WO2001077366A1 - Positive selection method, compounds, host cells and uses thereof - Google Patents

Abstract

This invention relates to a positive selection method, compounds useful for the positive selection and appropriate hosts. The method permits one to select a host, or auxotroph, which may be a prokaryote or an eukaryote, based on the ability of the host to express an enzyme(s) capable of catalyzing a reaction that converts a precursor molecule into a molecule or factor necessary for the host's survival. This invention encompasses methods useful to find new enzymes expressing a desired activity, methods of selecting host cells, methods of maintaining a plasmid within a host that do not utilize antibiotics, and methods of expressing proteins or other materials for commercial production purposes.

Description

POSITIVE SELECTION METHOD, COMPOUNDS, HOST CELLS AND USES THEREOF

This application claims priority to the United States provisional application serial no. 60/195,911, filed April 10, 2000, which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION This invention relates to a positive selection method, compounds useful for the positive selection and appropriate hosts. The method permits one to select a transformant of an auxotrophpic host cell, which may be prokaryotic or eukaryotic cell, based on the ability of the host to express at lease one enzyme capable of catalyzing a reaction that converts a precursor into a factor necessary for the host's survival, growth, and proliferation. This invention encompasses methods useful to find new enzymes expressing a desired activity, methods of selecting host cells, methods of maintaining a plasmid within a host that do not utilize antibiotics, and methods of expressing proteins or other materials for clinical and/or commercial production purposes.

2. BACKGROUND OF THE INVENTION

2.1 SELECTION METHODS

Cloning DNA into cells is now routine. See for example Cohen et al. U.S. Patent No. 4,237,224 entitled "Process for Producing Biologically Functional Chimeras," the contents of which is incorporated in its entirety into the present application. The technique for introducing DNA into cells typically employs a cloning vector, which is a circular fragment of DNA with an origin of replication, selection marker, and a number of other features. The selection marker is a short segment of DNA that will express an enzyme that confers resistance to an antibiotic. The selection marker must not already be present in the host organism. As a result, only those cells that take up DNA containing this expressible DNA segment will produce the enzyme that will allow the cells to survive in the presence of the antibiotic. This is the key to separating out the vast majority of cells that do not take up the cloning vector. It is referred to positive selection, for it yields only those cells that are desired, that is contain the cloning vector.

Unfortunately, the use of antibiotics for positive selection is suspected as increasing the creation of antibiotic resistant organisms. See for example, Klein, "Food as a potential vector for antibiotic resistances. 2: Relevance of lactic acid bacteria" Berl Munch Tierarztl Wochenschr 113(2): 46-52, 2000; van den Bogaard et al. "Antibiotic usage in animals: impact on bacterial resistance and public health" Drugs 58(4):589-607, 1999; Williams et al. "Containment of antibiotic resistance" Science 279: 1153-1154, 1998, the contents of which are incorporated in their entirety into the present application. This has led some to

5 look for other ways to make cloning vectors, for example Dickely et al. utilize a purine auxotroph to create a food grade cloning vector, Dickely et al. Mol. Microbiol. 15(5): 839- 847, 1995, the contents of which is incorporated in its entirety into the present application. A variety of disclosures have reported complementing autotrophs as a rapid method for isolating genes. Those disclosures are limited in scope because they only indicate that

10 the inserted gene encodes a protein that can replace the function of the auxotroph, examples include Gelpke et al. "Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium." Appl Environ Microbiol 65(4): 1670-4, 1999; Bajmoczi et al. "TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae." Biochem Biophys Res Commun 4;243(l):205-9, 1998; Williams et al. "Isolation by genetic

15 complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger" Curr Genet 30(4):305-l 1, 1996; Peng et al. "Genetic transformation of the pathogenic fungus Wangiella dermatitidis" Appl Microbiol Biotechnol 44(3-4):444-50, 1995; Pla et al. "Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double- ARS

20 shuttle vector" Gene 1995 Nov 7;165(1):115-20; Loubbardi et al. "Sterol uptake induced by an impairment of pyridoxal phosphate synthesis in Saccharomyces cerevisiae: cloning and sequencing of the PDX3 gene encoding pyridoxine (pyridoxamine) phosphate oxidase" J Bacteriol 177(7):1817-23, 1995; Saito et al. "cDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea" FEBS Lett 21;324(3):247-52,

25 1993; Toffaletti et al. "Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA" J Bacteriol 175(5): 1405-11, 1993; Saito et al. "Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase" Proc Natl Acad Sci U S A 89(17):8078-82, 1992; Gruber et al. "The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant

30 strain" Curr Genet 18(l):71-6, 1990; Gil et al. "Cloning and expression of a p- aminobenzoic acid synthetase gene of the candicidin-producing Streptomyces griseus" Gene 25(1):119-32, 1983; Hottinger et al. "Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae" Mol Gen Genet 188(2):219-24, 1982; Hertzberg et al. "Cloning of an EcoRI-generated fragment of the

35 leucine operon of Salmonella typhimurium" Gene 8(2): 135-52, 1980, the contents of which are hereby incorporated by reference in their entirety.

Others have reported methods of transforming eukaryotic cells for production of a desired protein by cotransfecting the cells with two DNA molecules, e.g., Axel et al. U.S. Patent No. 4,634,665 entitled "Processes for Inserting DNA into Eukaryotic Cells and for Producing Proteinaceous Materials," the contents of which is incorporated in its entirety into the present application. In Axel et al. a first DNA molecule encodes a gene or genes for a desired protein and a second DNA encodes a selectable marker, e.g. dihydrofolate reductase. Challenging the cotransfected cells with an appropriate selection agent, e.g., methotrexate, leads to the selection of cells that overexpress both dihydrofolate reductase and the gene encoding the desired protein.

2.2 ASSAYS FOR ENZYMATIC ACTIVITY

Currently, fluorogenic or chromogenic substrates are used to isolate enzymes or genes encoding enzymes having a desired activity such as that of an esterase or an oxidoreductase. For example, the reagent below (Molecular Probes, Eugene, Oregon) could be used to isolate an enzyme that cleaves esters of palmitic acid. Clones possessing the ability to cleave palmitic acid esters would produce the fluorescent product, 7-hydroxy-4- methylcoumarin. However, in this isolation method, each clone must be examined individually to detect those clones that actually produces the fluorescent molecule.

There are two major limitations to the current approach. First, the oxygen bound to the fluorogenic reagent is bound to sp² carbon, yet most ester bonds of biological interest are bound to a sp³ carbon. The fluorogenic reagent should be more labile than the desired substrate. This is an inherent feature of this kind of assay,, since the chromogenic or fluorogenic molecules in the literature require a cleavage or modification of a heteroatom attached to sp² carbon. The second limitation is that one need look at each clone in order to determine which is fluorescent. There is no real means of overcoming the major limitations of this approach, for in order to have a colorimetric or fluorimetric assay one needs a chromogenic or fluorogenic center which require an sp² center. Furthermore, each clone must be examined to see if it produces a fluorescent or colorimetric signal.

Harte et al. report on the enzymatic oxidation of a fluorophore precursor to a fluorescent compound in U.S. Patent No. 5,017,475 entitled "Fluorescent Detection Method Based on Enzyme Activated Conversion of a Fluorophore Precursor Substrate," the contents of which is incorporated in its entirety into the present application. In Harte et al. compounds such as 1,2-diaminobenzene (also known as o-phenylenediamine and referred to as OPD), 3,4-diaminobenzoic acid, or 2,3-diaminopyridine are enzymatically oxidized to their corresponding ortho-fused pyrazine such as aminophenazine which fluoresces. Thus, they provide an assay for enzymatic oxidizing agents. However, the methodology of Harte et al. is very limited in scope, in that it only works for detection of enzymes that oxidize specific substrates and thus a small subset of the oxidases may be detected. It cannot be used to detect other enzymatic activities, e.g. reductases, hydroxylases, etc. Morever it has the limitations discussed above regarding both the type of bond formed (an sp² center) and the need to look for the flourescent activity.

Similarly, Joo et al. disclose the evolution of cytochrome P450 hydroxylation by an assay in which the P450 hydroxylation products are further oxidized by horse radish peroxidase to fluorescent dimers and polymers. Joo et al. Nature 399: 670-673, 1999, the content of which is incorporated in its entirety into the present application. It is only a method for a specific oxidase. Here again, the product is an sp² center carbon oxygen bond and one must look at all of the samples to detect fluorescence.

Haugland et al. report an enzymatic screen based on a fluorophore covalently linked to a blocking group through an sp² center carbon oxygen bond, in U.S. Patent Nos. 5,316,906 and 5,443,986 entitled "Enzymatic Analysis Using Substrates that Yield Fluorescent Precipitates," the contents of which are incorporated in their entirety into the present application. Haugland forms a fluorescent phenolic product by hydrolysis of a phenolic ester or a phenolic glycoside, e.g., by phosphatase, sulfatase, glycosidase and esterase enzymes. Alternatively, the phenolic product may be formed by oxidation of aryl alkyl ethers, e.g. by cytochrome enzymes. Once again it has the limitations that it applies only to sp² centers and one must look for fluorescence.

Robertson et al. disclose an assay to detect esterase activity by forming a fluorescent coumarin product as described above. Robertson et al. SIM NEWS 46(1): 3-8, 1996, the contents of which is incorporated in its entirety into the present application. Here again, the assay is limited in that an sp² center carbon oxygen bond is formed and one must look for fluorescence. Thus, there is a need for a new positive selection method for biological activity. Ideally, the assay would yield exactly what was desired and nothing else, so many more clones can be screened. There would be no need to search for positives, for the assay would yield only positives. Moreover, the assay would not require chromophores or fluorophores, so the designed substrates can be much more like their targets. The sp² center can be replaced by the more likely sp³ center. In this way the assay will be more accurate and efficient with a million assays or more done on a 10-cm plastic petri dish, overnight.

3. SUMMARY OF THE INVENTION The present invention provides a method for detecting a desired enzymatic activity present in a population of cells, such as an expression library. The invention further provides novel enzyme substrates, vectors, host cells, and precursors for various enzymatic activities that can be used in the method.

In one embodiment, the method of the invention comprises providing a plurality of host cells that are auxotrophic for a factor and that are genetically engineered to express at least one enzymatic activity; contacting the genetically engineered host cells with a precursor of a factor, wherein the precursor cannot be converted to the factor by a non- genetically engineered host cell, and wherein the precursor is converted to the factor by the desired enzymatic activity; and (c) culturing the genetically engineered host cells of step (b) under auxotrophic condition such that survival of genetically engineered host cells indicates the expression of the desired enzymatic activity by the genetically engineered cells. In another embodiment, the method of the invention comprises contacting host cells that are auxotrophic for a factor with a precursor of the factor, wherein the precursor cannot be converted to the factor by the host cell, and wherein the precursor is converted to the factor by the desired enzymatic activity; and (b) transforming the host cells such that the host expresses at least one enzymatic activity; and (c) culturing the transformed host cells of step (b) under auxotrophic condition such that survival of transformed host cells indicates the presence of the desired enzymatic activity in or associated with the transformed host cells. In addition, the invention also includes a method for detecting a desired enzymatic pathway. The desired enzymatic activity or activities may, in non-limiting examples, be that of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. The desired enzymatic activity is associated with the making or breaking of, for example, an amide bond, an amine bond, a carbon carbon bond, carbon hydrogen bond, a carbon oxygen bond, a carbon nitrogen bond, a carbon phosphorous bond, a carbon sulfur bond, an ester bond, an ether bond, a nitrogen oxygen bond, a nitrogen phosphorous bond, nitrogen sulfur bond, an oxygen phosphorous bond or a phosphorous sulfur bond.

The factor is an amino acid, an amino acid biosynthetic intermediate, a carbohydrate, a cofactor, or a cofactor biosynthetic intermediate, a lipid, a lipid biosynthetic precursor, a nucleotide or a nucleotide biosynthetic intermediate.

For use with the methods of the invention are replicable vectors which can be used for introduction into a host cell that is auxotrophic to a factor. The vector may comprise a cloning site for insertion of a gene of interest, and an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor. Also encompassed in the invention are host cells that are auxotrophic for a factor and that are genetically engineered to express at least one enzymatic activity, i.e., gene expression libraries. Such host cells may comprise an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor.

The invention further provides a kit that comprises a first container comprising a host cell that is auxotrophic to a factor; and a second container comprising a replicable vector for introduction into the host cell, said vector comprising an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor. Optionally, the kit may comprise instructions for using the components of the kit according to the methods of the invention. The kit may also comprises a third container comprising a precursor to the factor.

In yet another embodiment of the present invention, cleavage of a non-toxic precursor molecule by an enzymatic activity releases a toxic compound. In this embodiment, compounds that inhibit the enzymatic activity prevent conversion of the non-toxic precursor into a toxic compound. Thus, in an alternative embodiment, the invention includes a method for detecting an enzyme inhibitor(s) which comprises (a) providing a plurality of host cells that are genetically engineered to express an enzyme; (b) contacting the genetically engineered host cells with a precursor of a toxin, wherein the precursor cannot be converted to the toxin by a non-genetically engineered host cell, and wherein the precursor is converted to the toxin by the enzyme; and (c) culturing the genetically engineered host cells of step (b) in the presence of a test composition that may comprise an inhibitor of the enzyme such that survival of genetically engineered host cells indicates the presence of an inhibitor of the enzyme. 4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a plot of the precursors fed to ATCC # 14561 M9 (Acetate). Abbreviations: P = pantetheine; S-Ac = S-acetyl P; S-Pro = S-propionyl P; S-Bu = S-butyryl P; Tri-Me Ac = S-trimethylacetyl P; t-Bu Ac = S-tbutylacetyl P. See Example 2 for the experimental details.

Figure 2 shows a plot of the precursor fed to ATCC # 14561 M9 (Glycerol). Abbreviations: P = pantetheine; S-Ac = S-acetyl P; S-Pro = S-propionyl P; S-Bu = S-butyryl P; Tri-Me Ac = S-trimethylacetyl P; t-Bu Ac = S-tbutylacetyl P; S-Me = S-methyl P. See Example 2 for the experimental details.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a positive selection method for detecting a desired enzymatic activity present in a population of cells, such as a gene expression library. The invention further provides novel enzyme substrates, vectors, host cells, and precursors for various enzymatic activities that can be used in the method.

In one embodiment, the method of the invention comprises providing a plurality of host cells that are auxotrophic for a factor and that are genetically engineered to express at least one enzymatic activity; contacting the genetically engineered host cells with a precursor of a factor, wherein the precursor cannot be converted to the factor by a non- genetically engineered host cell. As the host cell is auxotrophic to the factor, the factor is essential for the host to survive under auxotrophic condition. The precursor is converted to the factor by the desired enzymatic activity and by culturing the genetically engineered host cells under auxotrophic condition, survival, growth or proliferation of the genetically engineered host cells indicates the presence of the desired enzymatic activity in the genetically engineered cells. In this positive selection assay, only cells that are positive for the desired enzymatic activity can grow and thus be identified.

As used herein, the term "genetically engineered host cells" refers to any host cells which have been manipulated by recombinant DNA techniques, including but not limited to, transformation, transfection, site-directed mutagenesis, and other techniques by which foreign nucleic acid molecules can be introduced into the host cell.

As used herein the term "factor" refers to a molecule that satisfies an auxotrophic requirement of a cell or organism, enabling that auxotrophic cell to survive, grow, and proliferate. The factor can be an amino acid, an amino acid biosynthetic intermediate, a carbohydrate, a cofactor, or a cofactor biosynthetic intermediate, a lipid, a lipid biosynthetic precursor, a nucleotide or a nucleotide biosynthetic intermediate. As used herein, the term "precursor", or the phrase "precursor of the factor", which are used interchangeably, refers to a molecule that comprises a factor coupled with a protective group, or a molecule that can be enzymatically converted to the required factor, e.g., by oxidation or reduction etc.. That is, as used herein, a precursor per se cannot be cleaved or metabolized by the host organism, e.g. , an auxotrophic host cell requirement of a cell or organism, to provide the factor that will satisfy the auxotrophic requirement. However, contacting a precursor of the present invention with a desired enzymatic activity provides the factor that satisfies the auxotrophic requirement of a cell or organism, enabling that auxotrophic cell to survive, grow, and proliferate. The present invention encompasses such precursors, most of which are novel synthetic enzyme substrates and do not occur naturally in a host cell.

According to the invention, the method can also be carried out by first contacting a suitable host cell with a precursor of a factor that is essential for the host to survive, grow, and proliferate, followed by transforming the host cell such that the host expresses at least one new enzymatic activity; and culturing the transformed host cell such that the survival, growth, and proliferation of the transformed host cell indicates the presence of the desired enzymatic activity.

In addition, the invention can also be used for detecting a desired enzymatic pathway, especially in prokaryotes in which genes encoding enzymes for a pathway are colinear in a piece of genomic DNA which may be cloned into a host cell.

The invention also includes a method for producing a protein of interest comprising introducing into a host a first expressible gene encoding the protein of interest and a second expressible gene encoding an enzyme. The host cell is auxotrophic for a factor which is produced by the enzyme upon its reaction with a precursor of the factor which is added to the culture medium. The host cells are cultured under auxotrophic condition in the presence of the precursor, such that the enzyme converts the precursor to the factor thereby allowing the host to survive, grow, proliferate, and produce the protein. In specific embodiments, the first expressible gene and the second expressible gene are present in a single nucleic acid molecule. Alternatively, the first gene and the second gene(s) are on two separate nucleic acid molecules.

In yet another embodiment, the present invention provides a method for identifying inhibitors of an enzyme which utilizes the cleavage of a non-toxic precursor molecule by the enzyme to release a toxic compound. In this embodiment, compounds that inhibit the enzymatic activity prevent conversion of the non-toxic precursor into a toxic compound. The toxic compound is lethal to the host cells. Thus, the method comprises providing a plurality of host cells that are genetically engineered to express an enzyme; contacting the genetically engineered host cells with a precursor of a toxin, wherein the precursor cannot be converted to the toxin by a non-genetically engineered host cell, and wherein the precursor is converted to the toxin by the enzyme. The genetically engineered host cells are cultured in the presence of a test composition that may comprise an inhibitor of the enzyme. Survival of the genetically engineered host cells indicates the presence of an inhibitor of the enzyme in the test composition.

In the methods of the invention, the host cells may be a prokaryotic cell or an eukaryotic cell, including but not limited to an archaebacterial cell, an eubacterial cell, a cyanobacterial cell, a fungal cell, a plant cell, a mammalian cell, and a human cell. In various embodiments, the host cells are transformed with a nucleic acid molecule foreign to the host. Alternatively, the host cell is transformed by exposure to a physical or a chemical mutagen, whereby a new, desired enzymatic activity is generated in the mutagenized host cell. Accordingly, as used herein, the term "transformed" encompasses, inter alia, the introduction of a cloned gene into a host cell as well as the generation of a new, desired enzymatic activity in the host cell via mutagenesis using reagents and methods well known to those of skill in the art, such as radiation and carcinogens.

The desired enzymatic activity or activities may, in non-limiting examples, be that of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. The desired enzymatic activity is associated with the making or breaking of, for example, an amide bond, an amine bond, a carbon carbon bond, carbon hydrogen bond, a carbon oxygen bond, a carbon nitrogen bond, a carbon phosphorous bond, a carbon sulfur bond, an ester bond, an ether bond, a nitrogen oxygen bond, a nitrogen phosphorous bond, nitrogen sulfur bond, an oxygen phosphorous bond or a phosphorous sulfur bond. The desired enzymatic activity or activities may be selected, e.g., from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. The desired enzymatic activity is associated with the making or breaking of e.g., a bond selected from the group consisting of amide bond, an amine bond, a carbon carbon bond, carbon hydrogen bond, a carbon oxygen bond, a carbon nitrogen bond, a carbon phosphorous bond, a carbon sulfur bond, an ester bond, an ether bond, a nitrogen oxygen bond, a nitrogen phosphorous bond, nitrogen sulfur bond, an oxygen phosphorous bond, and a phosphorous sulfur bond.

More specifically, the bond made or broken may also be a carbon-carbon double bond, carbon-carbon triple bond, carbon-oxygen double bond, carbo- nitrogen double bond carbon-nitrogen triple bond, carbon-phosphorous double bond, nitrogen-nitrogen double bond, nitrogen-oxygen double bond, nitrogen-phosphorous double bond, nitrogen-sulfur double bond, oxygen-phosphorous double bond, or phosphorous-sulfur double bond. A variety of molecules can be used as the factor of the invention, such as but not limited to, an amino acid, an amino acid biosynthetic intermediate, a carbohydrate, a cofactor, or a cofactor biosynthetic intermediate, a lipid, a lipid biosynthetic precursor, a nucleotide or a nucleotide biosynthetic intermediate.

The amino acid or amino acid intermediate is selected, in certain embodiments, from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, D-proline, serine, threonine, tryptophan, tyrosine, valine, (S)-2- acetolactate, 2-aceto-2-hydroxy-butyrate, 3-amino isobutyrate, 5-amino valerate, anthranilate, anthranilic acid, N-carbamoyl aspartate, 3-carboxy-3-hydroxy-isocaproate, chorismate, cystathione, 5-dehydroquinate, 5-dehydro-shikimate, 2,3-dihydroxy-3-methyl- valerate, dimethylcitraconate, 3-enolpyruvyl-shikimate-5-phosphate, erythrose 4-phosphate, glutamic g-semialdehyde, gistamine, histidinol, histidinol phosphate, homocysteine, 3- hydroxy anthranilate, p-hydroxyphenyl-pyruvate, 4-hydroxy-D-proline, 4-hydroxy proline, 4-hydroxy-benzoate, imidazole acetol phosphate, imidazole glycerol phosphate, indole, isochorismate, α-etobutarate, oc-ketoglutarate, 3-mercapto pyruvate, 3-methyl aspartate, (S)- methylmalonate semialdehyde, oxaloacetate, 2-oxo-5-amino valerate, 2-oxobutyrate, 2-oxo- 4-hydroxy-5-amino valerate, 2-oxo-isocaprate, 2-oxo-isovalerate, 2-oxo-3-methyl-valerate, 5-oxoproline, phenyl-pyruvate, phosphoenopyruvate, 3-phosphoglycerate, 3- phosphohydroxypyruvate, 3-phosphoserine, prephenate, pyrrole-2-carboxylate, D'-pyrroline-5-carboxylate, pyruvate, ribose 5-phosphate, S-adenosylmethionine, S-adenosyl-homocysteine, shikimate or 2-succinyl benzoate.

The carbohydrate is galactose, D-galacturonate, D-gluconurate, D-gluconurate-1- phosphate, glucose, inositol, lactose, maltose or myoinisitol.

The cofactor, or cofactor biosynthetic intermediate is p-amino benzoic acid, 2- amino-3-carboxy-muconate semialdehyde, 2-amino-4-hydroxy-6-(D-erythro 1-2-3 - trihydroxypropyl-)dihydropterine, 2-Amino-4-hydroxy-6-(D-erythro 1 -2-3 - trihydroxypropyl-)dihydropterine-triphosphate, 2-amino-4-hydroxy-6-hydroxy-methyl- dihydropterine, 2-amino-4-hydroxy-6-hydroxy-methyl-dihydroρterine-diphosphate, (4- aminophenyl)-l,2,3,4-tetrahydroxypentane, D-aminolevulinate, 2-amino-muconate, 2- amino-muconate semialdehyde, L-ascorbate, biotin, α-carotene, β -carotene, catechol, coenzyme A, cyanocobalamin, α-cytochrome(s), 2-dehydropantoate, dephospho- coenzyme A, dethiobiotin, 7,8-dihydrofolate, 7,8-dihydromethanopterin, 7,8- dihydroopteroate, dihydro-nicotinamide adenine dinucleotide , dihydro-nicotinamide adenine dinucleotide phosphate, dihydrothymine, l,4-dihydroxy-2-naphthoate, dolichol, ergocalciferol, ergosta-5,7,22,24(28)-tetraen-3-ol, ergosterol, flavin mononucleotide, folate, folic acid, heme, homogestinate, 3-hydroxy-L-kynurenine, 6-hydroxy-nicotinic acid, 2- hydroxy-6-polyprenyl phenol, 3-isopropyl-pimylate, kynurenate, L-kynurenine, lipoic acid, menaquinol, menaquinone, 8-mercapto octinoic acid, nicotinamide, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, nicotinate, nicotinate- nucleotide, nicotinic acid, pantetheine, panthenic acid, pantoic acid, pantothenol, N- pantothenoyl-cysteine, 4'- phospho-pantetheine, 4'- phospho-pantothenate, 4'- phospho- pantothenoyl cysteine, phylloquinol, phylloquinone, pimelic acid, plastoquinone, 2- polyprenyl phenol, pyridoxal, pyridoxamine, pyridoxine, l-pyrroline-4-hydroxy-2- carboxylate, quinate, quinolinate, quinolinate-nucleotide, retinal, retinol, riboflavin, S- adenosylmethionine, 5,6,7,8-tetrahydrofolate, 5,6,7,8-tetrahydromethanopterin,'6(R)- 5,6,7,8-tetrahydrobiopterin, 6(R)-pyruvoyltetrahydropterin, 6(S)-5,6,7,8-tetrahydrofolate, thiamine, thiamine pyrophosphate, thymine, α-tocopherol, α-tocopherol(s), ubiquinol, ubiquinone or vitamin K 1.

The lipid or lipid biosynthetic intermediate is acetate, betaine aldehyde, betane, carnitine, ceramide, cholesterol, choline, creatine, cycloartenol, 7-dehydrocholesterol, 3- dehydro-sphinganine, delta-3-isopentyl-pyrophosphate, 1,4-desmethyl cycloartenol, 1,4- desmethyl lanosterol, dimethylallyl-pyrophosphate, dimethyl glycine, ethanol amine, farnesyl-pyrophosphate, geranol, geranyl-pyrophosphate, lanosterol, lathosterol, methyloxalo acetate, mevalonate, mevalonate-5 -phosphate, mevalonate-5-pyrophosphate, mevalonolactone, psychosine, sarcosine, sphinganine, sphingosine, squalene, (S)-squalene- 2,3-epoxide or zymosterol.

The nucleotide or nucleotide biosynthetic precursor is adenine, adenosine, adenosine-5'-diphosphate, adenosine-5'-phosphate, adenosine-5'-triphosphate, cytosine, cytidine, cytidine -5 '-diphosphate, cytidine -5'-phosphate, cytidine -5'-triphosphate, 2'- deoxy-cytidine, 2'-deoxy-cytidine -5'-diphosphate, 2'-deoxy-cytidine -5'-phosphate, 2'- deoxy-cytidine -5'-triphosphate, 4,5-dihydroorotate, 2,5-dihydroxy-pyridine, guanidine, guanosine, guanosine -5'-diphosphate, guanosine -5'-phosphate, guanosine -5'-triphosphate, inosine, orotate, orotidine-5'-phosphate, thymidine, thymidine -5'-diphosphate, thymidine - 5'-phosphate, thymidine -5'-triphosphate, uracil, 3-ureido-isobutyrate, uridine, uridine-5'- diphosphate, uridine-5'-phosphate, uridine-5'-triphosphate xanthanoside or xanthurenate. 5.1 SYNTHESIS OF THE PRECURSORS

In various embodiments of the present invention, a factor is used in the methods that can "break the auxotrophy," i.e. satisfy the auxotrophic requirement of a cell or organism when the cell or organism is cultured under auxotrophic condition. Structurally, the factor is coupled to a protective group to form a precursor. Functionally, as a result of the coupling, the factor in the precursor cannot be used by the auxotrophic cell or organism. Accordingly, the invention encompasses a wide varieties of precursors, i.e., novel molecules with unique structures and functional specificities that allow their use in the methods of the invention. In many instances, the precursor is a synthetic, non-naturally occurring enzyme substrate; the precursor is not a naturally-occurring metabolic intermediate of the host cell. In one non-limiting example, the factor comprises a hydroxyl moiety that is used to couple that factor to a protective group in a number of ways. For example, the hydroxyl moiety of the factor may be coupled to a carboxylic acid in the presence of DCC (dicycloxehylcarbodimide) to generate an ester. Such precursors are used to isolate esterases, wherein the specificity of the esterase is determined by the properties of the carboxylic acid.

In another example, a hydroxyl moiety of a factor is reacted with sodium hydride to generate the sodium alkoxide thereof, which is coupled with an alkyl halide to generate a precursor comprising the factor coupled to the protective group via an ether linkage. Such precursors are used to isolate etherases, which cleave the ether residue to yield the factor that would break the auxotrophy. The specificity of the etherase is determined by the properties of the two groups linked by the ether bond. Furthermore, such compounds could be used to isolate oxidoreductases. That is, the ipso ether carbon is oxidized to yield a hemiacetal, which would hydrolyse to yield the factor. Again, he specificity of the enzyme is determined by the properties of the groups linked by the ether bond.

In other embodiments, a hydroxyl moiety of a factor is oxidized to an aldehyde moiety, which can be used to couple the factor to protective groups. For example, such precursors are used for the isolation of oxidoreductases which reduce aldehydes to alcohols. In other embodiments, a hydroxyl moiety of a factor is oxidized to a carboxylic acid. Such precursors could be used for the isolation of oxidoreductases which reduce carboxylic acids to alcohols, thereby providing the factor that can be used by the auxotrophic cell or organism to satisfy the auxotrophic requirement.

In another embodiment, a hydroxyl moiety of a factor is converted to an alkyl halide. Such precursors are used for the isolation of enzymes which replace halides with hydroxy groups. In still another embodiment, the hydroxyl moiety of the factor is converted to a thiol moiety, and the resulting precursor used for the isolation of enzymes which replace thiols with hydroxy groups.

In yet another embodiment, a hydroxyl moiety of a factor is converted to a thioether moiety and the resulting precursor used for the isolation of enzymes which replace thioethers with hydroxy groups. In a further embodiment, a hydroxyl moiety of a factor, is converted to an olefin, and the resulting precursor used for the isolation of enzymes which convert olefins to hydroxy groups. For example, the hydroxyl moiety is eliminated to form an alkyl group, and the resulting precursor is used for the isolation of enzymes which replace alkyl hydrogens with hydroxy groups. In another embodiment, a hydroxyl moiety of a factor is converted to a phosphate ester and the resulting precursor used for the isolation of enzymes which convert phosphate esters to alcohols.

In a further embodiment, the hydroxyl moiety is converted to an amine, substituted amine, imine moiety, or subsituted imine, and the resulting precursors used, respectively, for the isolation of enzymes which convert amines to alcohols, substituted amines to alcohols, imines to alcohols, or substituted imines to alcohols.

In a further embodiment, the hydroxyl moiety is converted to a carbon silicon bond, and the resulting precursor used for the isolation of enzymes which convert carbon silicon bonds to alcohols. The following references, each of which is incorporated herein in its entirety, describe methods by which the precursor molecules may be synthesized: Various editors, Organic Syntheses (John Wiley & Sons: New York) 1921-2000 Volumes 1-79; Breuer, E., Aurich, H. G., Patai, S., and Rappoport, Z., Nitrones, nitronates, and nitroxides (John Wiley & Sons: New York) 1989; Fieser, L. F., Fieser M., Reagents for Organic Synthesis (John Wiley & Sons: New York) 1967-99 Volumes 1-19; Hartley, F. R., The Chemistry of organophosphorus compounds (John Wiley & Sons: New York) 1990; Larock, R. C, Comprehensive organic transformations : a guide to functional group preparations (VCH Publishers: New York) 1989; March, Jerry, Advanced organic Chemistry Reactions, Mechanisms, and structure, fourth edition (John Wiley & Sons, New York) 1992; Patai, S. Ed, Chemistiy of functional groups (John Wiley & Sons: New York); Patai, S., The

Chemistry of alkanes and cycloalkanes / (John Wiley & Sons: New York) 1992; Patai, S., The Chemistry of amidines and imidates / (John Wiley & Sons: New York) 1975-91; Patai, S., The Chemistry of cyanates and their thio derivatives (John Wiley & Sons: New York) 1977; Patai, S., The Chemistry of diazonium and diazo groups ( John Wiley & Sons: New York) 1978; Patai, S., and Rappoport Z., The Chemistry of enones ( John Wiley & Sons: New York) 1989; Patai, S., Chemistry of ethers, crown ethers, hydroxyl groups and their sulphur analogues (John Wiley & Sons: New York) 1980; Patai, S., The Chemistry of ketenes, allenes and related compounds (John Wiley & Sons: New York) 1980; Patai, S., The chemistry of organic arsenic, antimony, and bismuth compounds ( John Wiley & Sons: New York) 1994; Patai, S., and Rappoport Z.; The Chemistry of organic selenium and tellurium compounds (John Wiley & Sons: New York) 1986; Patai, S., The Chemistry of peroxides (John Wiley & Sons: New York) 1983; Patai, S., The Chemistry of sulphinic acids, esters and their derivatives (John Wiley & Sons: New York) 1990; Patai, S., Rappoport Z. and Stirling, C, The Chemistry of sulphones and sulphoxides (John Wiley & Sons: New York) 1988; Rappoport Z., The Chemistry of enols (John Wiley & Sons: New York) 1990.

5.2 METHODS OF THE INVENTION

The present invention is further illustrated by way of a non-limiting example comprising a cell or an organism that is auxotrophic for at least one factor, a non-limiting example of which is pantothenic acid. Such an organism, therefore does not survive, grow, and proliferate in the absence of the pantothenic acid factor. One of ordinary skill in the art will readily recognize that in addition to the specific factors listed above, host cells that are auxotrophic for any one of a wide variety of factors may be prepared. For example, a host that requires pantothenic acid may be isolated in a number of ways, such as deletion of at least one

Pantothenic acid

gene necessary for the biosynthesis of pantothenic acid, or the insertion of a transposon, or the introduction of a point mutation into one or more genes required for the biosynthesis of pantothenic acid. Although the used of auxotrophic cells or organisms generated by point mutations are within the scope of this invention, they are not preferred in certain embodiments, however, since they may be "leaky" and/or difficult to characterize. Gene deletions, which may be prepared for example by homologous recombination and allele exchange procedures well known to those of ordinary skill in the art, may be more laborious to prepare but are preferable for certain embodiments of the present invention. Such deletions are facilitated by the availability of the complete genomic sequence of a number organisms. Transposition mutagenesis and the generation of transposon-insert libraries are also used to generate auxotrophic mutants, which inserts are readily characterized, e.g., by using PCR analyses, and thus are often preferable in certain embodiments.

As would be appreciated by one of ordinary skill in the art, transformation of an auxotrophic cell or organism, as that term is used herein, also provide, e.g., a back-mutant or revertant of a point mutation by mutagenesis, or a genetically-complemented host wherein a heterologous gene complements the genetic defect in the auxotrophic host employed. However, such "background" results are readily distinguished from expression of a desired enzymatic activity that converts a precursor to a factor, since revertants and genetically-complemented cells or organisms are prototrophs; that is, they will survive, grow, and proliferate in the absence of the factor or the precursor. In contrast, auxotrophic hosts transformed to express a desired enzymatic activity that converts a precursor to a factor will not be able to survive, grow, and proliferate in the absence of the factor or a precursor that can be converted to the factor.

In an alternative embodiment, an auxotrophic host organism may be obtained from the ATCC (American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-220, www.atcc.org) or other commercial source. By way of example, available bacterial, yeast and fungal auxotrophs are listed in Tables 1-3 below. One skilled in the art will readily recognize that there are a variety of ways to prepare or obtain the auxotrophic hosts use in the present invention. Once the stable auxotroph has been constructed, obtained or purchased, then it can be tested for its ability to survive, grow and proliferate only if the medium is supplemented with the factor it is auxotrophic for, e.g., pantothenic acid. If the organism was grown on a medium containing the following precursor, it would not survive, unless it possessed

CujEsterasse

Designed reagent Pantothenic acid an enzyme that could cleave the palmitic acid ester. Without this ability, the auxotrophic organism will die.

Therefore, in one embodiment of the present invention, a cloned, genomic library is introduced into an auxotrophic host that requires pantothenic acid, and a clone expressing the desired esterase is selected by growth in the absence of exogenous pantothenic acid. Because this method provides a positive selection for those clones expressing the desired enzymatic activity, the method can be used to screen large numbers of clones for a desired enzymatic transformation. That is, in this assay, only those clones that produce the enzyme or enzymes that are capable of converting the precursor to the factor will survive. In certain preferred embodiments of the present invention, the following components are desirable:

1. A stable and well-defined auxotroph. Any organism or cell from which an auxotrophic isolate can be obtained or generated, can be used in the present invention, including, for example, bacterial, fungal, archaebacterial, cyanobacterial, plant, and mammalian cells, etc.

2. A precursor, comprising a factor and a protective group or a modified factor, (e.g. a factor that has been oxidized, reduced or otherwise chemically or enzymatically altered), that cannot be converted by the auxotrophic host to the factor. That is, if the organism possesses the ability to convert the precursor to the factor, then there will be no selection. 3. The precursor must be stable in the culture medium and not release the factor in the absence of the desired enzymatic activity. 4. The auxotrophic cell or organism is transformable, that is heterologous DNA or

RNA from other organisms can be introduced into and expressed by the host. If the expressed foreign DNA or RNA produces an enzyme or enzymes that convert the precursor to the factor, then a clone expressing that desired enzymatic activity will survive.

The claimed invention offers a number of advantages, for example, it permits the screening of extremely large numbers of clones, and the assay results in only positive clones. The method is not dependent upon fluorogenic or chromogenic substrates. Fluorogenic or chromogenic molecules are typically not natural substrates. Instead, they were chosen for their usefulness in visualizing the transformation. These typically involve cleavage of a heteroatom bound to sp² carbon, yet the transformation that is being assay for involves a heteroatom attached to a sp³ carbon. In contrast, the assay reagents of the invention may be designed so that they have a much closer chemical and biological resemblence to either the natural substrates or desired reagents of interest. The method is adaptable to a wide variety of cells and organisms, e.g. , fungi, bacteria including archaebacteria, eubacteria, and cyanobacteria, plant cells, mammalian cells etc. Any host cells that can be made auxotrophic and transformed with heterologous DNA may be used. The method of the invention is not dependent exclusively upon cleavage of a bond as is the case of fluorogenic reagents. Coupling enzymes and oxidases can be assayed for example:

d

Oxidase

Designed reagent Pantothenic acid

Using the example above, any of the designed reagents could be used to isolate enzymes that can cleave palmitic acid esters. Any of the following hydroxy containing compounds could be used: pantoic acid, pantothenic acid or pantothen, or the amino acids tyrosine or serine. Thus, one need only have the appropriate auxotroph and the designed reagent. The choice of auxotroph allows for the adjustment of the sensitivity of the assay. Typically, pantoic acid is required in the micromolar range while tyrosine and serine are required in the millimolar range.

Pantoic acid Pantothenic acid

Another advantage of the method is the possibility that this approach will allow for the isolation of functional subunits, which are inactive. In a natural system, without genetic modifications, the subunits of type I polyketide synthases do not function to produce molecules if all of the subunits and the appropriate starter units are not present. In principle, positive selection using designed reagents may allow these subunits to be isolated.

Positive selection as claimed herein can be used as a screen for the isolation of chemically synthesized or natural product enzyme inhibitors. In this alternative embodiment, the invention provides the following: a cell clone that expresses a target enzyme; a precursor molecule that will produce a toxic molecule that will kill the clone if the expressed target enzyme is active. In the method, all cells expressing the target enzyme grown in the presence of the precursor molecule will die. However, for the cells growing in the wells of an assay plate or inside a screening unit, such as gel droplets (see e.g., WO 98/41869), that contains an inhibitor of the enzyme, the inhibitor prevents the action of the target enzyme and the consequent production of the toxic molecule. As a result, the cells in such wells or screening unit survives and can be detected by any methods known in the art.

5.3 VECTORS, HOST CELLS OF THE INVENTION

The invention further provides replicable recombinant DNA vectors which can be introduced into a host cell that is auxotrophic to a factor. The vector may comprise a cloning site for insertion of a gene of interest, and an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor. Preferably, the precursor is not a naturally occurring metabolic intermediate of the host cell; and the enzyme is not one that is naturally occurring in the host cell.

The term "expression vehicle or vector" refers to a plasmid or phage or virus, for expressing a polypeptide from a nucleotide sequence. An expression vehicle can comprise a transcriptional unit, also referred to as an expression construct, comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and which is operably linked to the elements of (1); and (3) appropriate transcription initiation and termination sequences. "Operably linked" refers to a link in which the regulatory regions and the DNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation. Accordingly, the vectors of the invention can be used to build a new form of cloning vector wherein the selection is governed by enzymatic conversion of a precursor of a factor to a factor that breaks the auxotrophy of a host cell. Thus, no toxic antibiotics are needed to maintain selection. This system of selection and gene expression may be useful for large- scale production of proteins where the presence of antibiotics is undesirable. The precursor of the factor can be designed and tested such that it is non toxic and can ultimately be metabolized to form primary metabolites. The elimination of growth inhibitor, such as antibiotics, helps reduce the complexity and cost of downstream processing of large scale cell culture and fermentation. The use of such a vector-host cell system eliminates the need for antibiotic-based selection and may help reduce the number of antibiotic-resistant strains of bacteria.

The system can also be designed to allow the selection of an enzyme based on the biochemical and/or physical properties of the protein of interest. For example, one may choose an enzyme for conversion of the precursor that is much larger or smaller than the protein to be expressed thus making it easy to separate the enzyme form the desired protein. Many properties, such as differing affinity, solubility, heat stability, etc. could be exploited to make the separation steps easier.

In this system, a number of complementary vectors can be used for a given host as long as the enzymes used do not cross-react with the precursor of another enzyme. Another advantage of the vectors of the invention is due to the difficult in replicating the system without knowing the structure of the precursor, the enzyme used in converting it into the factor, and the genotype of the host. As a result, valuable DNA inserts ca be kept more secure, since the choice of selection markers, in this case enzyme/precursor combinations is more varied and not based upon commonly used natural antibiotic resistance.

Yet another advantage of this approach is its potential usefulness for designing a cloning system for organisms that could be released into the environment. Since the selection system is based on auxotrophy and synthetic non-toxic molecules, there is no selection pressure to favor further propagation of the vector in the wild. Yet another advantage of the invention is that the synthetic precursors are more stable than most naturally occurring antibiotics. These precursors may be useful in designing cloning vectors for organisms that live in extreme environments, e.g., high temperature, salinity, and pH, that may degrade natural antibiotics.

Also encompassed in the invention are host cells that are auxotrophic for a factor and that are genetically engineered to express heterologous genes which may encode an enzyme with a desired activity, activity profile, and/or substrate specificity. Such a collection of host cells are generally referred to as gene expression libraries, examples of which are described in U.S. Patent Nos. 5,783,431, 5,824,485. Such host cells may comprise an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth and proliferation of the host cell under auxotrophic condition in the presence of the precursor. Typically, such cultured host cells have a recombinant transcriptional unit stably integrated into chromosomal DNA or carry stably the recombinant transcriptional unit extrachromosomally. Recombinant host cells as defined herein will express heterologous polypeptides or proteins, and RNA encoded by the DNA segment or synthetic gene in the recombinant transcriptional unit. The invention further provides a kit that comprises a first container comprising a host cell that is auxotrophic to a factor; and a second container comprising a replicable vector for introduction into the host cell, said vector comprising an expressible gene , encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor. Optionally, the kit may comprise instructions for using the components of the kit according to the methods of the invention. The kit may also comprises a third container comprising a precursor to the factor.

5.4 EXAMPLES OF AVAILABLE AUXOTROPHS

The present invention also provides the use of a variety of auxotrophs in the methods of the invention.

Merely by way of example, Table 1 lists Escherichia coli K-12 cells prepared by Dr. E. A. Adelberg. Their production is described in Adelberg, E. A., et. al., Biochem Biophys Res Comm, 18:788, 1965, the contents of which is incorporated in its entirety into the present application.

TABLE 1 (E. coli Auxotrophs)

Merely by way of example, Table 2 lists available yeast auxotrophs, the reference corresponding to each of the citations listed in the table is incorporated in its entirety into the present application.

TABLE 2 (Yeast Auxotrophs)

Merely by way of example, Table 3 lists available fungal auxotrophs, the reference corresponding to each of the citations listed in the table is incorporated in its entirety into the present application.

TABLE 3 (Fungal Auxotrophs)

6. EXAMPLES

The following example illustrate the design of several positive selection methods, which include the selection of an auxotroph, synthesis of a precursor molecule, and testing of the methods.

6.1 Synthesis of Sodium 2-β-D-galactopyranoside- 4-hydroxy-3,3-dimethyl butanoate

Reference: M. Kunh und A. von Wartburg, Uber ein neues Glykosidierungsverfahren: Synthese von Epipodophyllotoxin-β-D-glucopyranosid, Helvetica Chimica Acta 51, 1631- 1641, 1968.

Unless stated all reagents were obtained from Sigma-Aldrich (P. O. Box 2060; Milwaukee, WI 53201 USA). The Escherichia coli strain number ATCC 14561 was obtained from the American Type Culture Collection. The NMR instrument is a Bruker 200 MHz instrument. B2 medium is composed of the following: 2.0 g NH₄C1, 6 g KH₂PO₄, 12 g Na₂HPO₄, 6g glucose (or other carbon source), 0.13g MgSO₄7H₂O, 0.074 g CaCl₂-2H₂O per liter and 10 μG thiamine per mL.

LB (Luria Broth) broth is from Difco laboratories (P. O. Box 331058; Detroit, MI 48232-7058 USA). All media are sterilized unless otherwise stated. Manipulation of E. coli was performed in a sterile fashion.

3.9 g of β-D-galactose pentaacetate and 1.3 g of pantolactone were added to 100 ml of dichloromethane. This solution was cooled to 0°C and 15 ml of boron trifluoride diethyl etherate was added over 1 minute. The solution was allowed to stir at room temperature for 5 days. The dichloromethane solution was extracted three times with a saturated solution of sodium bicarbonate and then twice with water. The resulting dichlorometane solution was dried over sodium sulfate and filtered. The dried dichloromethane was removed in vacuo to yield a pale yellow oil. The oil was recrystalized from aqueous methanol to yield white needles. 2-β-D-galactopyranosidetetraacetate-4-hydroxy-3,3-dimethyl butanoic acid (¹³C; δ; CDC1₃: 203.080, 170.269, 170.059, 169.881, 169.308, 168.908, 113.157, 106.717, 102.874, 102.805, 102.478, 92.099, 71.647, 70.771, 67.808, 66.785, 51.001, 20.757, 20.561, 15.688). 22 mg of 2-β-D-galactopyranosidetetraacetate-4-hydroxy-3,3-dimethyl butanoic acid

5 was dissolved in 1 ml of 0.1 M sodium hydroxide in methanol, placed in a sealed tube and heated to 60 degrees centigrade for 14 hours. The methanol was removed and the solid, sodium 2-β-D-galactopyranoside-4-hydroxy-3,3-dimethyl butanoate, was dissolved in water without further purification (¹³C; δ; CD₃OD; 199.971, 175.839, 82.538, 70.358, 69.065, 68.101, 67.464, 66.907, 59.764, 36.660, 20.290, 19.679).

*-v Assay of the designed reagent, Sodium 2-β-D-galactopyranoside-4-hydroxy-3,3- dimethyl butanoate. A strain of Escherichia coli that is auxotrophic for pantothenic acid or pantoic acid (ATCC 14561) was grown overnight in LB at 37 °C while being agitated in a circular shaker moving at 300 rpm. The culture was removed and centrifuged at 5000 x g for 3 minutes. The supernatant was removed and the resulting pellet was resuspended in B2

15 (2.0 g NH₄C1, 6 g KH₂PO₄, 12 g Na₂HPO₄, 6g glucose (or other carbon source), 0.13g MgSO₄7H₂O, 0.074 g CaCl₂2H₂O per liter and and 10 μg thiamine per mL) minimal medium containing glycerol as a carbon source. The resuspended culture was centrifuged at 5000 x g for five minutes. The supernatant was removed and the pellet was resuspended in B2 minimal medium containing glycerol as the carbon source. This was repeated two more

20 times to insure that none of the nutrients from the LB remained. After the washings were completed the cells were diluted 1 : 100,000 in B2 medium containing glycerol as the carbon source. 20 μl aliquots of this cell suspension were evenly spread on the following 25 ml agar plates having the following compositions:

A. B2 with glucose as the carbon source. 5

B. B2 with glucose as the carbon source and pantolactone at a concentration of lOμM.

C. B2 with glucose as the carbon source and supplemented with IPTG (isopropyl-β-D-fhiogalactopyranoside) and sodium 2-β-D- galactopyranoside-4-hydroxy-3,3-dimethyl butanoate both at a concentration

30 of 100 μM.

D. B2 with glucose as the carbon source and supplemented with sodium 2-β-D- galactopyranoside-4-hydroxy-3,3-dimethyI butanoate at a concentration of 100 μM.

32 E. B2 with glycerol as the carbon source.

F. B2 with glycerol as the carbon source and pantolactone at a concentration of lOμM. G. B2 with glycerol as the carbon source and supplemented with IPTG (isopropyl-β-D-thiogalactopyranoside) and sodium 2-β-D- galactopyranoside-4-hydroxy-3, 3 -dimethyl butanoate both at a concentration 5 of 100 μM.

H. B2 with glycerol as the carbon source and supplemented with sodium 2-β-D- galactopyranoside-4-hydroxy-3,3-dimethyl butanoate at a concentration of 100 μM. The plates were left to incubate in a 37° C for 40 hours. After this time they were ^ιυ examined. The glucose plates showed no growth except for the plate that was supplemented with pantolactone. All of the glycerol plates showed growth, except for the one that was not supplemented. The colonies grown on the agar plate containing B2 medium with glycerol and containing 100 μM of sodium 2-β-D-galactopyranoside-4-hydroxy-3,3-dimethyl butanoate were noticeably smaller than those observed on the analogous plate containing ¹⁵ 100 μM of IPTG.

E. coli grown in the presence of glucose will synthesize the enzymes necessary to process lactose at low levels. Ε. coli grown in the presence of an inducer of the lac operon, such as IPTG and in the absence of glucose will greatly increase the synthesis of the enzymes necessary to process lactose. The results of the experiments described above are

20 what would be expected if β-galactosidase was essential for the conversion of the designed reagent (sodium 2-β-D-galactopyranoside-4-hydroxy-3,3-dimethyl butanoate) to sodium pantoate or pantolactone. This experiment demonstrates positive selection for the Ε. coli strain described above which will survive if it expresses the enzyme necessary to convert the designed reagent to sodium pantoate or pantolactone, or not if it is prevented from doing so.

25

6.2 A Pantetheine-based Selection System 6.2.1 Background

The co-enzyme A molecule contains a pantetheine arm attached to a phosphorylated ADP residue. In fatty acid biosynthesis the thiol of co-enzyme A is used to form activated

30 thioester bonds. Pantetheine is an essential cofactor in the biosynthesis of polyketides, a class whose biosynthesis is related to that of fatty acids. Yue et. al., in 1987, synthesized the thioester (using the pantetheine mimic N-acetylcysteamine (NAC)) of a ¹³C-enriched intermediate of a polyketide. In the host organism, this intermediate is bound as the thioester of the pantetheine arm in the polyketide synthase. The NAC bound ¹³C-enriched intermediate

35 was fed to the host organism. Later, they isolated the biosynthesized polyketide. The ¹³C NMR of this polyketide showed an enrichment of the ¹³C signals corresponding to those of the fed thioester bound intermediate. N-acetylcysteamine has been used to perform a number of analogous experiements (see Cane, D. E., et. al., 1987, 1993a, 1993b, 1995; Pieder, R, et. al., 1995). These experiments show that a thioester pantetheine mimic is stable under physiological conditions and will transfer the acyl group in a manner analogous to the natural system.

The following literature related to fatty acid biosynthesis are incorporated herein by reference in their entireties:

S. Numa, ed., Fatty acid metabolism and its regulation (Elsevier, New York), 1984. Lubert Stryer, Biochemistry Third Edition (W. H. Freeman and

Company, New York), 1988. The following literature related to the use of thioesters for NMR incorporation experiments are incorporated herein by reference in their entireties: :

Yue, S., et. al, J. Am. Chem. Soc, 109, 1253-55 (1987) Cane, D. E. et. al., J. Am. Chem. Soc, 109, 1255-57 (1987)

Cane, D. E. and Luo, G., J. Am. Chem. Soc, 117, 633-4 (1995) Cane, D. E. et. al., G., J. Am. Chem. Soc, 115, 527-35 (1993a) Pieder, R, et. al., J. Am. Chem. Soc, 117, 11373-74 (1995) Cane, D. E., et. al., G., J. Am. Chem. Soc, 115, 522-26 (1993b) Unless stated all reagents were obtained from Sigma-Aldrich (P. O. Box 2060;

Milwaukee, WI 53201 USA). The Escherichia coli strains numbered ATCC 14561 andATCC 9637 were obtained from the American Type culture collection. The NMR instrument is a Varian 200 MHz instrument. M9 medium is composed of the following: 1.0 g NH₄C1, 3 g KH₂PO₄, 12.8 g Na₂HPO₄7H₂O, 0.5 g NaCl, 6g glucose (or other carbon source), 0.24g MgSO₄7H₂O, and 0.015 g CaCl₂2H₂O per liter and supplemented with thiamine (10 μg per mL). LB broth is from Difco laboratories (P. O. Box 331058; Detroit, MI 48232-7058 USA). All media are sterilized unless otherwise stated. Manipulation of E. coli was performed in a sterile fashion.

Pantatheine was obtained by the method of Overman et. al. from pantathine. Overman et al., Synthesis 1974: 59, 1974, the contents of which is hereby incorporated in its entirety.

6.2.2 Preparation of p-nitrophenol esters

One mM of a given carboxylic acid and 1.1 mM of p-nitrophenol were dissolved in 20 mL of ethylacetate. One mM of Dicyclohexylcarbodiimide (DCC) was added and the solution was allowed to stir overnight. The reaction mixture was filtered; the ethylacetate removed in vacuo; and resulting oil was chromatographed on silica gel to yield the p-nitrophenol ester.

p-nitrophenol ester of proprionic acid (¹³C, δ, CDC1₃; 171.892, 155.437, 145.079, 125.048, 5 122.333, 27.602, 8.732) p-nitrophenol ester oftri-methyl acetic acid (¹³C, δ, CDCl₃; 176.017, 155.867, 145.07, 124.794,

122.311, 39.186, 26.865) p-nitrophenol ester of t-butylacetic acid (¹³C, δ, CDC1₃; 169.619, 155.344, 145.121, 125.064,

122.446, 47.58, 29.511)

10

6.2.3 Conversion of the p-nitrophenol ester to the pantetheine thioester.

One equivalent of sodium hydride was added to a 100 mg solution of pantetheine dissolved in tetrahydrofuran (THF). This solution was stirred for 30 minutes and then 1 equivalent of one of the p-nitrophenol ester described above was added. This solution was left

15 to stir for 2 hours and then chromatographed on silica gel.

S-Methyl pantetheine (¹³C, δ, CD₃OD; 176.051, 173.799, 77.261, 70.338, 40.391, 39.463, 36.491, 36.404, 34.225, 21.347, 20.916, 15.126)

S-acetyl pantetheine (¹³C,δ, CD₃OD; 197.061, 176.038, 173.898, 77.219, 70.32,40.369,40.02,

20 36.388, 36.324, 30.511, 29.447, 21.337, 20.898) S-Proprionyl pantetheine (¹³C, δ, CD₃OD; 201.331, 175.995, 173.852, 77.192, 70.3, 40.351, 40.064, 38.133, 36.358, 36.307, 29.038, 21.307, 20.872, 9.921)

S-Butyryl pantetheine (¹³C, δ, CD₃OD; 200.529, 176.042, 173.872, 77.217, 70.318, 54.816, ₂₅ 46.668, 40.369, 40.105, 36.324, 29.085, 21.323, 20.888, 20.142, 13.751)

S-Trimethylacetyl pantetheine (¹³C, δ, CD₃OD; 207.680, 176.037, 173.843, 77.2141, 70.33, 40.377, 40.096, 36.41, 36.349, 28.816, 27.73, 21.343, 20.997, 20.91) S-t-Butylacetyl antetheine (¹³C, δ, CD₃OD; 199.009, 176.042, 173.857, 77.232, 70.333, 57.5, 40.368, 40.109, 36.415, 36.332, 32.343, 30.05, 29.365, 21.33, 20.895)

30

6.2.4 Assay of the designed reagent

A strain of Escherichia coli that is auxotrophic for pantothenic acid or pantoic acid

(ATCC 14561; American Type culture collection; 12301 Parklawn Drive; Rockville. Maryland

20852- 1776) was grown overnight in LB at 37 degrees Celsius while being agitated in a circular

35 shaker moving at 300 rpm. The culture was removed and centrifuged at 5000 xg for 3 minutes.

The supernatant was removed and the resulting pellet was resuspended in M9 minimal medium. The resuspended culture was centrifuged at 5000 x g for five minutes. The supernatant was removed and the pellet was resuspended in the same M9 minimal medium. Three sets of 2 mL solutions listed below were prepared in 15 mL sterile plastic capped tubes. A 20 μl aliquot of the E. coli culture described above was added to each tube comprising the following 5 compositions:

A. M9 with glycerol as the carbon source

B. M9 with glycerol as the carbon source and 45 μM S-acetyl pantetheine

C. M9 with glycerol as the carbon source and 4.5 μM S-acetyl pantetheine

D. M9 with glycerol as the carbon source and 0.45 μM S-acetyl pantetheine

^{1 υ} Ε. M9 with glycerol as the carbon source and 45 μM S-proprionyl pantetheine

F. M9 with glycerol as the carbon source and 4.5 μM S-proprionyl pantetheine

G. M9 with glycerol as the carbon source and 0.45 μM S-proprionyl pantetheine H. M9 with glycerol as the carbon source and 45 μM S-butyryl pantetheine I. M9 with glycerol as the carbon source and 4.5 μM S-butyryl pantetheine

15 J. M9 with glycerol as the carbon source and 0.45 μM S-butyryl pantetheine

K. M9 with glycerol as the carbon source and 45 μM S-tri-methylacetyl pantetheine

L. M9 with glycerol as the carbon source and 4.5 μM S-tri-methylacetyl pantetheine 20

M. M9 with glycerol as the carbon source and 0.45 μM S-tri-methylacetyl pantetheine

N. M9 with glycerol as the carbon source and 45 μM S-t-butylacetyl pantetheine

N. M9 with glycerol as the carbon source and 4.5 μM S-t-butylacetyl pantetheine

_ _ O. M9 with glycerol as the carbon source and 0.45 μM S-t-butylacetyl pantetheine

P. M9 with glycerol as the carbon source and 5 μM S-methyl pantetheine

Q. M9 with glycerol as the carbon source and 4.5 μM S-methyl pantetheine

R. M9 with glycerol as the carbon source and 0.45 μM S-methyl pantetheine

S. M9 with acetate as the carbon source _Q T. M9 with acetate as the carbon source and 45 μM S-acetyl pantetheine

U. M9 with acetate as the carbon source and 4.5 μM S-acetyl pantetheine

V. M9 with acetate as the carbon source and 0.45 μM S-acetyl pantetheine

W. M9 with acetate as the carbon source and 45 μM S-proprionyl pantetheine

X. M9 with acetate as the carbon source and 4.5 μM S-proprionyl pantetheine

35 Y. M9 with acetate as the carbon source and 0.45 μM S-proprionyl pantetheine Z. M9 with acetate as the carbon source and 45 μM S-butyryl pantetheine

AA. M9 with acetate as the carbon source and 4.5 μM S-butyryl pantetheine

AB. M9 with acetate as the carbon source and 0.45 μM S-butyryl pantetheine

AC. M9 with acetate as the carbon source and 45 μM S-tri-methylacetyl pantetheine

AD. M9 with acetate as the carbon source and 4.5 μM S-tri-methylacetyl pantetheine

AE. M9 with acetate as the carbon source and 0.45 μM S-tri-methylacetyl pantetheine

AF. M9 with acetate as the carbon source and 45 μM S-t-butylacetyl pantetheine

AG. M9 with acetate as the carbon source and 4.5 μM S-t-butylacetyl pantetheine AH. M9 with acetate as the carbon source and 0.45 μM S-t-butylacetyl pantetheine

Additionally, the strain of Escherichia coli (ATCC 9637) that was used to create the auxotroph described above (ATCC 14561) was used as acontrol. Itwas growninLB at 37° C while being agitated in a circular shaker moving at 300 rpm. The culture was removed and centrifuged at 5000 x g for 3 minutes. The supernatant was removed and the resulting pellet was resuspended in M9 minimal medium. The resuspended culture was centrifuged at 5000 x g for five minutes. The supernatant was removed and the pellet was resuspended in the same M9 minimal medium. Three sets of 2 mL solutions listed below were prepared in 15 mL sterile plastic capped tubes. A 20 μl aliquot of this E. coli culture was added to each tube:

1. M9 with glycerol as the carbon source

2. M9 with acetate as the carbon source

Each day for three days a 0.5 ml aliquot was removed and the absorbance at 600 nm was recorded vs. sterile M9 medium. At the end of three days 50 μl was removed and plated on M9 agar plates (1.5 % agar), with glycerol as the carbon source. The plates were examined the next day to determine if any showed a lawn of growth, which would indicate that the mutant auxotrophs had reverted to the wild type. If there was evidence of reversion, then the results for that tube were removed from the data set. The results of these experiments are summarized in the Figure 1 and 2. Figure 1 shows precursor fed to ATCC # 14561 M9 (Acetate) P = pantetheine; S-Ac = S-acetyl P; S-Pro = S-propionyl P; S-Bu = S-butyryl P; Tri-Me Ac = S-trimethylacetyl P; t-Bu Ac = S-tbutylacetyl P. Figure 2 shows a plot of the precursor fed to ATCC # 14561 M9 (Glycerol) P = pantetheine; S-Ac = S-acetyl P; S-Pro = S-propionyl P; S-Bu = S-butyryl P; Tri-Me Ac = S-trimethylacetyl P; t-Bu Ac = S-tbutylacetyl P; S-Me = S-methyl P. 6.2.5 Results

The results show clearly that the thioester bond of the various S-esters are required to be cleaved to yield pantetheine, the factor that breaks the auxotrophy. Furthermore, this compound is not a degradation product of pantetheine. This is shown clearly in the experiment using S-methyl pantetheine in M9 medium with glycerol as a carbon source. There is no growth, for the methyl group capping the thiol is difficult to remove and the S- ' methyl pantetheine is not degraded to yield a factor that will break the auxotrophy, since there is no observed growth above background. Thus, any observed effects are due to the cleavage of the thioester bond.

The results also show that the growth of E. coli is dependent upon the substrate specificity of the fatty acid synthase (or other pantetheine requiring enzyme) for a given designed reagent. The cells grown in M9 (glycerol carbon source) and the presence of the S-acetyl, S-proprionyl, or S-butyryl pantetheine grow at comparable rates. This is not true for the S-trimethylacetyl or S-t-butylacetyl pantetheine designed reagent, for at low concentrations 0.45 μM the growth is essentially that of background. The difference is more dramatic in the set of analogous experiments where the cells are grown in M9 with acetate as the carbon source. These results show that non-enzymatic hydrolysis of the thioester bond is not a significant problem in this experiment. These experiments also show that growth of auxotrophic cells can be determined through the interaction of a precursor of a factor and an enzymatic activity present in the cell.

6.3 A Precursor For An Esterase

A Saccharomyces cerevisae strain (ATCC 44378) that is auxotrophic for histidine and leucine is chosen for used in this example. An expression vector pC504 has been used for cloning in this organism. The strain and plasmid are available from the ATCC. Saccharomyces cerevisiae ATCC 44378 M.C. Kielland-Brandt C78-H26. Transformant of ATCC 44377. Genotype: MATalpha his4-24 leul-1 [HIS4] carries the plasmid pC504 (Carlsberg Res. Commun. 44: 77-87 and 269-282, 1979). The cell will lose the plasmid if grown in presence of histidine. The cell requires leucine. Growth Conditions are Medium 1049 at 30°C.

6.3.1 Synthesis of a precursor for the purpose of isolating an esterase that cleave esters of hexanol. l equivalent N-(tert-Butoxycarbonyl)-L-leucine and hexanol is dissolved in ethyl acetate and then 1 equivalent of dicyclohexyldicarbodimide is added. The resulting solution is left to stir overnight. The ethylacetate is filtered and then removed in vacuo. The resulting material is chromatographed to yield the hexanol ester. The t-BOC protecting group is removed by dissolving the ester in dry trifluoroacetic acid and allowing it to stand for 30 minutes. The trifluoroacetic acid is removed in a stream of nitrogen to yield the amino ester, the designed reagent. This procedure may be used to synthesize a number of analogous designed reagents.

Using standard cloning techniques the pC504 or other appropriate cloning vector is used to make an expression library from organisms that are likely to possess ester hydrolyases. The yeast is transformed and plated out on medium containing without histidine and leucine. Those colonies that are able to grow on these plates possess the ability to convert the hexanol ester of leucine to leucine. The activity of the enzymes is examined to see if they are suitably specific.

The result of this experiment is an enzyme that is specific for the cleavage of the ester bond of a hexanol ester.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WE CLAIM:

1. A method for detecting a desired enzymatic activity comprising the steps of:

(a) providing a plurality of host cells that are auxotrophic for a factor and that are genetically engineered to express at least one enzymatic activity;

(b) contacting the genetically engineered host cells with a precursor of the factor, wherein the precursor cannot be converted to the factor by a non-genetically engineered host cell, and wherein the precursor is converted to the factor by the desired enzymatic activity; and

(c) culturing the genetically engineered host cells of step (b) under'auxotrophic condition such that survival of genetically engineered host cells indicates the expression of the desired enzymatic activity by the genetically engineered cells.

2. A method for detecting a desired enzymatic activity which comprises the steps of:

(a) contacting host cells that are auxotrophic for a factor with a precursor of the factor, wherein the precursor cannot be converted to the factor by the host cell, and wherein the precursor is converted to the factor by the desired enzymatic activity; and

(b) transforming the host cells such that the host expresses at least one enzymatic activity; and (c) culturing the transformed host cells of step (b) under auxotrophic condition such that survival of transformed host cells indicates the expression of the desired enzymatic activity by the transformed host cells.

3. The method of claim 1 or 2 wherein the host cells are animal cells, bacterial cells, fungal cells or plant cells.

4. The method of claim 1 or 2 wherein the host cell is genetically engineered with or transformed with a nucleic acid molecule foreign to the host.

5. The method of claim 2 wherein the host is transformed with a physical or a chemical mutagen.

6. The method of claim 1 or 2 wherein the enzymatic activity or activities is that of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase.

7. The method of claim 1 or 2 wherein the desired enzymatic activity is associated with the making or breaking of an amide bond, an amine bond, a carbon carbon bond, carbon hydrogen bond, a carbon oxygen bond, a carbon nitrogen bond, a carbon phosphorous bond, a carbon sulfur bond, an ester bond, an ether bond, a nitrogen oxygen bond, a nitrogen phosphorous bond, nitrogen sulfur bond, an oxygen phosphorous bond or a phosphorous sulfur bond.

The method of claim 1 or 2 wherein the factor is an amino acid, an amino acid biosynthetic intermediate, a carbohydrate, a cofactor, or a cofactor biosynthetic intermediate, a lipid, a lipid biosynthetic precursor, a nucleotide or a nucleotide biosynthetic intermediate.

9. The method of claim 8 wherein the amino acid or amino acid intermediate is

Alanine, Arginine, Asparagine, Aspartate, Cysteine, Glutamine, Glutamate, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Ornithine, Phenylalanine, Proline, D-proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, (S)-2-Acetolactate, 2-Aceto-2-hydroxy-butyrate, 3-Amino isobutyrate, 5-Amino valerate, Anthranilate, Anthranilic acid, N-Carbamoyl aspartate, 3-Carboxy-3-hydroxy-isocaproate, Chorismate, Cystathione, 5- Dehydroquinate, 5-Dehydro-shikimate, 2,3 -Dihydroxy-3 -methyl- valerate, Dimethylcitraconate, 3 -Enolpyruvyl-shikimate-5 -phosphate, Erythrose 4- phosphate, Glutamic g-semialdehyde, Histamine, Histidinol, Histidinol phosphate, Homocysteine, 3 -Hydroxy anthranilate, p-Hydroxyphenyl- pyruvate, 4-Hydroxy-D-proline, 4-Hydroxy proline, 4-Hydroxy-benzoate, Imidazole acetol phosphate, Imidazole glycerol phosphate, Indole, Isochorismate, a-Ketobutarate, a-Ketoglutarate, 3-Mercapto pyruvate, 3- Methyl aspartate, (S)-methylmalonate semialdehyde, Oxaloacetate, 2-Oxo- 5-amino valerate, 2-Oxobutyrate, 2-Oxo-4-hydroxy-5-amino valerate, 2- Oxo-isocaprate, 2-Oxo-isovalerate, 2-Oxo-3 -methyl- valerate, 5-Oxoproline, Phenyl-pyruvate, Phosphoenopyruvate, 3-Phosphoglycerate, 3- Phosphohydroxypyruvate, 3-Phosphoserine, Prephenate, Pyrrole-2- carboxylate, D'-Pyrroline-5-carboxylate, Pyruvate, Ribose 5-phosphate, S-

Adenosylmethionine, S-Adenosyl-homocysteine, Shikimate or 2-Succinyl Benzoate.

10. The method of claim 8 wherein the carbohydrate is Galactose, D- Galacturonate, D-Gluconurate, D-Gluconurate-1 -phosphate, Glucose,

Inositol, Lactose, Maltose or Myoinisitol.

11. The method of claim 8 wherein the cofactor, or cofactor biosynthetic intermediate is p- Amino benzoic acid, 2-Amino-3-carboxy-muconate semialdehyde, 2-Amino-4-hydroxy-6-(D-erythro 1 -2-3-trihydroxypropyl-

)dihydropterine, 2-Amino-4-hydroxy-6-(D-erythro 1 -2-3 -trihydroxypropy 1- )dihydropterine-triphosphate, 2-Amino-4-hydroxy-6-hydroxy-methyl- dihydropterine, 2-Amino-4-hydroxy-6-lιydroxy-methyl-dihydropterine- diphosphate, (4-Aminophenyl)-l,2,3,4-Tetrahydroxypentane, d- Aminolevulinate, 2-Amino-muconate, 2-Amino-muconate semialdehyde, L-

Ascorbate, Biotin, a-Carotene, b-Carotene, Catechol, Coenzyme A, Cyanocobalamin, a Cytochrome(s), 2-Dehydropantoate, Dephospho- Coenzyme A, Dethiobiotin, 7, 8 -Dihydrofolate, 7,8-Dihydromethanopterin, 7,8-Dihydroopteroate, Dihydro-nicotinamide adenine dinucleotide , Dihydro-nicotinamide adenine dinucleotide phosphate, Dihydrothymine, l,4-Dihydroxy-2-naphthoate, Dolichol, Ergocalciferol, Ergosta- 5,7,22,24(28)-tetraen-3-ol, Ergosterol, Flavin mononucleotide, Folate, Folic acid, Heme, Homogestinate, 3-Hydroxy-L-kynurenine, 6-Hydroxy-nicotinic acid, 2-Hydroxy-6-polyprenyl phenol, 3-Isopropyl-pimylate, Kynurenate, L- Kynurenine, Lipoic acid, Menaquinol, Menaquinone, 8-Mercapto octinoic acid, Nicotinamide, Nicotinamide adenine dinucleotide , Nicotinamide adenine dinucleotide phosphate, Nicotinate, Nicotinate-nucleotide, Nicotinic acid, Pantetheine, Panthenic acid, Pantothenic acid, Pantoic acid, Pantothenol, N-Pantothenoyl-cysteine, 4'- Phospho-pantetheine, 4'- Phospho-pantothenate, 4'- Phospho-pantothenoyl cysteine, Phylloquinol, Phylloquinone, Pimelic acid, Plastoquinone, 2-Polyprenyl phenol, Pyridoxal, Pyridoxamine, Pyridoxine, l-Pyrroline-4-hydroxy-2-carboxylate, Quinate, Quinolinate, Quinolinate-nucleotide, Retinal, Retinol, Riboflavin, S-Adenosylmethionine, 5,6,7,8-Tetrahydrofolate, 5,6,7,8- Tetrahydromethanopterin, 6(R)-5,6,7,8-Tetrahydrobiopterin, 6(R)-

Pyruvoyltetrahydropterin, 6(S)-5,6,7,8-Tetrahydrofolate, Thiamine, Thiamine pyrophosphate, Thymine, a-Tocopherol, a Tocopherol(s), Ubiquinol, Ubiquinone or Vitamin K 1.

12. The method of claim 8 wherein the lipid or lipid biosynthetic intermediate is

Acetate, Betaine aldehyde, Betane, Carnitine, Ceramide, Cholesterol, Choline, Creatine, Cycloartenol, 7-Dehydrocholesterol, 3-Dehydro- sphinganine, Delta-3-isopentyl-pyrophosphate, 1,4-Desmethyl cycloartenol, 1 ,4-Desmethyl lanosterol, Dimethylallyl-pyrophosphate, Dimethyl glycine, Ethanol amine, Farnesyl-pyrophosphate, Geranol, Geranyl-pyrophosphate,

Lanosterol, Lathosterol, Methyloxalo acetate, Mevalonate, Mevalonate-5- phosphate, Mevalonate-5-pyrophosphate, Mevalonolactone, Psychosine, Sarcosine, Sphinganine, Sphingosine, Squalene, (S)-Squalene-2,3-epoxide or zymosterol.

13. The method of claim 8 wherein the nucleotide or nucleotide biosynthetic precursor is Adenine, Adenosine, Adenosine-5'-diphosphate, Adenosine-5'- phosphate, Adenosine-5'-triphosphate, Cytosine, Cytidine, Cytidine -5'- diphosphate, Cytidine -5 '-phosphate, Cytidine -5'-triphosphate, 2'-Deoxy- cytidine, 2'-Deoxy-cytidine -5'-diphosphate, 2'-Deoxy-cytidine -5'- phosphate, 2'-Deoxy-cytidine -5'-triphosphate, 4,5-Dihydroorotate, 2,5- Dihydroxy-pyridine, Guanidine, Guanosine, Guanosine -5'-diphosphate, Guanosine -5'-phosphate, Guanosine -5'-triphosphate, Inosine, Orotate, Orotidine-5'-phosphate, Thymidine, Thymidine -5'-diphosphate, Thymidine -5'-phosphate, Thymidine -5'-triphosphate, Uracil, 3-Ureido-isobutyrate,

Uridine, Uridine-5'-diphosphate, Uridine-5'-phosphate, Uridine-5'- triphosphate Xanthanoside or Xanthurenate.

14. A method for producing a protein comprising the steps of: (a) introducing into a host cell a first expressible gene encoding the protein and a second expressible gene encoding an enzyme, wherein the host cell is auxotrophic for a factor; and (b) culturing the host cell of (a) under auxotrophic condition and in the presence of a precursor of the factor, wherein the precursor is converted to the factor by the enzyme expressed by the host cell, thereby allowing the host cell comprising the first and second expressible gene to grow and produce the protein.

15. The method of claim 14 wherein the host cell is an animal cell, a bacterium, a fungal cell, or a plant cell.

16. The method of claim 14 wherein the first expressible gene and the second ' expressible gene are present in a single nucleic acid molecule.

17. The method of claim 14 wherein the first gene and the second gene are on two separate nucleic acid molecules.

18. A replicable vector for introduction into a host cell that is auxotrophic to a factor, said vector comprising an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor.

19. A host cell that is auxotrophic to a factor, said host cell comprising an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor.

20. The replicable vector of claim 18, wherein the precursor is not a metabolic intermediate of the host cell.

21. The replicable vector of claim 18, wherein the enzyme is not one that is naturally occurring in the host cell.

22. The host cell of claim 19, wherein the precursor is not a metabolic intermediate of the host cell.

23. The host cell of claim 19, wherein the enzyme is not one that is naturally occurring in the host cell.

24. A kit that comprises a first container comprising a host cell that is auxotrophic to a factor; and a second container comprising a replicable vector for introduction into the host cell, said vector comprising an expressible gene encoding an enzyme that converts a precursor of the factor to the factor in the host cell, thereby allowing the growth of the host cell under auxotrophic condition in the presence of the precursor.

25. The kit of claim 24 further comprising a third container comprising the precursor to the factor.

26. A method for detecting an enzyme inhibitor which comprises the steps of:

(a) providing a plurality of host cells that are genetically engineered to express an enzyme; (b) contacting the genetically engineered host cells with a precursor of a toxin, wherein the precursor cannot be converted to the toxin by a non-genetically engineered host cell, and wherein the precursor is converted to the toxin by the enzyme; and (c) culturing the genetically engineered host cells of step (b) in the presence of a test composition that may comprise an inhibitor of the enzyme such that survival of genetically engineered host cells indicates the presence of an inhibitor of the enzyme.