as the procarcinogen. A TA98 Salmonella tester strain containing a mutation in the histidine operon was preincubated with benzo(a)pyrene and a 10% S9 mixture for 30 minutes at 37 0 C prior to the Ames assay.

Following the preincubation, a histidine/biotin top agar was added to the contents listed above and plated on minimal glucose plates. Spontaneous reversion of TA98 to histidine independence by B(a)P was measured by counting the number of colonies on the minimal glucose plates after a 48 hour incubation at 37 0

C.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel compositions and methods for supplementing the body's natural system for detoxifying various ingested procarcinogenic and other toxic substances. The compositions of the invention comprise recombinant probiotic bacteria engineered to express one or more isoforms of two critical mammalian detoxification enzymes, cytochrome P450 (Cyt P450) and glutathione-Stransferase (GST), as well as the important contributor to the Phase I metabolism step, NADPH-P450 reductase.

Though other native bacterial reductases have been shown to marginally support rec cytochrome P450-catalyzed oxidation in bacteria, co-expression of the mammalian NADPH-P450 reductase with the rec cytochrome P450 has been shown to confer the greatest metabolic capacity to the expressing cells.

The Cyt P450 enzyme catalyzes the first step in the detoxification, converting a non-reactive procarcinogen to a reactive species that can be further processed. The GST enzyme then conjugates the intermediate product of the Cyt P450-catalyzed reaction WO 99/27953 PCT/US98/25315 to glutathione, which is normally present in bacterial cells at millimolar concentrations to yield a biologically-inert, water-soluble metabolite. One possible step in further engineering the probiotic bacteria would be to integrate and stably express two cDNAs encoding the heavy and light subunits of the enzyme gamma-glutamylcysteine synthetase (y-GCS). Levels of this enzyme are typically rate-limiting in glutathione biosynthesis in both bacterial and mammalian cells, and augmented levels of the enzyme have been shown to stably increase the baseline concentration of glutathione in cells with no apparent deleterious effects.

Other recombinant enzymes can also be expressed in probiotic bacteria to direct the detoxification of procarcinogens; examples include one or more isoforms of N-acetyltransferase, aldehyde dehydrogenase, and aldehyde reductase to metabolize aflatoxin. This would broaden the scope of environmental toxins that could be safely detoxified by probiotic bacteria of the invention.

These recombinant gene products are stably expressed in strains of probiotic bacteria, such as Lactobacillus acidophilus, which are generally accepted as being "friendly floral" strains, strains that grow well within the human or other mammalian GI ecosystem without producing or secreting detrimental bacterial toxins.

The substrates for the two-step drug metabolism catalyzed by the fortified recombinant bacteria of the invention are molecules that are generally referred to herein as "procarcinogens." These are environmental contaminants that humans come in contact with, most commonly, through smoking or gastrointestinal absorption.

Some examples of procarcinogens that commonly appear in the daily human diet are illustrated in Figure 1. These and additional procarcinogens that commonly occur in the production and packaging of human foods were recently studied and listed in a prioritized manner in a book WO 99/27953 PCT/US98/25315 -11recently published by a study group of the U. S. National Academy of Sciences (Carcinogens and Anticarcinoqens in the Human Diet, National Academy Press, Washington,

D.C.,

1996).

The procarcinogens consumed in a daily diet usually are not highly water soluble. Therefore, they are readily absorbed from the lumenal contents of the stomach, as well as the small and large intestine. Once absorbed, the procarcinogens distribute throughout the body, where they are stored or metabolized by cellular Cyt P450s to yield highly reactive, electrophilic "proximate" carcinogens which can now, because of their highly reactive nature, attack DNA and induce mutation (see Figure The accrued result of this chronic DNA modification can be either cell death (a cytotoxic result) or transformation to a cancer cell (a neoplastic transformational result).

The fortified bacteria of the present invention that stably express the mammalian detoxification enzymes can aid in averting systemic distribution of procarcinogens by detoxifying them in the GI lumen, before they can be absorbed into the system. The fortified bacteria residing in the GI lumen can routinely mix with ingested, partially digested, hydrated food.

After absorbing the procarcinogens and converting them to safe, biologically inert metabolites, the bacteria will secrete the innocuous metabolites back into the GI lumenal contents to be excreted.

Lessening the daily dose of procarcinogen absorbed into the general circulation lowers the overall risk for cancer formation at all organ sites. Thus, the fortified bacteria of the invention will serve not only to lower the risk of GI associated cancers, but also will contribute to lower risks of all types of cancer.

The following basic steps should be followed in order to practice the present invention: select the detoxifying enzymes desired for expression in probiotic WO 99/27953 PCT/US98/25315 -12bacteria; clone the enzyme-encoding genes in a form suitable for expression in the selected probiotic bacteria; transform the bacteria with the cloned genes, such that the bacteria express functional enzymes; and feed the recombinant bacteria to animal or human subjects for which protection from procarcinogens is desired. Each of these steps is described in greater detail below.

The recombinant bacteria of the present invention are engineered to express enzymes involved in the two-step detoxification mechanism described above.

Such enzymes include, but are not limited to: (1) selected substrate-specific isoforms of Cyt P450; one or more isoforms or recombinant variants of GST; a form of NADPH-P450 oxidoreductase, an enzyme that produces reducing equivalents consumed by Cyt P450 during its oxidation of the procarcinogen molecule; one or more isoforms of N-acetyltransferase, aldehyde dehydrogenase, aldehyde reductase, and heavy and light subunits of the enzyme y-GCS.

A small group of P450 isozymes are capable of metabolizing the large majority of environmental procarcinogens encountered by humans (Guengerich, 1996).

Candidate cytochrome P450 isozymes include CYP 1B1, CYP 1A1, 1A2 and 2E1. CYP 1B1, a form that was first described several years ago (Gehly, Fahl, et al., J.

Biol. Chem. 254: 5041-5048, 1979) because of its unique specificity for metabolizing polycyclic aromatic hydrocarbon (PAH) substrates, has now been cloned and can be efficiently expressed in bacterial cells (Savas et al., Arch. Bioch. Biophys. 347: 181-192, 1997). The rec CYP 1B1 still retains its broad specificity and catalytic efficiency for PAH substrates. CYP 1A2 is a logical candidate because of its capacity to oxidize the carcinogenic arylamines and heterocyclic amines found in broiled meats. CYP 2E1 is a logical candidate because of its ability to metabolize nitrosamines and benzene.

WO 99/27953 PCT/US98/25315 -13- Certain P450s like CYP 2D6 will need to be approached cautiously in clinical trials because its substrate specificity includes several drugs, such as antidepressants, which adult humans fairly commonly consume.

Cytosolic members of mammalian GSTs are dimeric proteins that have four classes (Alpha, Mu, Pi, and Theta). The ability of Alpha-class GSTs to detoxify alkylating compounds is well documented. GSTs catalyze the nucleophilic attack of the thiolate anion of the tripeptide glutathione (GSH) on the electrophilic functional group of a hydrophobic substrate. The hydrophobic electrophilic compounds that serve as substrates for GSTs undergo catalytic conjugation to GSH to become more polar, less toxic, and more readily excretable from the cells as glutathionyl conjugates.

Because of their evolutionary heritage of conjugating large numbers of structurally diverse electrophilic metabolites that mammals encounter, the spectrum of GST isoforms required to accommodate the Phase I metabolites generated by the above CYP450s is smaller (Hayes Pulford, Crit. Rev. Bioch. Mol. Biol.

445-600, 1995). Generally, all GST isoforms are capable of catalyzing the conjugation of most electrophiles, but there can be sizable differences in the rates at which the isoform-catalyzed reactions proceed. The GST pi isoform is capable of conjugating very diverse groups of organic electrophiles including PAH epoxides and diol-epoxides. The GST alpha class isoform, Yc, is very efficient at conjugating aflatoxinepoxide, while other alpha class isoforms are efficient at detoxifying alkylating drugs like melphalan and cytoxan. Other broad rules of specificity are described in the GST review by Hayes (Hayes Pulford, 1995, supra).

In one embodiment of the invention, a gene encoding a wild-type GST is used. In a preferred WO 99/27953 PCT/US98/25315 -14embodiment, recombinant GSTs having increased enzymatic activity are used. Such recombinant GSTs are known in the art (Gulick Fahl, Proc. Natl. Acad. Sci. USA 92: 8140-8144, 1995; and are described in commonly-owned

U.S.

Patent Application Serial No. 08/297,431, incorporated by reference herein.

In a preferred embodiment of the invention, a panel of, six to eight recombinant plasmids are generated, which express, three or four different cDNAs encoding Cyt P450s with different substrate specificity and, two or three different cDNAs encoding GSTs with different substrate specificity.

These different cDNAs also may be expressed in different probiotic bacteria having complementary biological features. Mixing clones of different bacteria expressing different forms of detoxifying enzymes will enable metabolism of broad mixtures of procarcinogens, or very specific procarcinogens, depending on the desired application.

Probiotic bacteria suitable for the present invention include any non pathogenic, preferably physiologically beneficial, bacteria that colonize or are otherwise associated with or resident in the gastrointestinal tract of the selected subject animal or human. Preferably, these bacteria are species of the genus Lactobacillus. However, other genera may be utilized, including, but not limited to: Lactococcus, Bifidobacteria, Eubacteria and non-pathogenic strains of Escherichia coli (see Gibson and Roberfroid, J. Nutrition 125: 1401-1412, 1995).

The Lactobacilli are a large and diverse group of Gram-positive bacilli that are common components of the normal indigenous flora of humans and other animals.

Lactobacilli are considered "health promoting," but in any event are rarely pathogenic, making them good candidates for use in the present invention. Indeed, Lactobacilli have been exploited recently as vaccine WO 99/27953 PCT/US98/25315 delivery vehicles, i.e. expressing antigens in the GI tract of immune-functional individuals for purposes of eliciting an immune response (see, Rush et al., Chapter 6 in Gram-Positive Bacteria as Vaccine Vehicles for Mucosal Immunization, Pozzi J.M. Wells, eds.), Landes Bioscience, 1997).

Different strains and species of Lactobacillus are differentially capable of colonizing or becoming otherwise associated with the GI tract of a particular animal. Two approaches can be used in selecting appropriate strains for delivery of detoxifying enzymes to a particular animal species (including humans): (1) selection of strains known to be capable of colonizing mucosal surfaces of the animal Lactobacillus GG is known to colonize the human GI tract); and/or (2) selection of strains naturally found in foods, which may have limited interactions with the host GI tract, but nevertheless may inhabit the GI tract for significant periods of time.

The fate of viable Lactobacillus cells within the GI system of mammals depends on a number of variables. At one extreme, the bacterial cells simply mix with the undigested fiber of ingested food and move along the GI system. In this situation there is little colonization by the Lactobacillus cells. At the opposite extreme, cells will find host sites within the mucosal matrix, stay there and replicate "colonize"). The factors that determine the ability of cells to colonize or merely populate sites within the GI system are not fully understood. Some parameters that affect the ability of probiotic bacteria to colonize include: the pH of the particular GI site, acidophilus means "acid lover" meaning these strains can accommodate the low pH of the stomach and duodenum; the oxygen content of the site; the strain of bacteria; and the ability to compete for nutrients with other types of bacteria that are encountered on the mucosal surface of GI epithelium.

WO 99/27953 PCT/US98/25315 -16- Inasmuch as transient or colonizing strains will be able to perform the intended function of producing detoxifying enzymes, selection of appropriate strains can be made on the basis of how long such production is desired in the individual undergoing the treatment. Alternatively, combinations of different strains, some transient and others colonizing, can be used to provide short and longterm protection.

Many Lactobacillus species have been demonstrated amenable to transformation and expression/secretion of recombinant proteins.

Lactobacilli can be genetically modified by way of two mechanisms: via introduction of an ectopic plasmid carrying a foreign gene; or via stable integration of a foreign gene into the genome. One advantage to this latter approach is that it avoids potential loss of the foreign gene, which can result from plasmid shedding during expansion of the bacterial cultures. Thus, the potential lower expression associated with stable integration of the foreign gene could be offset by the more stable retention of that gene and resulting ability of the bacteria to express the detoxifying enzymes for an extended period of time.

There are numerous examples in the art of cloning and expression vectors designed for use in Lactobacillus species. Moreover, the art describes the successful introduction and expression of foreign genetic material in a variety of Lactobacillus species, including L. casei, L. plantarum, L. paracasei, L. acidophilus,

L.

fermentum and L. zeae, among others (Rush et al., 1997, supra; Rush et al., Microbiol. Biotechnol. 47: 537-542, 1997; Hols et al., Microbiology 143: 2733-2741, 1997).

Example 2 below describes vector design and a protocol for transformation of an L. gasseri strain that colonizes the murine upper GI tract. This protocol follows standard protocols for transformation of Lactobacillus by electroporation (see, Aukrust et al., Chapter 20 in WO 99/27953 PCT/US98/25315 -17- Methods in Molecular Biology, Vol. 47, Nickoloff, Humana Press Inc., Totowa, The stable integration of foreign genes into the Lactobacillus genome via the temperate phage mv4 has also been described (Dupont et al., J. Bact. 177: 586- 595, 1995; Avuray et al., J. Bact. 179: 1837-1845, 1997).

This bacteriophage, which naturally occurs in L.

delbruckii subsp. bugaricus has been modified for use in integrating foreign genes into the genome of L.

plantarum, L. casei and L. lactis.

The vectors presently available in the art for transforming Lactobacillus were not designed for consumption by humans or animals. In particular, most of the vectors utilize antibiotic resistance as a selection means. Since it is undesirable to introduce antibiotic resistance into an animal or human, the currently available vectors should be modified to comprise a different selection means, such as a color indicator.

Selection means that do not rely on antibiotic resistance are known in the art and are available.

Another group of probiotic bacteria preferred for use in the present invention are non-pathogenic strains of Escherichia coli. Example 1 describes the transformation of E. coli with a recombinant plasmid encoding a Cyt-P450, NADPH-P450 reductase and a GST.

Once the recombinant probiotic bacteria are produced, they are used to protect against ingested procarcinogens and other toxic agents. The mode of administration is by oral or nasal injection, or by feeding (alone or incorporated into the subject's feed or food). In this regard, it should be noted that the Lactobacillus acidophilus that is presently widely available commercially is sold as gel capsules containing a lyophilized mixture of bacterial cells and a solid support such as mannitol. When the gel capsule is ingested with liquid, the lyophilized cells are rehydrated and become viable, clonogenic bacteria. Thus, WO 99/27953 PCT/US98/25315 -18in one embodiment, the recombinant Lactobacillus cells are added to food by sprinkling in the powdered, lyophilized preparation. The re-hydrated, viable Lactobacillus cells will then populate and/or colonize at sites throughout the upper and lower gastrointestinal system. In a particularly preferred embodiment, the recombinant bacteria are used to produce fermented milk products, such as yogurt and kefir, in the same manner that non-recombinant lactobacilli would be used.

In another embodiment, the fortified probacteria are mixed into a product such as chewing tobacco. As the procarcinogens in the roasted tobacco leaves are extracted into the saliva, the bacteria are mobilized to begin scavenging the procarcinogens to preemptively detoxify them before they are absorbed by oral, esophageal, gastric and intestinal epithelial cells.

Another application of this invention in humans is directed to populated environments that are heavily contaminated with environmental contaminants whose identities are known, whose consumption by inhabitants is known, and the toxic impact upon the inhabitants consuming contaminated groundwater and produce is also known. In many settings, such as Gladowice, Poland, and other industrialized Eastern bloc sites, the level of environmental contamination has been estimated to be greater than that which could be environmentally remediated within the next 100 years.

In an embodiment involving animals other than humans, the fortified bacteria are mixed into feed for agriculturally important animals, such as cows, pigs and chickens. In this regard, there is a growing realization that, although some human food is tested to regulate its content of procarcinogens resulting from mold and bacterial growth on harvested crops aflatoxin from Aspergillus flavus in peanut butter), there is little or no regulation of the same procarcinogens coming into WO 99/27953 PCT/US98/25315 -19human food via the animals that consume mold-contaminated feeds and then concentrate the fat-soluble procarcinogens in the milk or meat that is produced from them.

Decreasing the procarcinogen "load" to the animals should be easier and more cost-effective than attempting to remove them from the milk or meat consumer products.

Additional applications are envisioned for this invention in livestock settings where the goal is to reduce or eliminate acute forms of toxicity, including death, resulting from oral consumption and GI absorption of naturally-occurring plant toxins. In several settings in the world, where cattle and sheep are raised by open grazing, naturally occurring foliage has been shown to contain toxins lethal to grazing animals. Two examples of this include Leucaena and Tansy Ragwort (Allison et al., Appl. Envir. Microbiol. 56: 590-594, 1990; Science News 153: 364, 1998), which are lethal to grazing cattle.

In this case, the toxic element in Leucaena, the amino acid analogue mimosine, can be targeted for degradation by probiotic strains of bacteria engineered to specifically degrade mimosine using either naturallyoccurring forms of P450 or forced-evolution derivatives of P450 and GST that we develop specific to the required detoxification need.

The following examples are provided to describe the invention in greater detail. They are not intended to limit the invention in any way. Unless otherwise specified, general cloning, biochemical and molecular biological procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) ("Sambrook et or Ausubel et al.

(eds) Current Protocols in Molecular Biology, John Wiley Sons (1998) ("Ausubel et are used.

WO 99/27953 PCT/US98/25315 EXAMPLE 1 Concurrent Expression of recCytochrome P450 1A1 or 1A2, recNADPH-P450 Reductase and recGST pi in Escherichia coli: Detoxification of Benzo(a)pyrene, a Common Food Procarcinoqen Benzo(a)pyrene (BP) has long been known to be tumorigenic when fed or applied topically to experimental animals, and has served as a prototype for chemical carcinogens. It is metabolized by the cytochrome P450 enzymes to form the 7,8-epoxide, which is then metabolized by epoxide hydrolase to form the BP-7,8-diol.

The compound is further oxidized by cytochrome P450s to BPDE. BPDE was demonstrated to be a substrate of GST for glutathione conjugation. The ability of GST expression in cells to confer BPDE resistance is known. However, co-expression of Cyt P450 and GST in recombinant bacteria heretofore has not been reported.

In this example, we describe the expression of two isoforms of cytochrome P450, CYP450 1A1 and 1A2, separately with NADPH-P450 reductase and GST pi in E.

coli, and demonstrate the ability of the transformed cells to metabolize benzo(a)pyrene and convert it into non-reactive, water-soluble conjugates. We also demonstrate that bacterial cells expressing all three recombinant enzymes are able to significantly deplete an incubation mixture of benzo(a)pyrene and thus lower the mutagenic risk to cells subsequently exposed to the incubation mixture.

Materials and Methods: Abbreviations. The following abbreviations are used in this example: bp Base pair BPDE Benzo(a)pyrene-7,8-diol-9,10-epoxide CDNB l-Chloro-2,4-dinitrobenzene CIP Calf intestinal (alkaline) phosphatase DTT Dithiothreitol ECL Enhanced chemiluminescence WO 99/27953 PCT/US98/25315 -21-

EDTA

G6P G6PDH kDa LB-amp

NADP

phosphate,

NADPH

phosphate,

PAGE

PCR

PMSF

SDS

TB

Ethylenediamine tetraacetic acid Glucose-6-phosphate Glucose-6-phosphate dehydrogenase Kilodalton Luria broth with ampicillin (100g/ml) Nicotinamide adenine dinucleotide oxidized form Nicotinamide adenine dinucleotide reduced form Polyacrylamide gel electrophoresis Polymerase chain reaction Phenylmethyl sulfonyl fluoride Sodium dodecyl sulfate Terrific broth Construction of the Construct 1 and Construct 2 Expression Plasmids. The design and structure of two examples of expression plasmids, Construct 1 and Construct 2, are presented in Figure 2. Using Construct 1 as an example, we started with the pCW1A1OR vector constructed by Parikh, Guengerich et al. (Nature Biotech.

784-788, 1997). This bicistronic expression vector containing a full length cDNAs for both CYP 1A1 and human NADPH P450-reductase was kindly provided by Dr. Fred Guengerich, Vanderbilt University. Dr. Guengerich has likewise provided the same vector in which the P450 site contains cDNAs encoding human CYP 1A2, 2E1, 2C9, 2D6, or 3A4. A cDNA encoding human CYP 1B1, modified for efficient expression in bacteria, has been provided by Dr. Colin Jefcoate, University of Wisconsin. With these cDNAs, the sequences of which are publicly available, a variety of P450 expression vectors can be constructed using standard methods. An expression cassette containing a lac i promoter sequence joined to a cDNA encoding the human GST pi isozyme was prepared by PCR amplifying this roughly 700 bp sequence from the pGFLEX vector created in our laboratory (Manoharan et al., Gene WO 99/27953 PCT/US98/25315 -22- 193: 229-237, 1997) and ligating it into a unique restriction site on the pCW1A1OR vector. The resulting plasmid, Construct 1 (or Construct 2 where the lac i-GST cassette is ligated into pCW1A20R) was transformed into DH5a cells, and the transformed cells were streaked on a LB-amp plate. Viable colonies were picked and the plasmids were prepared using the PERFECTprep Plasmid

DNA

Preparation kit. The plasmids were checked for correct incorporation by diagnostic restriction digests, and the successful constructs were sequenced to further confirm correct incorporation, using AmpliTaq DNA polymerase.

Expression of plasmids in E. coli. The procedure of Gillam et al. (Arch. Biochem. Biophys. 305: 123-131, 1993) was used. A single ampicillin-resistant colony of DH5a cells transformed with Construct 1 and Construct 2 plasmid DNA was grown overnight at 37 :C in LB-amp. A 5-ml aliquot was then diluted 1:100 in modified TB [12g bactotryptone, 24g yeast extract, 2g bactopeptone, and 4ml glycerol (liter containing 100mg ampicillin liter 1.0mM thiamine, and 0.25ml of trace elements solution (liter Composition of the trace elements solution: [27g FeCl,.6H-O, 2g ZnCl *4HO, 2g CoC1,*6HO, 2g Na 2 MoO,.2H:O, 1g CaCl 2HO, Ig CuCl

H

3 BO3, and 100ml concentrated HC1 (liter-~)] Incubation was carried out at 30°C for 48 hours.

Membrane fraction preparation. The procedure of Gillam et al. (1993, supra) was used. Cells were chilled on ice and harvested by centrifugation at 5,000g for 10 minutes. The cell pellet was weighed and resuspended in 100mM Tris-acetate buffer (pH 7.6) containing 500mM sucrose and 0.5mM EDTA (15ml buffer per g of wet weight cells). Following the addition of lysozyme (0.2mg the suspension was diluted twofold with chilled HO, and then incubated on ice for minutes. The resulting spheroplasts were pelleted at 10,000g for 10 minutes at 4"C, and resuspended (0.5g ml-) in 100mM potassium phosphate buffer (pH 7.6) containing WO 99/27953 PCT/US98/25315 -23- 6mM magnesium acetate, 20% glycerol and 0.1mM DTT.

At this stage the spheroplast suspension was frozen at 0 C for later use. The suspension was treated with several protease inhibitors: 1.OmM PMSF, 2.0M leupeptin, 0.04U aprotinin and 1.OgM bestatin right before sonication. Spheroplasts were lysed in an ice-salt bath using a Branson Cell Disruptor 185 (Branson Sonic Power Co., Danbury, CT), and centrifuged at 10,000g for minutes at 4 0 C. Supernatant was removed and centrifuged at 180,000g for 65 minutes at 4 0 C. The membrane fraction was resuspended in 50mM Tris-acetate buffer (pH 7.6) containing 250mM sucrose and 0.25mM EDTA using gentle homogenization.

Detection of recombinant enzyme expression by immunoblotting. Bacterial cells transformed with either the Construct 1 or Construct 2 plasmids were grown and harvested, and subcellular fractions were electrophoresed and immunoblotted to demonstrate the expression of each the three recombinant enzymes encoded by the Construct 1 and 2 plasmids. Following is a brief description of how the electrophoresis and immunoblotting was done, but specifics of antibodies used are provided in the descriptions of Figures 3-6.

Cell lysates or subcellular membrane fractions were loaded onto a 12.5% SDS-PAGE. Proteins were separated by a constant current of 10mA for 15 hours at 4 0 C, and were transferred onto a nitrocellulose membrane (Schleicher Schuell, Keene, NH) using a semi-dry blotting aparatus (100mA for 2 hours at 4 0 Nonspecific binding sites on the membrane were blocked overnight at room temperature by 25ml PBST with 10% dry milk. Specific dilutions of primary and secondary antibody are given in the descriptions of the drawings.

After washing the blots three times with PBST, detection of bands was done using the ECL kit (Amersham International plc., Buckinghamshire, England) according to the supplier's instructions.

WO 99/27953 PCT/US98/25315 -24- Results: Functional assays of recombinant enzymespresent in bacterial cells. In Figure 7 and Tables 1-3 below, we show a series of enzyme assays that demonstrate that the recombinant enzymes produced by the Construct 1and Construct 2-transformed cells are catalytically active. Specific details of the individual assays to demonstrate activity of the rec-cytochrome P450 1A1 and 1A2, recNADPH-P450 reductase and recGST pi are provided in the description of Figure 7 and in the tables.

Table 1. Ethoxyresorufin O-deethylation Catalyzed by recCytochrome P450 and recNADPH P450 reductase in Bacterial Cells Containing Construct 1 or 2 Construct Catalytic Activity (fluorescence units/min/ug protein) 1 581.9 1 223.0 2 319.0 2 320.0 Ethoxyresorufin O-deethylation was measured by fluorescence spectroscopy as described by Parikh et al.

(1997). Replicate clones of washed bacterial cells containing either pCW 1Al/reductase/GST pi (Construct 1) or pCW 1A2/reductase/GST pi (Construct 2) were mixed with 2.7 um 7-ethoxyresorufin in 50 mM Tris, 0.1 M NaCl, pH 7.8 buffer. There was no discernible background reaction in washed bacterial cells that did not contain a plasmid.

Table 2. Reduction of Cytochrome C Catalyzed by recNADPH P450 Reductase in Bacterial Cells Containing Construct 1 or 2 Construct Catalytic Activity (Cytochrome C reduced/min/~g protein) 1 24.9 1 11.5 2 87.7 2 41.8 The level of recNADPH P450 reductase in bacterial membranes was measured by its ability to catalyze the reduction of cytochrome c. Bacterial membranes isolated from replicate clones of bacterial cells containing either Construct 1 or Construct 2 were preincubated with 0.5 mM horse heart cytochrome c in 0.3 M potassium phosphate buffer, pH 7.7 for 2 min at 30 0

C

before the addition of 10 mM NADPH. The formation of reduced cytochrome c was monitored at 550 nm.

WO 99/27953 PCT/US98/25315 Table 3. GSH Conjugation of Chloro-dinitro-benzene (CDNB) by recGlutathione-S-Transferase (GST) in Bacterial Cells Containing Construct i or 2 Construct Rate Rate (OD/min/~g protein) (CDNB/GSH conjuqation units) 1 2.24 0.234 1 2.24 0.234 2 2.015 0.21 2 ND

ND

recGST-catalyzed conjugation of glutathione (GSH) to CDNB was measured by monitoring absorbance of the conjugate formed at 340 nm in replicate clones of washed cells containing either Construct 1 or Construct 2. Two mM l-chloro-2,4-dinitrobenzene was added to washed cell suspensions.

Conversion of benzo(a)pyrene to water soluble metabolites by bacterial cells transformed with Construct 1 or Construct 2. The ability of bacterial cells concurrently expressing each of the three recombinant enzymes to take up and metabolize the model procarcinogen benzo(a)pyrene to water soluble metabolites is shown in Table 4. Assays were done using BP as the substrate by the method described by Nesnow, Fahl Jefcoate (Anal.

Biochem. 80: 258-266, 1977). Previous prolonged storage of the [H 3 ]BP at -20°C resulted in some auto-oxidation of the BP and the higher than usual background of DPMs that were present in the aqueous phase in tubes that received no bacterial cells. In bacterial cells expressing the three genes on Construct 1 or Construct 2, large and significant increases were seen in the conversion of the hydrophobic substrate BP to water soluble metabolites.

WO 99/27953 PCT/US98/25315 -26- Table 4. Conversion of Benzo(a)pyrene to Water Soluble Metabolites by Bacterial Cells Containing Construct 1 or Construct 2 that are Concurrently Expressing CYP450 1Al or CYP450 1A2, NADPH-P450 Reductase and GST pi Recombinant enzymes Percent B(a)P Group produced by bacterial cells present as sol.

metabolites postreaction No cells 7.9% none 10.8% Construct 1 Cytochrome P450 1Al, 17.0% NADPH P450 reductase, GST pi Construct 2 Cytochrome P450 1A2, 19.1% NADPH P450 reductase, GST pi ml incubations containing phosphatebuffered saline, washed bacterial cells and 18 nmol (2 x dpm 3 H) benzo(a)pyrene (delivered in 25 pl acetone) were incubated at 37 0 C for 30 minutes. Incubation, separation and quantification of unmetabolized benzo(a)pyrene and water-soluble metabolites were done as previously described (Nesnow, 1977).

Diminished mutagenic potential of a benzo(a)pyrene-containing "meal" after co-incubation with bacterial cells transformed with Construct 1. In a first step for this experiment, conditions were optimized to yield linear, dose-dependent increases in mutant colonies in Ames assay incubations using benzo(a)pyrene as the procarcinogen and TA98 as the tester strain. The mutagenesis assays were performed according to the most recent published protocol (Maron &Ames, Mutat. Res. 113: 173-215, 1983), and the results in Figure 8 show linear, dose-dependent mutation induction at concentrations of benzo(a)pyrene ranging from 10-96 uM.

In Table 5 we show the results of preincubating a benzo(a)pyrene solution with washed bacterial cells transformed with Construct 1 in physiologic buffer in the WO 99/27953 PCT/US98/25315 -27stomach and duodenum of an anesthetized mouse. To perform this experiment, mice were anesthetized with nembutal and then surgically opened with a ventral, midline incision. Ligatures (untied) were placed around the esophagus near the stomach entry and around the small bowel just distal to the duodenum. The small bowel was cut, the feeding needle was inserted orally into the mouse and threaded down to the esophageal opening to the stomach. 3 X 1 ml aliquots of room temperature PBS were injected into the stomach via the feeding needle, and the liquid was gently effluxed from the cut small bowel by exerting mild pressure on the distended stomach. After 3 X 1 ml, the discharged PBS was essentially clear.

Following the third 1 ml rinse, the ligature distal to the duodenum was tied. The 0.9 ml bacteriabenzo(a)pyrene mixture was then delivered to the rinsed, drained stomach-duodenum pouch via the feeding needle, and the esophageal ligature was tied. The feeding needle was withdrawn, the abdominal organs were gently re-positioned in the abdominal cavity, and the midline incision was closed. The continually anesthetized animals were placed beneath warming lamps for the next 30 minutes. After the minute incubation period, the stomach-duodenum pouches were surgically removed, and the contents gently drained into an eppendorf tube.

The incubation material was filtered through a 0.2 A sterile filter, and the filtrate was then used to reconstitute cofactor/TA98 Ames assay incubations. The carcinogen/mutagen component of the Ames incubations was contributed by the benzo(a)pyrene that had been carried through the preceding bacterial incubations in the mouse stomach. No metabolism of the benzo(a)pyrene (originally, 24 nmol) by the bacterial cells in the mouse stomach incubation the pIC20r control cells) would mean a largely intact carryover of the benzo(a)pyrene to the Ames assay and a resulting high TA98 colony count.

Significant metabolism of the benzo(a)pyrene by the WO 99/27953 PCT/US98/25315 -28bacterial cells in the mouse stomach incubation the Construct 1-expressing cells) would mean a diminished carryover of the benzo(a)pyrene to the Ames assay and a resulting lower TA98 colony count.

Table 5. Diminished Mutagenic Potential of a Benzo(a) pyrene-Containing "Meal" After Co-Incubation in the Mouse Stomach/Duodenum of the "Meal" with Bacteria Containing Construct 1 that were Concurrently Expressing CYP4501A1, NADPH-P450 Reductase and GST pi Group Recombinant Enzymes Ames Assay Colony Produced by Bacterial Cells Counts none 288 Construct 1 Cytochrome P4501A1 NADPH P450 reductase GSTpi 172 12 (p<0.01) 0.9 ml incubations containing phosphatebuffered saline (PBS), washed bacterial cells, and 24 nmol benzo(a)pyrene (delivered in 25 gl acetone) were delivered to rinsed, stomach-duodenum pouches in anesthetized C3H mice using an 18 gauge animal feeding needle attached to a 3 cc disposable syringe.

Ames assay incubations were done as described above, and demonstrates that the above colony counts were within the linear part of the mutation induction curve for TA98 and benzo(a)pyrene.

The means and standard deviations of TA98 colony counts were obtained using Microsoft Excel, and the Student t-test was used to determine whether a statistical difference existed between the Construct 1- S containing and pIC20r-containing populations.

The results shown in Table 5 demonstrate a sizable, significant (p<0.01) decrease in the mutagenic potential of the benzo(a)pyrene solution. Basically, there is a positive correlation between conversion of the procarcinogen benzo(a)pyrene to water soluble metabolites (Table 4) and depletion of the mutagenic potential of the benzo(a)pyrene solution. This is an encapsulated example of the present invention.

WO 99/27953 PCT/US98/25315 -29- EXAMPLE 2 Transformation of Lactobacillus gasseri and Lactobacillus brevis Cells with Plasmid-Encoded Genes that Enable Metabolism of the Exogenous Drug Molecules Nitrocefin, Chloramphenicol and Erythromycin Lactobacillus species are amenable to transformation by electroporation. In this example we describe protocols devised for the transformation of Lactobacillus gasseri and Lactobacillus brevis cells.

Media: 1) MRS broth: 10.0 g peptone, 8.0 g meat extract, 4.0 g yeast extract, 20.0 g glucose, 1 mL monooleate (Tween 2.0 g KHPO 4 5.0 g sodium acetate 3H20, 2.0 g citrate, 0.2 g MgS0O,7H,0, 0.05 g MnSO,"4H 0, distilled water to 1 L.

2) MRS plates: MRS broth solidified with 1.5% agar.

3) MRSSM: MRS with 0.5 M Sucrose, 0.1 M MgC..

Electroporation solutions: 1) SM: 326 g sucrose (952 mM), 0.71 g MgC1 6H.O (3.5 mM), distilled water to 1 L.

2) DNA: Dissolve plasmid pGK12 in TE (10mM Tris-HCl, 1 mM EDTA, pH 7.5) to 0.10-1.0 Ag/iL. Ligation mixtures should be ethanol precipitated and washed, then dissolved in TE before electroporation.

Methods: A 25- mL preculture of bacteria gasseri or L. brevis) in exponential growth phase was used to inoculate 100 mL of MRS or MRS supplemented with glycine.

Inoculation was to A6 00 0.25; cultures were incubated at 0 C until the Ao 00 0.6. Cells were harvested by centrifugation at minimum speed (1500 g for 5 min) and the supernatant decanted.

Cells were resuspended in 100 mL of SM (or 100 mL of 1 mM MgCl2 according to an alternate procedure), then washed and re-pelleted as above. Pellets were again resuspended in 100 mL of SM or, in an alternate procedure, 30% PEG, then washed and re-pelleted as above.

WO 99/27953 PCT/US98/25315 Cells were resuspended in 1 mL of SM (or PEG according to an alternate procedure). Aliquots containing 109-10' cells/mL were transferred to micro tubes for electroporation.

The DNA in a volume 5 1/20 of the cell suspension volume was added immediately before electroporation. The mixture was transferred to an ice cold electroporation cuvette with a 2-mm electrode gap.

Different electric pulses were delivered as shown in Table 6.

Immediately following the discharge, 1 mL of MRSSM was added to the cuvette and the resulting diluted cell suspension transferred to a microfuge tube and incubated at 30 0 C for 2 hrs. Undiluted and serial dilutions of the cell suspension were spread on MRS plates containing appropriate selection ingredients antibiotics, color indicators). Plates were incubated at 30 0 C for up to 3 days, to allow for growth of colonies of transformed cells.

Results: Results of the electroporation of L. gasseri are shown in Table 6. These results demonstrate efficient delivery of a plasmid-encoded drug resistance gene to a probiotic bacterial strain, Lactobacillus gasseri, that is reported in the literature to successfully colonize within mouse intestinal sites.

Table 6. Electroporation of Lactobacillus gasseri Strain 100-5.

Resistance Settings Chloramphenicol" colonies 2000 400Q 13 600Q 800Q >100 a. 5.0 Ag/mL chloramphenicol was used for selection of plasmid-containing transformants.

WO 99/27953 PCT/US98/25315 -31b. 2-mm electrode gap cuvettes were used in all electroporations.

c. The resistance setting was the only variable parameter. The other settings were: voltage, 1.5 kV and capacitance, d. 3 Ag of pGK12 plasmid and 100 gL of competent 100-5 cells were used in all electroporations.

e. No colonies were seen on negative control plates, which consisted of 100-5 cells that were not mixed with pGK12 but were subjected to the same electroporation conditions as stated above.

Table 7 below shows the results of electroporation experiments demonstrating efficient delivery and expression of a plasmid-encoded drug resistance gene in a probiotic bacterial strain, Lactobacillus brevis, that is reported in the literature to successfully colonize human intestinal sites.

Expression of the erythromycin resistance gene from the pKTH2121 plasmid expression cassette is of special significance because this vector and its expression cassette configuration provide the likely model of how the drug metabolizing enzyme cDNA sequences of the invention are expressed.

Table 7. Electroporation of Gram-Positive Shuttle Vectors into Lactobacillus brevis cells.

Plasmid Erythromycin" colonies pGK12 1000 pKTH2121 pGKnucMCS a. 2.0 gg//mL erythromycin was used for selection of plasmid-containing transformants.

b. 2-mm electrode gap cuvettes were used in the electroporations.

c. The electroporation settings were 2.5 kV, 200Q, and 25 MF.

d. Optimal number of transformants were obtained using the following DNA concentrations: pGK12, 2 gg; pKTH2121, ng; pGKnucMCS, 4.5 Ag.

e. No colonies were seen on negative control plates, which consisted of L. brevis cells in the absence of plasmid DNA, subjected to the same electroporation conditions as stated above.

WO 99/27953 PCT/US98/25315 -32- The results shown in Table 8 demonstrate that probiotic L. brevis cells transformed with the pKTH2121 plasmid and which stably express the encoded B-lactamase gene are able to metabolize an organic substrate, nirocefin, to yield the red-colored metabolite.

Table 8. /-Lactamase Activity in L. brevis cells Transformed with a plasmid carrying the 3- Lactamase gene Plasmid Presence of red product of B-lactamase substrate Negative control Positive control pKTH2121 Lactobacillus brevis cells were electroporated as described above and by Raya et al.(1992). Following selection of colonies for 48 hours on Lactobacilli

MRS

plates containing 2 Ag/mL erythromycin, portions of the colonies were picked and smeared onto small discs impregnated with the P-lactamase substrate, nitrocefin (Becton Dickinson). Following a 15 minute incubation at room temperature, the discs undergo a color change from yellow to red as the amide bond of the 3-lactam ring is hydrolyzed by P-lactamase. The positive control consisted of E. coli DH5a cells electroporated with pUC119 plasmid; the negative control consisted of L.

brevis cells electroporated with pGK12 plasmid. Each value represents the results of duplicate experiments.

The results shown in Table 8 clearly demonstrate the ability of probiotic bacterial cells to take up and express an exogenous gene that enables the cells to metabolize an exogenous drug molecule. In this case the drug metabolism gene, here encoding B-lactamase, is positioned on a plasmid in the L. brevis cells. For the practice of the present invention, we will take the two recombinant expression cassettes (CYP450/NADPH-P450 reductase and GST) from Construct 1 and all other P450 and GST variants of this plasmid and stably integrate the genes into the genome of the various probiotic host cells WO 99/27953 PCT/US98/25315 -33that are used. This final gene integration and screening to isolate clones with the desirable metabolism phenotypes will be done without using a linked drug resistance marker gene. Such clones, containing integrated copies of the three recombinant enzyme genes, which are efficiently expressed to enable procarcinogen metabolism, are then grown in mass culture for sales and use in the marketplace.

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification without departure from the scope of the appended claims.

Claims (20)

1. A composition that augments the ability of mammalian gastrointestinal cells to detoxify or eliminate procarcinogenic or toxic substances, which comprises at least one probiotic microorganism genetically modified to express one or more mammalian enzymes that catalyze detoxification or elimination of said substances formulated for delivery to a mamnalian gastrointestinal tract.

2. The composition of claim 1, wherein the probiotic microorganism is selected from the group consisting of Lactobacillus, Lactococcus, Bifidobacteria, Eubacteria and non-pathogenic strains of Escherichia coli.

3. The composition of claim 2, wherein the Lactobacillus is a species selected from the group consisting of L. gasseri, L. brevis, L. casei, L. plantarum, L. paracasei, L. acidophilus, L. fermentum and L. zeae.

4. The composition of claim 1, wherein the enzymes are selected from the group consisting of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione-S-transferase, gamma-glutamylcysteine synthetase, N-acetyltransferase, aldehyde dehydrogenase, and aldehyde reductase.

The composition of claim 4, wherein the probiotic microorganisms express at least one form of 30 cytochrome P450, at least one form of NADPH-P450 reductase and at least one form of glutathione-S- transferase.

6. The composition of claim 5, wherein the cytochrome P450 is selected from the group consisting of SCYP 1B1, CYP 1Al, CYP 1A2 and CYP 2E1. WO 99/27953 PCTUS98/25315

7. The composition of claim 5, wherein the glutathione-S-transferase is selected from the group consisting of GST alpha, GST mu, GST pi, and GST theta.

8. The composition of claim 7, comprising a recombinant form of glutathione-S-transferase having improved catalytic activity as compared with an equivalent wild-type glutathione-S-transferase.

9. The composition of claim 1, wherein the procarcinogenic or toxic substances are selected from the group consisting of polycyclic aromatic hydrocarbons, mycotoxins, arylamines, heterocyclic amines, nitrosamines and benzene.

The composition of claim 1, comprising a plurality of different probiotic microorganisms and expressing a plurality of different enzymes. 20

11. The composition of claim 1, formulated for •oral administration.

12. The composition of claim 11, formulated in a fermented milk product.

13. The composition of claim 1, formulated for intranasal administration.

14. A composition that augments the ability 30 of mammalian gastrointestinal cells to detoxify or eliminate procarcinogenic or toxic substances, which comprises at least one species of Lactobacillus genetically modified to express at least one form of cytochrome P450, at least one form of NADPH-P450 reductase and at least one form of glutathione-S- transferase, formulated for delivery to a manmalian gastrointestinal tract.

WO 99/27953 PCT/US98/25315 -36- A method for augmenting the ability of an individual to detoxify or eliminate procarcinogenic or toxic substances from the gastrointestinal cells, the method comprising administering to the individual a probiotic microorganism genetically modified to express one or more mammalian enzymes that catalyze detoxification or elimination of said substances, in a manner enabling the microorganism to produce the enzymes in the gastrointestinal tract of the individual, for a time and in an amount effective to partly or fully detoxify or eliminate the substances.

16. The method of claim 15, wherein the individual is a human.

17. The method of claim 15, wherein the individual is an animal.

18. The method of claim 15, wherein the composition is administered orally.

19. The method of claim 18, wherein the composition is incorporated into food or feed.

20. The method of claim 15, wherein the composition is administered intranasally.