ENHANCEMENT OF INDUSTRIAL PRODUCTION BY INCREASING SUBSTRATE TRANSPORT

Abstract: A method for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway of said host cell is described. Such method comprises selecting a host cell that has an at least partially intracellular synthetic pathway which utilizes an extracellular substrate; increasing the transport of said extracellular substrate into said host cell while maintaining the integrity of the host cell, culturing the host cell to produce said extracellular substrate; and producing the desired compound. The transport of the substrate is increased by transforming into the host cell DNA encoding for one or more enzymes transporting the substrate into the host cell.

Description: ENHANCEMENT OF INDUSTRIAL PRODUCTION BY INCREASING SUBSTRATE TRANSPORT FIELD OF THE INVENTION The present invention generally relates to enhancing the bioconyersion of a desired end- product and specifically to the overexpressing transporters to facilitate transport of a substrate from one cellular location to another across the membrane. The present invention provides expression vectors, methods and systems for the enhanced production of a desired end-product in microorganisms. BACKGROUND OF THE INVENTION The lipid bilayer of biological membranes is generally impermeable to ions and polar molecules. These biological membranes compartmentalize a cell, separating different sections of cell from one another. Thus substrates utilized by the cell to synthesize various products as well as metabolites utilized by the cell for generating energy or growth may be separated from the synthetic and/or catabolic reactions which utilize them. With respect to product synthesis, different synthetic pathways or portions thereof, can be found in different portions of the cell. Some oxidative reactions can occur outside of the cytosol. For example, membrane bound proteins can be used to oxidize a carbon source to another intermediate. Cystolic reactions or pathways, for example some reductions or dehydrogenations, can also be utilized to convert a substrate or intermediate into another product. When the substrate and the synthetic machinery are on opposite sides of a membrane, production of the desired end-product may require translocation of the substrate to the situs of the synthetic reaction to enable its conversion to the desired end-product . Alternatively, end-products generated inside of the cell membrane may require translocation from within the cell. Since the partitioned sections of the cell may have different environmental parameters, e.g., solute, ion, end-product, etc., concentrations, or may require translocation across a normally impermeable barrier, some form of active transport may be required. Responsive to these problems, investigation related to increasing the transport of materials across membranes has occurred. Solvents or lyzing agents have been used to rupture the membranes, enabling the crossing of the materials across membrane. However, such methods have adverse effects upon the viability of the host cell. If the synthetic or metabolic pathways are dependent or energized by a host cell's own metabolic or catabolic pathways, such as requiring co-factors such as NADH or NADPH, destroying the viability of the cell halts further or continued synthetic production by the host cell. In addition, while the increase in the transport of glucose or other saccharides has been explored in increasing the growth of the cell (Parker, C, et al, Mol. Microbiol 15(5)795-802 (1995)), altering cellular transport systems to increase the industrial production of chemical end-products or intermediates through biosynthetic pathways utilizing recombinant microorganisms has not been recognized. Cornish (J. of Gen. Microbiol., 134:3111-3122 (1988)) discusses the relationship between glucose transport and the production of a succinoglucan exopolysaccharide by Agrobacteriyum radiobacter. Cornish proposed that glucose uptake was a major kinetic control point for succinoglucan production, and that it ought to be possible to obtain even higher rates of succinoglucan production by using recombinant DNA methods to obtain even higher rates of succinoglucan production. However the production rates of Cornish were not on the scale of industrial needs. Furthermore, the high levels of energy expended and complex regulatory mechanism involved in transporting glucose could discourage rather than encourage its use. Volschenk, H., et al (Nat. Biotechnol. 15:253 (March 1997) describes the introduction of malate degradation pathways into Sacchaomyces cerevisiae by the cloning and expression of heterologous DNA encoding the same for the purpose of depleting the malate levels present in wine. Volschenk was primarily concerned with the removal of malate from the surrounding medium, not the production of any desired end product on an industrial scale. Furthermore, the mere knowledge that a transport system is involved does little to guarantee the enhanced production of the desired end product or intermediate. The synthetic machinery may already be saturated and thus an increased presence of the substrate won't necessarily result in the increased production of the desired end product. In addition, increased transport of a substrate that is utilized directly or indirectly as a metabolite in addition to its use as a production substrate may not result in the increased production desired. Merely increasing the expression level of the enzyme converting the substrate to the desired end product may not result in an increased production of chemical compounds using recombinant microorganisms. There is a need for a means to increase the production of chemical compounds using recombinant microorganisms by means other than increasing the expression level of substrates within the cell. SUMMARY OF THE INVENTION The capacity of the substrate transport machinery of a microorganism may become a limiting factor or bottleneck to a desired end-products production, in particular when the end- product, intermediate or precursor production is compartmentalized in different regions of the cell. The present invention provides a means for alleviating that bottleneck. The present invention is based, in part, upon the discovery that increasing the transport of a substrate across the cell membrane increases the overall production of the desired substrate. The present invention also provides improved methods for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway in microorganisms. Accordingly, a method is provided for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway of said host cell, the method comprising selecting a host cell that has an at least partially intracellular synthetic pathway which utilizes an extracelluar substrate; increasing the transport of said extracellular substrate into said host cell while maintaining the integrity of the host cell; culturing the host cell to produce said extracellular substrate; and producing the desired compound. In one preferred embodiment the transport of said substrate into said host cell is a rate- limiting step in said host cell's biosynthetic productions. In the preferred embodiment, the host cell is selected from Kleibsiella and Pantoea. In one embodiment of the present invention, increasing the transport of the extracellular substrate into the host cell includes the step of transforming into the host cell DNA encoding for one or more proteins increasing the transport of the substrate into the host cell. In one embodiment the protein is a transport promoter. In another embodiment, the protein is a major superfamily facilitator. In yet another embodiment, the protein is a anion/cation:H+ transporter. The present invention further provides a method of overexpressing a substrate/H+ symporter comprising the steps of selecting a host cell and transforming into said host cell, DNA encoding for one or more substrate/H+ symporters. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the DNA and amino acid sequencing of YiaX2 of Klebsiella oxytoca; PE1 (environmental permease);PE6 (environmental permease);PermA of Pantoea citrea; PermB of Pantoea citrea; YiaX2 of Pantoea citrea. FIG. 2 is a flow diagram showing the synthetic pathway for the production of ascorbic acid precursor 2-KLG from glucose. FIG. 3 is a diagram showing the synthetic pathway of the ascorbic acid precursor 2-Keto-L- gulonic acid (2-KLG). Fig. 4 is a flow chart showing the various synthetic pathways through which glucose can be converted to 2-KLG. Boudrant, J., Enzym Microb. Tech., 1990, 12, 322-329 FIG. 4A is a diagram showing the synthetic pathways of D-sorbitol to 2-KLG showing the cellular location of the reactions relative to other reactions within the pathway and the transport of substrates across cell membranes. Saito, Y, et al Biotechnol. Bioeng. 58(2/3):305-315 (1998). FIG. 5 shows the synthetic pathway of D-glucose (G) to 2,5-DKG to 2-KLG, showing the location of the reactions relative to other reactions within the pathway and the transport of the respective substrates across the cell membrane. FIGS. 6 is a line graph comparing the DKG transport rate with the KLG production rate showing the amount of 2,5-DKG uptake (nmoles/OD 600) v. time (-♦- = fructose feed 139- 2a, 0.0486x + 0.67; - A- = glucose feed 139-2a, y= 0.0497 + 0,5877; -•- = seed flask 139- 2a, y=o.oo75x+0.0569 ). FIG.7 is a schematic of the yia operon of ascorbic acid catabolism in Klebsiella oxytoca . FIG. 8 is a graph showing the amount of 2,5 DKG uptake (nmol) by Klebsiella oxytoca measured by the silicone oil transport assay (-♦- = Tester + isopropyl β-D- Thiogalactopyranoside [IPTG]) ; - A- = ΔyiaaX2 + 1PTG); - ■ - = Tester - IPTG) FIG. 9 is a schematic drawing showing the selection design to close permeases from P. citrea, K. oxytoca and environmental sources. FIG. 10 is a bar graph showing 2,5-DKG uptake activity in K.oxytoca strains (YiaX2, pcpl , pcp10, pcp32, pK1 , Environmental #1 ; and Environmental #6). FIG. 11 is a bar graph showing 2,5-DKG uptake assay of shaker flask having various DKG permeases (139-2A, 139-2A + PCP32; 139-2A + PCP10; 139-2A + PK1 ; 139-2A+ PCP1) and 139-2A + PE6.in the same plasmid construct (pBCL 1920) measuring the DKG Uptake rate (g/l/hr) at 28 degrees C.9A-9B . FIG. 12 is a line graph showing the specific productivity increase with overexpressed DKG permease/ Spec Production rate (g/L/hr) (-♦ - = wild-type; -x- = WT, prmA) FIG. 13 is a schematic drawing of the PermA transporter in a membrane surface. The putative membrane spanning domains numbered l-XI. The positions of the conserved residues are indicated in bold. N is the amino-terminus and C is the carboxyl-terminus. Putative membrane-spanning domains of Pantoea citrea Permease A (SEQ ID: ), were deduced using the tool available to http;//sosui.proteome,bio,tuat.ac.ip/sosuiframe0.html FIG. 14 is a conserved amino acid sequence corresponding to residues G119s through142. DETAILED DESCRIPTION Definitions Transporter definitions Bacterial channel transporters refers to those transporters generally in the TC classification of #1.A (Saier, M., et al.,1998, Advances in Microbial Physiology (Poole, R.K., ed.) pp. 81- 136, Academic Press, San Diego, CA.). ( "TC" stands for "Transport Council", a classification system which takes into consideration the phylogenetic aspects of the transporter.) These generally transport substrates, ions or other material via an energy independent facilitated diffusion mechanism employing a transmembrane pore. Primary transporters refers to those transporters generally in the TC classification of (TC #3.A)(Saier, ML, et al, 1998), and are those that utilize chemical energy, typically in the form of ATP hydrolysis as a mode of energy coupling for the active uptake and transport extrusion of substrates. Group translocation systems refers to transporters in the TC classification of TC #4.A. (Saier, M., et al, 1998) are transporters that concommittantly transport and phoshorylate their substrates during transport. The members of this category generally are part of the bacterial specific phosphotransferase system (PTS) and are characterized by the coupling to the oxidation of phosphoenol pyruvate (PEP) utilization. Secondary transports refers to those transporters generally in the TC classification of #2.A,( Saier, M., et al, 1998) those that generally use chemiosmotic energy, for instance in the form of a proton gradient, to provide energy to transport the substrate, ions or end products across the membrane. Major facilitator superfamily (MFS) refers to secondary transporters that are generally in the TC classification of #2 (Saier, M., et al, 1998). A transporter refers to any macromolecule that allows the translocating of a chemical compound or substrate across a cell membrane and into or out from a cell or cellular compartment. Transporters are also known or referred to as permeases. While not being limited to a specific theory, it is thought that the transporter is a protein that interacts with a membrane, with portions of the protein extending from the outer surface of the membrane, through the membrane, and from the inner surface of the membrane. Active transport refers to transport that is coupled with an expenditure of energy, for example the hydrolysis of adenosine tri-phosphate (ATP) or phosphophenolpyryvate (PEP). An anion/cation symporter refers to a transporter that utilizes an chemoisomotic gradient to transport the substrate across the membrane (TC class 14). They are also refered to as substrate/H+ symporters. TMS refers to transmembrane spanning domains Pathway definitions Cytoplamic refers to being within the inner cell membrane. Exogenous substrate refers to a material, found on the opposite side of the separating membrane from the synthetic reaction, e.g., outside of the inner cell membrane when the substrate is to be converted by an intracellular synthetic pathway or an intracellular portion of a synthetic pathway to the desired end product or intermediate. Examples of exogenous substrates include hexoses, including glucose, pentoses, including xylose, arabinose, ribose, lyxose, nucleosides, neurotransmitters, drugs, vitamins, amino acids, intermediates or precursors of various pathways, including ascorbic acid. Extracellular or outside the inner cell membrane refers to cell locations on the opposite side of a membrane from the cytoplasm, including, but not limited to the periplasm. Inner cell membrane refers to the barrier that separates the cytoplasm from the periplasm. Membrane refers to a lipid bilayer that is intrinsically impermeable to the substrate. Intracellular refers to the portion of the cell on the side of the membrane that is closest to or of the cytosol. Intracellular also includes cystolic. Intracellular reaction refers to a synthetic reaction or bioconversion located within the cystolic cell material, i.e., material enclosed inside of the inner cell membrane. Rate limiting step refers to the step within the synthetic pathway where an increase in the conversion across that step results in an increase in the production by the synthetic pathway. Enhancing the production refers to increased titer (total amount) of the desired intermediate, end-product or precursor of a synthetic reaction, generally measured by an increase in the gm/l/hour obtained through the process. It may also refer to an increase in the rate at which the desired products are made, generally measured in g/l per unit time.of the recombinant production, wherein the amount of end-product, intermediate or precursor produced increases as a result of the transforming of DNA encoding the at least one protein increasing the transport of the substrate across a membrane in the presence of the overexpressed transporter. Increased yield refers to the increased weight percent calculated by the division of the amount of desired end-product or intermediate produced by the amount consumed. A substrate refers to the compound that is bioconverted by a synthetic reaction, the reaction situs being generally separated from the substrate by a membrane and requiring a transport mechanism to bring the substrate to the reaction situs. The substrate may itself be the end-product, intermediate or precursor of a subsequent synthetic reaction or subsequent pathway. Synthetic reaction refers to the bioconversion of a substrate to an intermediate or an end- product. The reaction may be endogenous to the organism or can be incorporated within the cell machinery via genetic engineering. 2,5-DKG reductase refers to a protein which is capable of catalyzing the conversion of 2,5- DKG stereoselectively to 2-KLG. 2,5-DKG transporter refers to a protein which is capable of transporting the 2,5-DKG across the inner cell membrane for conversion to 2-KLG by a 2,5-KLG reductase. Expression definitions Promoter refers to DNA elements that guide the RNA polymerase to start the transcription of a gene at the appropriate site to generate a messenger RNA capable of forming a polypeptide once it is translated by the translational machinery of the cell. An "upstream activating sequence" is a binding position for a positively-acting DNA binding regulator. As indicated by its name, the upstream activating sequence is upstream of the transcription start site and is a nucleic acid. Regulatory regions refers to regions on the DNA that modulate the expression of genes. One mechanism for this modification is that some regulatory regions serve a binding sites for proteins (also known as repressors). Once bound, a repressor interferes with the ability of RNA polymerase to transcribe a gene. An expression system includes one or more proteins and/or nucleic acids which, when acting together, can increase the expression of a protein in a host cell. The expression system can be encoded on one or more plasmids and may or may not be on the same plasmid as the gene encoding the protein of interest. The phrase "functionally linked" or "functionally coupled" means that the regulating elements (DNA or protein) interact physically in order to exert their function. This can be a protein/protein, DNA/protein or a DNA/DNA interaction. For example, the DNA binding regulator interacts with the promoter but genes encoding them may be at different sites on the chromosome. As such, the genes encoding the elements can be on different plasmids from each other and from the gene encoding the protein of interest and still work together to regulate expression of the protein. Commonly, when describing proteins and the genes that encode them, the term for the gene is not capitalized and is in italics, i.e., permA. The term for the protein is generally in standard type letters and the first letter is capitalized, i.e., PermA. Organism definitions Fungal refers to being related to any number of numerous plant organisms which lack chlorophyll, including the yeasts, molds and mushrooms. Mammalian refers to being related to any of various warm-blood vertebrate animals, marked by a covering of hair on their skin. Botanical refers to being related to plants, including monocots, dicots, etc. "Bacteria" include microorganisms of the class Schizomycetes. Bacteria can be either Gram-negative or Gram-positive. Gram-negative bacteria include members of the genera Escherichia, Hemophilus, Klebsiella. Proteus, Pseudomonas, Salmonella, Gluconobacter, Acetobacter, Yersenia, Shigella, Vibrio, Acinetobacter, Pantoea and Serratia. Gram-positive bacteria include members of the genera Bacillus, Clostridium, Staphylococcus, Streptomyces, Lactobacillus and Lactococcus. Gram-negative bacteria can be pantoeans which are strains that are members of the genus Pantoeas. A preferred bacterial is Pantoea citrea. Pantoea citrea is also sometimes referred to as Erwinia herbicola. The terms "isolated" or "purified" as used herein refer to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 5 The present invention provides novel methods for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway of said host cell, by increasing the transport of the exogenous or extracellular substrate to ameliorate the bottleneck to pathway synthesis and the enhanced production of desired o intermediates or end-products, in particular when the transporters are recombinantly introduced and overexpressed by the host cell. One embodiment of the invention is directed to a method of transforming a host cell with a plasmid that includes the nucleic acid encoding the expression system. Another embodiment of the invention is directed to a method of transforming a host cell with a s plasmid that includes DNA encoding for one or more proteins increasing the transport of the substrate across themembrane. A host cell is a cell into which a plasmid of the present invention can be inserted through, for example, transformation. The host cell is preferably a bacteria and more preferably in the group of Pantoea citrea, Escherichia coli; Klebsilla or Bacillus. o In another embodiment, if regulating elements are incorporated, such elements of the expression system are from E. coli and B.subtilis In one embodiment, the host cell is preferably a Gram-negative bacteria. In another preferred embodiment, the host cell is a Pantoean. The same host cell can be transformed with a further plasmid that includes a nucleic acid that encodes one or more transporters. Preferably, the transporters are 5 encoded MFS transporters, more preferably anion/cation symporters. Exemplary transporters include those encoded or expressed from yiaX2, permA, perm B, pe6, pel from Pantoea citrea or Klebsiella oxytoca and heterologous sources. Synthetic Reaction o The present invention provides for the increased transport of a substrate across a membrane to enhance the output of a synthetic reaction. The increased transport provides for translocation of the substrate across a membrane separating the substrate from the cellular location of the synthetic reaction. The synthetic reaction can be a single enzymatic conversion, a portion or all of a synthetic pathway. The present invention is especially useful in synthetic pathways comprising both cystolic and extracellular synthetic reactions, where the end-product generated by one reaction is transported for utilization by another reaction on the other side of the membrane. The inventors contemplate a synthetic reaction that may occur inside the cytosol and require the transport of the substrate into the cytosol; or reactions that may occur outside the cytosol and require the transport of a substrate across the membrane to an extracellular location. Thus the present invention is especially useful in combination with reactions that require a co-factor that is present in the reaction, e.g., NADH or NADPH reactions, reactions that generally require a reduction of a substrate or intermediate, or reactions that are oxidized to provide a reducing equivalent. Exemplary reactions include, but are not limited to the reduction of 2,5-DKG to 2-KLG; the reduction of L-sorbosone to 2-KLG; the reduction of 5-keto-D-Gluconic acid (5-KDG) to L-idonic acid (IA); and the reduction of 5-KDG to L-gulonic acid (GA). Additional reactions include: Phosphatase (1 ) Glycerol-3 -phosphate + NADH ► Glycerol (2) Gluconate several enzymatic steps D-ribose 5-Phosphate In reaction (1) above, it is possible to envision that in the bioconversion of Glycerol-3 - phosphate into glycerol, the transport of Glycerol-3 -phosphate could be a rate limiting step. In reaction (2) above, gluconate is converted into D-ribose 5-Phosphate through a series of enzymatic steps. D-ribose 5-Phosphate is a valuable compound that can be converted into ribose, nucleotides, riboflavin or flavor enhancers like inosine monophosphate (Wulf & Vandame, 1997. Appl. Microbiol. Biotechnol. 48: 141-148). The bioconversion of gluconate into other products could reach a point where the intracellular transport could be a limiting step. Another reaction particularly useful in the practice of this invention is the bioconversion of pentose sugars into ethanol. Recently xylose has been has been used for engineering a homoethanol pathway in E.colL Tao, H, et al J. bact. 2001 , 183, 2979-2988. Increased transport of xylose into the cytosol could result in the enhanced production of ethanol . It is possible to envision, coupled with the production of these sugars by plant derived biomnass, a inexpensive source for the enhanced production of chemical compounds derived from these pentose sugars. Increased transport of these sugars by bacterial proton linked transport systems Henderson, P.J. Bioeng. Biomem 1990, 4, 525-569, or similar products, pathway linked to these pentoses. The inventors herein appreciate that the reactions provided above are merely illustrative of the invention and are provided as useful examples by which to understand the invention. One of skill in the art will be readily capable of identifying other synthetic reactions whereby the use of the present invention will be effective to increase production of a desired intermediate or end-product from a substrate. Similarly, the selection of suitable chemical production or biosynthetic pathways will be readily determined by one of skill in the art using the teachings provided herein. For example, where the end-product is related to a pathway or reaction that is part of the endogenous metabolism of an organism, it is often a simple matter to determine the critical transport steps. Similarly, where the pathway to the desired end-product is engineered into the cell, it is possible, using the techniques provided herein, to determine the critical pathway elements and a which point optimizing substrate transport will be of benefit to the production of end-product. In one embodiment, the synthetic pathway may comprise at least one synthetic reaction involving either a substrate or a product which is to be transported into or out of the cell. For example in this embodiment, the reaction to be enhanced comprises at least a portion of the pathway being intracellular or within the cytosol; and at least a portion of the pathway being extracellular. An exogenous substrate is utilized by the synthetic reaction or portion of the synthetic pathway residing within the cytosol. By way of example, the production of ketosugar derivatives, some of which can be used as ascorbic acid precursors or intermediates may be optimized using the present invention. Others like 5-keto-D-gluconic acid can be used in the preparation of tartaric acid (US patent 5,763,656). The present invention is particularly useful in conjunction with ascorbic acid intermediate synthesis, for example the conversion of 2,5 DKG to 2-KLG; the conversion of sorbose or sorbitol to 2- KLG via sorbosone; the reduction of 5-keto-D-Gluconic acid (5-KDG) to L-idonic acid; and the reduction of 5-Keto-D-Gluconic acid to L-gulonic acid . Each of these pathways is characterized by a portion of the synthetic pathway, a synthetic reaction, that resides within the cytoplasm, e.g. the reduction of 2,5-DKG by 2,5 DKG reductase; the reduction of L- sorbosone to 2-KLG by a sorbosone dehydrogenase; the reduction of 5-keto-D-Gluconic acid (5-KDG) to L-idonic acid by 5-KDG dehydrogenase; and the reduction of 5-Keto-D- Gluconic acid to L-gulonic acid by 5-KDG reductase. These pathways are also characterized by the necessity of transporting the substrate, e.g., 2,5-DKG; L-sorbosone, etc. across the membrane for bioconversion by the synthetic reaction residing in the cytoplasm. The substrate is generally one that can not pass through the membrane efficiently without some sort of active transport mechanism. Preferably these can include, but are not limited to ascorbic acid intermediates (2,5-DKG, sorbosone). Others substrates contemplated by the inventors include those described in Table 2 and Table 3. In addition, the substrate is a material that is transported for synthetic use on an industrial scale and generally not for metabolic use by the host cell. Industrial scale refers to the titer and volumetric productivity of being greater than 1 gm/liter/hour, preferably greater than 2 g/l/h, more preferably greater than 3 g/l/h and still more preferably greater than 5 g/l/h. In another embodiment, the productivity titer is between 2 and 14 gm/l/hour, preferably between 3 and 12 g/l/h, and still more preferably between 5 and 10 g/l/h to be an economically viable industrial production process. In addition, the yield of the synthetic pathway may also increase as a result of the present invention. In one embodiment, the yield is greater than 50 weight%, in another embodiment greater than 60% and in another embodiment greater than 70%. Especially preferred substrates include 2,5-DKG; and sorbosone. The inventive aspects of the present invention are especially useful in these embodiments, A reaction especially useful in the practice of this invention is the transport of 2,5-DKG across the inner cell membrane for the cystolic reduction of the same to 2-KLG by the cystolic dehydrogenase 2,5-DKG reductase. 2,5-DKG is converted from 2-keto-D-gluconate (2-KDG) by membrane bound 2-ketogluconate dehydrogenase. 2-KDG is converted from glucose through oxidation of D-gluconate (GA). Transport of 2,5-DKG across the inner cell membrane to the site of the cytolic reduction to 2-KLG can by achieved by the DNA encoding an increase in the transport of 2,5-DKG. Boudrant, J. (1990) Enzyme Microb, Technol., 1990:322-329) Another reaction especially useful in the practice of this invention is the transport of sorbosone, an intermediate in the production of 2-KLG through sorbose, sorbitol (Saito, Y, et al, Biotechnol. Bioeng. 58(2/3):309-315 (19987). The conversion of sorbitol and /or sorbose to sorbosone is a step in the pathway of converting sorbitol or sorbose to 2-KLG (Boudrant, J., 1990; Saito (1997)). The following is a discussion of engineering of sorbosone transporters according to the present invention. As shown in Fig.4a, the pathway of D-sorbose to 2-KLG includes the oxidation of L- sorbose to L-sorbosone by L-sorbose dehydrogenase (SDH), followed by the oxidation of L-sorbosone to 2KLG by L-sorbosone dehydrogenase. One recombinant host cell has been described which converts D-sorbitol to L-sorbosone by membrane bound dehydrogenases (Saito, Y., et al (1997)). L-sorbosone is then transported from the periplasm for reduction by L-sorbosone-dehydrogenase in the cytoplasm. Overexpression of the sorbosone transporter to facilitate the transport of the sorbosone intermediate to the pathway for conversion to 2-KLG is perceived as having a beneficial effect. As shown in Fig. 4, an alternative pathway of D-glucose to 2-KLG includes the oxidation of D-gluconic acid to 5-Keto-D-Gluconic acid (5KDG) which in turn is reduced to L-idonic acid (IA) or L-gulonic acid for oxidation to 2KLG. (Boudrant, J. (1990)). Transport of 5-Keto-D- Gluconic acid into the cytosol for reduction by keto-reductase could also be facilitated by the overexpression of the 5KDG transporter. In another embodiment, the synthetic reaction may be extracystolic or located outside the membrane relative to the substrate. The end-product of the first reaction may be the an intermediate substrate for a second reaction on the opposite side of a membrane. For example, in the synthesis of 2-KLG from D-sorbitol in Gluconobacter oxydans T-100 from Japanese persimmon (FERM BP-4188), the conversion of D-sorbitol to L-sorbose by cytosolic L-sorbitol dehydrogenase results in an intermediate that is transported out of the cytoplasm, across the cell membrane for conversion to L-sorbosone by the membrane bound L-sorbose dehydrogenase. Thus the inventors contemplate increasing the transport of the cytosolic intermediate, a substrate, from the cytosolic side of the inner membrane across the membrane to outside the membrane for subsequent conversion. Saito, Y., (1997) While a preferred embodiment includes the synthetic reaction or the pathways including the same as being within a single organism, having separate reactions in separate organisms is also contemplated by the inventors. For example, in a mixed culture system for the production of 2-KLG from glucose, the conversion of glucose to an intermediate 2,5-DKG, may occur within one organism (Acetomonas, Acetobacter, Gluconobacteer or Erwinia) while the conversion of that intermediate to the desired ascorbic acid intermediate 2-KLG occurs within the second organism (Brevibacterium, or Corynebacterium) see US Patent No. 3,963,574 to Sonoyama (1976). See also Hoshino, US Patent No. 5,312,741. In another example, the amino acid L-tryptophan has been produced by converting indole or anthranilic acid into L-tryptophan. In this case the product is partially utilized by cells for protein biosynthesis (US Patent No 3,801 ,457). Therefore the synthetic reaction may generate an intermediate that itself may be converted at another cellular location separated by a cell membrane. The end-product, in this embodiment, may be an intermediate substrate for a subsequent reaction. Methods for Determining the Rate limiting step of the To maximize the effect of increased transport of the substrate from a first cellular location to a second cellular location, in a preferred embodiment, the transport of substrate across the membrane constitutes a rate limiting step of the pathway. The determination of whether such a step is a rate limiting step is determined by analyzing the pathway, assessing the production of each step and altering the amount of substrate available for conversion by the pathway to ascertain whether such increased presence of the substrate results in an increase in the overall production of the pathway. Upon determining that increasing the presence of the substrate increases the yield of the desired end-product, if the substrate is one that requires translocation from one cellular location to another across a cell membrane, increasing the transport of the substrate will enhance the production by the pathway. There are several methods to identify the bottleneck(s) that may limit the production of a desired molecule. In general, most of them depend on the variation of the level of one or more components of the biosynthetic pathway and measuring the effect that those variations have in the process. These variations can be on the rate of synthesis, the total amount accumulated or the yield on which the substrate is converted into product. (Walsh & Koshland., 1985. PNAS 82: 3577-3581) (Jensen et al., 1993. Eur. J. Biochem. 211 : 181- 191). One method for determining the rate limiting status of the pathway portion is to compare the intermediate productivity at various points of the pathway, before and after increasing the presence of a particular bioconverter. If there is no increase in the production of the end-product despite increased presence of an intermediate or the overexpression of the converting pathway, the step may not be rate limiting, and thus overexpression of the particular enzyme effecting the synthetic reaction may not result in an enhanced production. The amounts of the individual intermediates can by determined by various indirect or direct means. Indirect means includes measuring the consumption or production of respiratory parameters, e.g. carbon dioxide production, oxygen consumption, by in-line measurements, such as gas partial pressures. Direct measurement of the intermediates can be achieved by various analytical techniques known in the artas described by Lazarus, Analyt. Biochem 157, 360-366 (1986) and references cited therein, which are incorporated by reference herein, including, but not limited to paper, gas, liquid and thin-layer chromatography as well as chemical and enzymatic assays. High performance liquid chromatography methodologies, especially as set forth in Lazarus, 1986, are especially helpful. One method used by the inventors includes the Waters 2690 HPLC and Waters 410 differential refractermeter , settings, using a 50mM acetate buffer, 1 ml/minute flow rate, as the eluting medium and Dyonex lonpac AS-10 ion-exchange column (4 x 250 mm) for separating and quantifying the chemical compounds present. Another method used by the inventors to determine the purity of the 2-klg produced in the broth was by total carbon analysis. Thus in one embodiment, the transport activity can be measured in any cell in which the substrate can be converted to a product, by measuring production of the product in the presence of extracellular substrate. For example, in a cell naturally expressing, or recombinantly expressing, a 2,5-DKG reductase, intracellular 2,5-DKG is converted to 2-KLG. The ability of the bacterial cell to produce 2-KLG when provided with extracellular 2,5-DKG, upon expression of a 2,5-DKG permease, is a measure of the ability of the expressed permease to transport 2,5-DKG into the cell, and is thus a measure of its 2,5-DKG permease activity. Intracellular 2-KLG can be detected, for example, using HPLC or other sensitive detection methods known in the art. Other metabolic products of 2,5-DKG can also be detected, by similar methods. The transporter of the desired substrate Transporters as used in the present invention comprise compounds that facilitate the translocation of materials across the cell membrane. In one embodiment, the type of transporter is selected from the group of channels proteins, primary active transporters, secondary active transporters, and group translocators. In another embodiment, the type of transporter is a secondary transporter, preferably a major facilitator superfamily member, more preferably an anion:cation symporter (ACS) member, most preferably a transporter selected from the group encoded from yiaX2, permA, permB, pel and pe6. There are four distinct types of functionally characterized transport systems based upon mode of transport and energy coupling mechanisms. The first are bacterial channel proteins (TC#1.A, Saier, 1998) , which transport via an energy independent facilitated diffusion mechanism utilizing a transmembrane pore. A second transport system, the facilitators and/or secondary transporters (the second class, TC#2.A), represent the largest category of transporters. A third group ATP driven primary active transporters contsitute a use ATP hydrolysis as a mode of energy coupling for the active uptake and or extrusion of solutes. The last group consist of group transports that phosphorylate their substrates during transport (TC #4.A). Of the secondary families, the largest of the class II families are the major facilitator superfamily (MFS) and the amino acid polyamine choline (APC) family (Reizer et al. Protein Sci 2(1):20-30 (1993)). These secondary transporter families can be further divided into three groups (1) proton motive form (pmf driven), (2) sodium motive force (smf)-driven, and (3) other ion or solute driven exchangers. These transport systems catalyze uni, anti and/or symport of solutes. Secondary active transporters have been identified in E.coli, H.influenzae, H. pylori, B. subtilis, M. genitalium, Synechocystis, M.Jannaschii (Paulsen et al 1998 J. Mol. Biol. 277:573-592). Secondary transporters are typically polytopic membrane proteins, frequently with 12 TMS. With most primary carriers, a chemical form of energy drive the group translocation, be it ATP-dependent systems as most ATP-binding cassette (ABC) superfamily members are, or PTS, which use PEP as the phosphyoryl donor for sugar uptake and phosphorylation. Secondary transporters differ from primary (ABC transporters) in that the primary transporters use ATP, taking energy away from the cell. In addition, the use of ABC transporters generally requires a more complex transporter system, one that can comprises two hydrophobic integral membrane domains, and two ATP-binding domains (Hosie, et al Molecul. Microbiol (2001) 40(6), 1449-1459. Those ABC transporters responsible for the uptake of solutes also require the presence of a solute- binding protein (SBP). Thus genetic engineering of an improved ABC transport system would require the expression and transformation of a more complex nature than one of a secondary transporter. Transporters within the scope of the present invention include those described in Table I and II: Table 1. Transport protein families identified by bacterial genome analysis Name of Family Abbreviation TC no. a Example I. Channel proteins Major intrinsic protein MIP 1.1 GlpF Eco Large conductance mechano-sensitive ion MscL 1.3 MscL Eco channel Voltage-sensitive ion channel VIC 1.5 Kch Eco Chloride channel CIC 1.10 Gefl See II. Secondary active transporters Major facilitator superfamily MFS 2.1 LacY Eco Glycoside-pentose-hexuronide:cation symporter GPH 2.2 MelB Eco Amino acid-polyamine-choline APC 2.3 PheP Eco Cation diffusion facilitator CDF 2.4 CzcD Aeu Resistance-nodulation-cell division RND 2.6 AcrB Eco Small multidrug resistance SMR 2.7 Smr Sau Gluconate:H + symporter GntP 2.8 GntP Eco L-rhamnose transporter RhaT 2.9 RhaT Eco 2-keto-e-dexoygluconate transporter KdgT 2.10 KdgT Eco Citrate-Mg 2+ :H + symporter CitMHS 2.11 CitM Bsu C4-dicarboxylate uptake Dcu 2.13 DcuA Eco Lactate permease LctP 2.14 LctP Eco Betaine/carnitine/choline transporter BCCT 2.15 BetT Eco Tellurite-resistance/dicarboxylate transporter TDT 2.16 TehA Eco Proton-dependent oligopeptide transporter POT 2.17 Ptr2 Eco Ca 2+ :cation antiporter CaCA 2.19 YrbG Eco Inorganic phosphate transporter PiT 2.20 PitA Eco Sodium:solute symporter SSS 2.21 PutP Eco Neurotransmitteπsodium symporter NSS 2.22 Gat1 Hsa Dicarboxylate:cation (Na + or H + ) symporter DCS 2.23 GltP Eco Citrate:Na + symporter CSS 2.24 CitS Kpn Alanine:Na + symporter ASS 2.25 DagA Aha Branched chain amino acid: Na + symporter LIVSS 2.26 BrnQ Sty Glutamate:Na + symporter ESS 2.27 GltS Eco Bile acid: Na + symporter BASS 2.28 NabA Rno NhaA Na + : H + antiporter NhaA 2.33 NhA Eco NhaB Na + : H + antiporter NhaB 2.34 NhB Eco NhaC Na + : H + antiporter NhaC 2.35 NhC Eco Monovalent cation:proton antiporter-1 CPA-1 2.36 Nhe1 Rno Monovalent cation:proton antiporter-2 CPA-2 2.37 KefC Eco K + :H + transporter Trk 2.38 TrkHAE Eco Nucleobase:cation symporter-1 NCS1 2.39 CodB Eco Nucleobase:cation symporter-2 NCS2 2.40 UraA Eco Nucleoside uptake permease NUP 2.41 NupC Eco Aromatic amino acid permease ArAAP 2.42 Mtr Eco Serine/threonine permease STP 2.43 SdaC Eco Formate-nitrite porter FNP 2.44 FocA Eco Metal ion transporter MIT 2.45 CorA Eco Benzote:H + symporter BenE 2.46 BenE Aco Divalent anion:Na + symporter DASS 2.47 NadC1 Has Ammonium transporter Amt 2.49 HoxN Reu Ni 2+ -Co 2+ transporter NiCoT 2.52 HoxN Reu Sulfate transporter SulP 2.53 *Sul1 See Name of Family Abbreviation TC no. a Example Tripartite ATP-independent periplasmic TRAP-T 2.56 DctPQM Rca transporter Phosphate:Na + symporter PNaS 2.58 NapT Rno III. Primary active transporters ATP-binding cassette ABC 3.1 MalEFGK Eco H + or Na + -translocating F-type, V-type and A-type F-ATPase 3.2 UncA-H Eco ATPase Cation-translocating P-type ATPase P-AtPase 3.3 CadA Sau Arsenical efflux Ars 3.4 ArsAb Eco IV. Group Translocators PTS glucose-glucoside Glc 4.1 PtsG/Crr Eco PTS fructose-mannitol Fru 4.2 FruAB Eco PTS lactose-cellobiose Lac 4.3 Cel ABC Eco PTS glucitol Gut 4.4 GutAB Eco PTS galactitol Gat 4.5 GatABC Eco PTS mannose-fructose-sorbose Man 4.6 ManXYZ Eco V. Unclassified Polysaccharide transporter PST 99.1 RfbX Eco MerTP mercuric ion (Hg 2+ ) permease MerTP 99.2 MerTP Sau Nicotinamide mononucleotide uptake permease NMN 99.4 PnuC Eco K + uptake permease Kup 99.5 Kup Eco L-lysine exporter LysE 99.6 LysE Cgl Chromate ion transporter CIT 99.7 ChrA Aeu Ferrous iron uptake FeoB 99.8 FeoB Eco Mg 2+ transporter Mgt 99.19 MgtE Pst Table 2. Transport protein families identified by bacterial genome analysis Name of Family Typical substrates Dist. c References I. Channel proteins Major intrinsic protein Glycerol, water BE Park & Saier (1996) Large conductance mechano-sensitive Ions B Sukarev et al. ion channel (1996) Voltage-sensitive ion channel Potassium BAE Alexander & Peters (1997) Chloride channel Chloride BAE Saier et al. (unpublished); Jentsch et al. (1995) II. Secondary active transporters Major facilitator superfamily Sugars, drugs, BAE Pao et a/. (1998) metabolites, (unpublished nerotransmitters, results); Paulsen organic cations, & Skurray (1993); nucleosides, Paulsen et al. carboxylates, (1996a,b) organic and inorganic anions Glycoside-pentose-hexuronide:cation Glycosides, B Poolman et al. symporter pentoses, (1996) hexuronides Amino acid-polyamine-choline Amino acids, BAE Reizer et al. choline, polyamines (1993) Cation diffusion facilitator Metal ions BAE Paulsen & Saier (1997) Resistance-nodulation-cell division drugs, metal ions, B Paulsen et al. lipooligosaccharides (1996b) Small multidrug resistance Drugs B Paulsen et al. (1996b) Gluconate: H + symporter Gluconate B Peekhaus et al. (1997) L-rhamnose transporter Rhamnose B Tate et al. (1992) 2-keto-e-dexoygluconate transporter 2-Keto-3- B Allen et a/. (1989) deoxygluconate Citrate-Mg 2+ :H + symporter Citrate G+ Boorsma et al. (1996) C4-dicarboxylate uptake Dicarboxylates G- Saier et al. (unpublished results) Lactate permease Lactate BA Saier et al. (unpublished results) Betaine/carnitine/choline transporter Betaine, carnitine, B Saier et al. choline (unpublished results) Tellurite-resistance/dicarboxylate Tellurite, BA Saier et al. transporter dicarboxylates (unpublished results) Name of Family Typical substrates Dist. c References Proton-dependent oligopeptide Peptides BE Saier et. al. transporter (unpublished results) Ca 2+ :cation antiporter Ca 2+ /Na + or H + : BAE Saier et al. (unpublished results) Inorganic phosphate transporter Phosphate BAE Saier et al. (unpublished results) Sodium:solute symporter Amino acids, BE Reizer et al. sugars, vitamins, (1993) nucleosides, inorganic anions Neurotransmitteπsodium symporter Neurotransmitters BAE Reizer et al. (1993) Dicarboxylate:cation (Na + or H + ) Dicarboxylates, BE Saier et al. symporter amino acids (unpublished results) Citrate: Na + symporter Tri and B Kawai et al. dicarboxylates (1997) Alanine:Na + symporter Alanine B Reizer et al. (1993 Branched chain amino acid: Na + Branched chain B Reizer et al. symporter amino acids (1993 Glutamate:Na + symporter Glutamate G- Reizer et al. (1993 Bile acid: Na + symporter Bile acids BE Reizer et al. (1993 NhaA Na + : H + antiporter Na + /H + G- Gerchman et al. (1993) NhaB Na + : H + antiporter Na + /H + B Pinner et al. (1992) NhaC Na + : H + antiporter Na + /H + B Ivey et al. (1991 ) Monovalent cation:proton antiporter-1 Na/H + BAE Saier et al. (unpublished results); Orlowski et al. (1992) Monovalent cation:proton antiporter-2 K + ,Na + /H + BAE Saier et al. (unpublished results) Stumpe et al. (1996) K + :H + transporter K + /H + BAE Saier et al. (unpublished results) Stumpe et al. (1996) Nucleobase ation symporter-1 Cytosine, uracil, BE Saier et al. allantoin (unpublished results) Nucleobase:cation symporter-2 Nucleosides BE Saier et al. Name of Family Typical substrates Dist. c References (unpublished results) Nucleoside uptake permease Nucleosides BE Saier et al. (unpublished results) Aromatic amino acid permease Amino acids G- Sarsero & Pittard (1997) Serine/threonine permease Amino acids G- Shao et al. (1994) Formate-nitrite porter Formate, nitrite BAE Saier et al. (unpublished results) Metal ion transporter Mg 2+ ,Co 2+ ,Mn 2+ ,A1 3+ BA Smith et al. (1993) Benzote:H + symporter Benzoate G- Neidle et al. (1991) Divalent anion: Ha + symporter NadC1 Hsa BAE Markovich et al. (1993) Ammonium transporter Ammonium BAE Saier et al. (unpublished results) Ni 2+ -Co 2+ transporter Ni 2+ ,Co 2+ B Wolfram et al. (1995) Sulfate transporter Sulfate BE Saier et al. (unpublished results); Smith et al. (1995) Tripartite ATP-independent periplasmic Dicarboxylates B Forward et al. transporter (1997) Phosphate: Na + symporter Phosphate BE Magagnin et al. (1993) III. Primary active transporters ATP-binding cassette Sugars, amino BAE Kuan et al. (1995) acids, drugs, metal ions, Metabolites, peptides, vitamins, proteins, complex carbohydrates H + - or Na + -translocating F-type, V-type i H + /Na + BAE Blair et al. (1996) and A-type ATPase Cation-translocating P-type ATPase M + ,M^ + BAE Fagan & Saier (1994) Arsenical efflux Arsenite, antimonite BAE Tsai et al. (1997) IV. Group Translocators PTS glucose-glucoside Glucose, sucrose, β- B Reizer et al. glucosides, N- (1996c) acetylglucosamine PTS fructose-mannitol Fructose, mannitol B Reizer et al. (1996c) PTS lactose-cellobiose Lactose, cellobiose B Reizer et al. (1996a) Name of Family Typical substrates Dist. c References PTS glucitol Glucitol B Reizer et al. (1996b) PTS galactitol Galactitol G- Nobelmann & Lengeler (1996) PTS mannose-fructose-sorbose Mannose, sorbose, B Reizer et. al. fructose (1996d) V. Unclassified Polysaccharide transporter Polysaccharides B Paulsen et al. (1997) MerTP mercuric ion (Hg 2+ ) permease Mercury and heavy B Silver & Phung metal ions Nicotinamide mononucleotide uptake Nicotinamide G- Foster et al. permease mononucleotide (1990) K + uptake permease Potassium BE Banuelos et al. (1995) L-lysine exporter Lysine B Vrljic et al. (1996) Chromate ion transporter Chromium ions BA Nies & Silver (1995) Ferrous iron uptake Ferrous iron BA Kammler et al. (1993) Mg 2+ transporter* Mg + ,Co 2+ BA Townsend et al. (1995) a TC no., classification number of the transport commission (Saier, 1998) b The cited example indicates the abbreviation of the protein, based on the encoding gene designation (first four letters), followed by a three letter abbreviation of the source organism (e.g. Eco, E. coli; See, S. cerevisiae) c The occurrence of members of the family within the following phylogenetic groups of organisms is indicated as follows: Gram-negative bacteria, G-; Gram-positive bacteria, G+; bacteria, B; Archea, A; Eukaryotes, E (Paulsen, I ., et al., J. Mol. Biol. 277, 573-592 (1998) and the references cited therein are incorporated by reference. Secondary transporters within the scope of this invention are described in Tables 3 and 4: Table 3 Abbreviation Description Phylum* ExuT Eco Hexuronate transporter B YcbE Bsu Putative glucarate transporter B YcbE Ppu Putative glucarate transporter B HpaX Eco 4-Hydroxyphenylacetate transporter B Orf 1450 Eco Putative glucarate transporter B Pht1 Ppu Phthalate transporter B TtuB Avi Tartrate transporter B TtuB1 Avi Tartrate transporter B YhaU Eco Putative glucarate transporter B YidT Eco Galactonate transporter B YybO Bsu Glucarate transporter B C11 D3.18c Spo C11D3.18c (putative Na7phosphate cotransporter) Y Dal5 See Allantoate permease Y L0578 See Unknown function Y Yal0670 See Unknown function Y Ycr28c See Unknown function Y Ygr260w See Unknown function Y Yil 166c See Unknown function Y Ylr004c See Orf Ylr004c (function unknown) Y Table 4 Abbreviation Organism Size (no. of Accession Dataresidues) no. base* ExuT Eco Escherichia coli 472 P42609 SP YcbE Bsu Bacillus subtilis 455 P42237 SP YcbE Ppu Pseudomonas putidas 456 P42205 SP HpaX Eco Escherichia coli 458 Z37980 GB Orf f450 Eco Escherichia coli 450 U29581 GB Pht1 Ppu Pseudomonas putida 451 Q05181 SP TtuB Avi Agrobacterium vitis 433 U25634 GB TtuB1 Avi Agrobacterium vitis 449 U32375 GB YhaU Eco Escherichia coli 444 P42613 SP YidT Eco Escherichia coli 445 P31457 SP YybO Bsu Bacillus subtilis 435 P37489 SP C11D3.18c Spo Schizosaccharomyces pombe 498 Z68 66 GB Dal5 See Saccharomyces cerevisiae 543 P15365 SP L0578 See Saccharomyces cerevisiae 531 S50965 PIR Yal0670 See Saccharomyces cerevisiae 593 P39709 SP Ycr28c See Saccharomyces cerevisiae 512 P25621 SP Ygr260w See Saccharomyces cerevisiae 534 Z73044 GB Yil 166c See Saccharomyces cerevisiae 542 P40445 SP Ylr004c See Saccharomyces cerevisiae 523 Z73176 GB Pao, S.S., et al., 1998, Microbiol Mol. Biol. Rev. 62(1): 1-34. In one embodiment, the DNA encoding the at least one protein for increasing the transport of the substrate across the inner cell membrane is selected from a mammalian, plant, fungal or bacterial cell. As examples of plant cells, the following are herbs, trees, seagrasses. As examples of fungal cells, the following are useful: yeasts, filamentous fungi, white-rot fungi and slime molds. As examples of bacterial cells, the following are usefukAcetobacter, Pseudomonas, Bacterium, Cyanococcus, Micrococcus, Brevibacterium, Arthrobacter, Staphylococcus, Bacillus, Corynebacterium, Acetomonas, Gluconobacter and Erwinia. Prefered organisms are selected from the group consisting of E.coli, Pantoea and Kleibsiella. II. Expression Systems The present invention provides expression systems for the enhanced production and transport of desired heterologous or homologous proteins in microorganisms, including bacteria and yeast. a. Coding Sequences In the present invention, the vector comprises at least one copy of nucleic acid encoding a transporter and preferably comprises multiple copies. The coding sequences for the transporters will be obtained pursuant to routine and standard techniques available to the skilled worker in the field. Appropriate start and stop codons, and issues related to introns, 5 exons and other gene construction optimizations will be readily applied by methods of standard molecular biology. b. Vector Sequences o Expression vectors used in expressing the transporters of the present invention in microorganisms comprise at least one promoter, termination sequence and other regulatory sequences which are essential or desirable for expression or overexpression of the transporter factor, which promoter is functional in the host cell. In one embodiment of the present invention, the promoter is the wild-type promoter for the selected transporter and in s another embodiment of the present invention, the promoter is heterologous to the transporter, but still functional in the host cell. Additional promoters associated with heterologous nucleic acid encoding desired proteins or polypeptides may be introduced via recombinant DNA techniques. In one embodiment of o the present invention, the host cell is capable of overexpressing a heterologous protein or polypeptide and nucleic acid encoding one or more transporter(s) is(are) recombinantly introduced. In one preferred embodiment of the present invention, nucleic acids encoding the at least one protein or more proteins increasing the transport of the substrate maybe stably integrated into the microorganism genome. In another embodiment, the host cell is 5 engineered to overexpress DNA encoding for one or more proteins increasing the transport of said substrate into said host cell of the present invention and nucleic acids encoding the heterologous protein or polypeptide is introduced via recombinant DNA techniques. The present invention encompasses host cells that are capable of overexpressing other transporters known to those of skill in the art, including but not limited to, those identified in o Tables 1 , 2 or 3 or other transporters known to those of skill in the art or identified in the future. In a preferred embodiment, the expression vector contains a multiple cloning site cassette which preferably comprises at least one restriction endonuclease site unique to the vector, 5 to facilitate ease of nucleic acid manipulation. In a preferred embodiment, the vector also comprises one or more selectable markers. As used herein, the term selectable marker refers to a gene capable of expression in the gram-positive host which allows for ease of selection of those hosts containing the vector. Examples of such selectable markers include but are not limited to antibiotics, such as, erythromycin, aspectinomycin, chloramphenicol and tetracycline. Also provided are embodiments in which a transporter encoded by a nucleic acid having at least 90% homology with the DNA sequence shown in SEQ ID NOS:[ ]. Preferably, the homology is at least 95%, more preferably at least 98%. Homology can be determined by lining up the claimed amino acid or DNA sequence with another sequence and determining how many of the amino acids or nucleotides match up as a percentage of the total. Homology can also be determined using one of the sequence analysis software programs that are commercially available, for example, the TFastA Data Searching Program available in the Sequence Analysis Software Package Version 6.0 (Genetic Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis. 53705). One can screen for homologous sequences using hybridization as described herein or using PCR with degenerate primers. Chen and Suttle (1995) Biotechniques 18(4):609-610, 612. Also, in several embodiments of the invention, there are provided nucleic acids that can hybridize with the DNA or fragments thereof, shown in FIGS, and , SEQ ID NOS:, respectively, under stringent conditions. Stringent hybridization conditions include stringent hybridization and washing conditions as is known to one of ordinary skill in the art. Hybridization and appropriate stringent conditions are described in Sambrook et al. 1989 Molecular Cloning 2d ed., Cold Spring Harbor Laboratory Press, New York. Methods for Increasing Production of Recombinant Peptides from Host Cells having transformed DNA encoding transport proteins A particularly powerful method of increasing the transport of the substrate from one cellular location to another involves the deletion of metabolic diversions genomes from the transformed host cell and the concomitant provision of DNA encoding which increases the transport of the desired substrate. This is advantageously achieved by providing to the cell a deletion-transporter chimera or fusion protein, in which the metabolic diversions of the deleted portion are minimized, and in which the transporter ability portion is overexpressed. Chemically-fused polypeptides, or shuffled sections of the genenome are a possibility, but recombinant proteins are naturally most preferred for use in this manner. The identification of appropriate permease fragments for use in such a chimera has been described herein above. In terms of the transport portions of these fusion proteins, any permease-derived sequence that contains enough primary sequence information to confer transport activity to the chimera will be useful in this context. However, it will often be preferred to use the entire transporter enzyme as this is more straightforward in terms of methodology. Again, one may look to the extensive information available in various published references in order to assist with the identification of appropriate transporters or fragments thereof. Transformation The present invention also contemplates augmenting or increasing the capabilities of cells to produce biologically active polypeptides, such polypeptides increasing the transport of a substrate from a first location of the cell across a membrane to a second location of the cell, This can be accomplished, in some instances, by overexpressing the proteins involved in the transport of a substrate to another cellular location for additional bioconversion, such as a secondary transporter of the anion/cation symporter (Saier, 1988), in one embodiment an anion/cation H+ symporter. Expression of transporters involved in maintaining the viability and productive qualities of host cells, especially their transport capacity, is important. In the event that the pathways are NADPH or NADH requiring reactions, the continued viability of the host cells is a necessity for successful continuous production by the desired pathway. Certain considerations and factors can be kept in mind while determining the number of multiple copies or promoters that can be overexpressed while maintaining the viability of the organism. In general, excessive expression of transporters can be detrimental to the cells because the available space to incorporate the transporter in the membrane is limited. Therefore, very excessive overproduction of one transporter may decrease the incorporation in the membrane of other transporters that may be involved in the transport of other nutrients from the media . Engineering the overexpression of a cell type-specific transcription factor could increase or stabilize the transporter capabilities of engineered host cells. Stable overexpression of H+ symporters in bacterial host cells will serve several purposes. It will increase transgene expression under while maintaining the viability of the microorganism. The overexpression of the symporters is simpler than overexpression of ABC transporters since symporters do not require the extensive encoding for the multiple components of the ABC transporter. Thirdly, by overexpressing a secondary transporter instead of ATP or PTS coupled transports diversion of energy requirements from the host cell is minimized. In one embodiment of the present invention, nucleic acid encoding one or more transporters of the present invention is introduced into a host cell via an expression vector capable of replicating within the host cell. Suitable replicating plasmids for Pantoea are described in Sambrook, et al, 1989, Molecular Cloning 2d ed., Cold Spring Harbor Laboratory Press, New York),hereby expressly incorporated by reference, based on the fact that Pantoea sustain the replication of the same plasmids that E.coli. In another embodiment, nucleic acids encoding one or more transporters is stably integrated into the microorganism genome. Preferred host cells are from the genus Pantoea. Another preferred host cell is K. oxytoca. Several strategies have been described in the literature for the integration of DNA into the chromosome (see for example Balbas, et al., 1996, Gene 172:65-69; LeBorge, et al, 1998, Gene 223: 213-219 Transformation of P.citrea can be accomplished by the electroporation method, using the protocol developed for E.coli (Potter, H., 1988, Anal.Biochem. 174:361-373). III. Identification of Transformants After introducing the DNA into the host, the transformants are selected by antibiotic resistance encoded in the vector or in general by selecting for a function coded within the plasmid. Once transformants have been differentiates form non-transformed cells, the presence of the plasmid with an intact structure can be confirmed using standard protocols (Sambrook, et al, 1989) The presence of the transporter encoding polynucleotide sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using appropriately designed probes, portions or fragments based on sequences or homology . IV. Transport Assays Means for determining the levels of substrate transported in a host cell and detecting the transported substrate are readily determined by one of skill in the art for a specific pathway and include, using either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox DE et al (1983, J Exp Med 158:1211). For the purposes of illustration of the present invention, a method for determining the levels of the ascorbic acid intermediates pursuant to the 2-KLG pathway are provided below. However, the general concepts and techniques disclosed, together with those known to the skilled worker in the field, would be useful in designing a strategy for the determination of levels of substrate, intermediates and end-products in biosynthetic pathways generally. By way of example a method for determining the levels of the ascorbic acid intermediates in the KLG product pathway, those of skill in the art will recognize that HPLC methodology is one method for determining the levels of chemical compounds and assessing there production and consumption relative each other. In addition, a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting specific polynucleotide sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the nucleotide sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567 and incorporated herein by reference. Also, recombinant immunoglobulins may be produced as shown in U.S.Pat. No. 4,816,567 and incorporated by reference. EXAMPLES Example 1 materials and methods a. Plasmid, bacterial strain and media: Plasmid pBCL1920, K. oxytoca, P. citrea 1392A, Murphy III medium contained fructose 0.5%, Phosphate 1.6%, MgSO4.7H2O 0.2%, Soytone 0.2%, citrate 0.01%, (NH4)2SO4 1%, Trace salts in ppm range such as Fe, Co, Mn, Zn and vitamins such as nicotinic acid, folate and B12 ; M9 medium, 0.9% Phosphate, 0.1% NaCl, 0.1% NH4CI, MgSO4 0.0005%, CaCI2 0.025%; Fermentation medium Potassium. Phosphate1%, MgSO4 0.15%, glutamate 1.5%, fructose 2.5%, ammonium sulfate 0.1%, and vitamin blend having biotin, thiamin, pyridoxine, riboflavin, nicotinic acid, folic acid and B12 ,and trace metals cocktail solution having iron, Zn, and Mn ions in 10-100 ppm level; Transport assay medium 100 mM Potassium Phosphate pH 6.9 ; Antibiotics Spectinomycin 50 ug/ml and Tetracyclin 20 ug/ml, IPTG 100 uM. b. DNA techniques c. Growth of cells : Strains constructed using recombinant methods and wild-type P. citrea and K. oxytoca were grown in either M9 medium containing DKG as the sole carbon source or Mill medium with fructose as the sole carbon source or with Mill having mixed carbon source such as fructose, Gluconate and DKG in the range from 0.1 % to 1%. Cells were grown between 20-37°C but preferably below 30°C. Cells were preferably grown at neutral pH but the range comprised of from pH 5-8. P. citrea cells grown in a fermenter from a seed flask used fermentation medium using either fructose or glucose feed. d. DKG uptake biochemical assay: Samples of fermentation broth containing cells were withdrawn from respective growth apparatus and were quenched on ice-water bath. The fermentation broth was centrifuged and supernatant was discarded. The cell pellet was washed using 0.95 ice-cold saline solution followed by 2 washes by DKG uptake assay buffer 100 mM ice-cold Potassium phosphate pH 6.9. Cells were resuspended in the same assay buffer to an OD of 12 at 550 nm and were incubated at room temperature or preferably at 28°C. DKG uptake assay was started by mixing the cells with C-14 enriched radio-isotoped 2,-5 DKG. Time course of DKG uptake was performed using vaccum/filter based quenching using ice-cold assay buffer. DKG uptake measurement were done by radioisotope incorporation in the cells and the data obtained was plotted against time to give the DKG uptake rate. Another assay using silicone oil for cellular DKG uptake and metabolism study was also performed (Johnson, J.D. et al., J. Lab. Clin. Med., 1980, 95: 429-439). 100ul of silicon oil was added to epitubes ready for the assay. Cells and the 14C (U) enriched 2,5-DKG was mixed and then at a regular time intervals 100ul of the cell/substrate mixture was withdrawn and added to the epitubes containing silicon oil, centrifuged for 15 seconds and then immediately frozen in dryice/ethanol bath. After five minutes, tip of the epitube containing cell pellet is cut directly into a scintillation vial. Rest of the tube is recut just above the frozen portion and dropped into another vial containing scintillation fluid. Counts were measured after after an overnight to facilitate cell pallet loosening from the epi tip. The difference between the loss of counts from top and appearance of counts in the pallet is due to the cell metabolism and released CO 2 . At the end of the uptake assay, when cells were permeabilized, the low molecular cell contents leaked out and provided information about the accumulated imported substrate and fate of substrate futher metabolized to become cellular component. e. DKG reductases assay: Cell pellets from each fermentor were collected and frozen at - 70°C for approx. 24 hours. Pellets at the 15 and 25 hour time points were thawed on ice and French pressed in 50 mM PIPES buffer, pH 6.5. The extracts were spun for 2 min. X 14 K rpm on a bench-top centrifuge, and subsequently measured for total protein concentration and reductase activity. All samples were measured by Bradford and BCA assays for protein, and diluted as needed for accurate rate assays. The assays were measured against background rates which contained all but the 2,5-DKG (and were all less than 10% of the total rates). The buffer contained: 50 mM PIPES pH 6.5, 150 uM NADPH and 5 mM 2,5-DKG. Another assay using silicon oil for cellular DKG uptake and metabolism study was also performed (Johnson, J.D., et al., J. Lab. Clin. Med. 1980, 95:429-439). 100 ul of silicon oil was added to epitube ready for the assay. Cells and 14C (U) enriched 2,5-DKG was mixed and then at a regular time intervals (for example about every 10 seconds) 10Oul of the cell/substrate mixture was withdrawn and added to the epitubes containing silicon oil, centrifuged for 15 minutes and then immediately frozen in dry ice/ethanol bath. After 5 minutes, the tip of the epitube containing the cell pellet is cut directly into a scintillation vial. The rest of the tube is recut just above the frozen portion and dropped into another vial containing the scintillation fluid. Counts were measured after an overnight to facilitate cell pallet loosening from the epi tip. The difference between the loss of counts from top and appearance of counts in the pallet is due to the cell metabolism and released CO 2 . At the end of the uptake assay, when cells were permeabilized, the low molecular cell contents leaked out and provided information about the accumulated imported substrate and fate of substrate further metabolized to become cellular components. Example II Example II provides the basis that transporters of substrate may be rate limiting in a whole- cell bioconversion This example narrates the key steps of 2KLG formation from glucose which can be compartmentalized into four parts (Fig. 5) . Production of the key intermediate 2,5-DKG using three periplasmic enzymes in P. citrea at 14-15 g/l/hr rate (Sonoyama, et al, Appl. Environ. Microbiol., 1982, 43:1064-1069). The second part is the rate by which DKG is needed to be transported in the cell's cytoplasmic space. The third is the rate of conversion of DKG to 2KLG using DKG reductases (US Patent No. 5,032,514). DKG to 2KLG conversion is not the rate limiting when DKG reductase is overexpressed. When in our current 2KLG production fermentations, inducible plasmids were used to both increase and decrease reductase specific activities relative to our typical fermentations, which presently use pD92, in which DKG reductase in under a constitutive trp promoter. The inducible plasmid, pD23, is under a taq promoter, and can be induced with IPTG. Three fermentors were run, one with pD92, and two with pD23, one of which was induced with IPTG. The control pD92 and the induced pD23 produced nearly identical levels of 2-KLG, while the uninduced pD23 made significantly less. Assays of reductase specific activities show that relative to the control pD92, the uninduced plasmid made less than half the levels of reductase, while the induced pD23 made more than twice as much. These results indicate that the level of reductase activity in our 2KLG producing P. citrea fermentation is not the bottleneck for the production of 2-KLG. Strains Protein Concentration Reductase Activity Specific Activity pD92 (control) 3.2 mg/ml 25.0 u/ml 7.8 u/mg pD23 (uninduced) 3.7 mg/ml 12.0 u/ml 3.2 u/mg pD23 (induced) 3.9 mg/ml 73 u/ml 19 u/mg The fourth part is the transport of 2KLG which is intracellularly made and need to be exported out. The production rate of 2KLG in the fermentation ( 2.2 g/l/hr - 2.7 g/l/hr) is considered to be equal to the export rate of 2KLG from the cell. It is argued, if the rate of export of 2KLG is limiting then cells will accumulate 2KLG in side the cell and cells will not be able to function at their metabolic potential and eventually die. However, 2KLG production cells of P. citrea do not exhibit either of these conditions and intracellular measurements of 2KLG remain 10-20 fold below the maximum concentration of 2KLG produced. It is thus conceived that 2KLG export is also not a rate limiting step in the production of 2KLG. Example III Example III provides the proof that indeed the transport rate of substrate in to the cell for bioconversion can be the rate limiting step. Cell pallets from three different times of the fermentation process, seed-flask stage, fructose feed stage and glucose feed stage were collected and processed as described in the experimental. The DKG uptake assay using these cell-types gave 0.5 g/l/hr, 2.7 g/l/hr and 2.75 g/l/hr DKG uptake rate of bring in the DKG to get converted to KLG (Fig 2). The rate of DKG import is same as KLG export. This result thus demonstrate that the rate of bioconversion of DKG to KLG is dependent upon the import rate of 2,5-DKG into the cell. Thus this invention will demonstrate that by increasing the DKG uptake transporters by overexpression will enhance the import rate of 2,5-DKG and thus will enhance the 2KLG production rate. Those expert in this art, can visualize that the key limiting factor in various biotransformations using whole cell conversion methods may indeed be the import and the import rate of substrate into the cell for the biotransformation. Example IV Example IV provides the discovery of a 2,5-DKG transporter in K. oxytoca using DKG uptake assay WO 002170 describes the identification and sequencing of an operon from Klebsiella oxytoca, designated the yia operon, which contains eight putative open reading frames. The functions of these polypeptides encoded by the individual open reading frames in the yia operon are not described in WO002170. Disruption of this Operon removed the ability of K. oxytoca to use ascorbic acid as sole carbon source. It is known that ascorbic acid is an oxidatively unstable substance and it decomposes to 2,3-DKG by air oxidation (Kimaya, S., J. Vitaminol., 1961, 7:19-26). It was thus reasonable to suggest that it is 2,3- DKG which is the real substrate for growth. One of the open reading frames in the yia operon , designated as yiaX2, encoded a transporter type transmembrane protein and was thus considered a candidate for 2,3-DKG permease. In light of the parallel search of finding 2,5-DKG permease, it was thus realized that 2,3-DKG and 2,5-DKG being analogous molecules, it may be possible that yiaX2 can transport 2,5-DKG and other sugar keto acids such as 2KLG. In order to determine whether yiaX2 can transport 2,5-DKG, and 2-KLG this gene was deleted from the chromosome of K. oxytoca strain M5a1 (see, for example, Streicher et al., Proc. Natl. Acad. Sci. 68: 1174-1177 (1971)). The yiaX2 deletion mutant designated as MGK002 was created using standard molecular biology protocols known in the art (see for example, Hamilton et al., J. bacterial. 171 :4617-4622 (1989)). Another K. oxytoca strain designated as Tester strain was created by adding back the plasmidly encoded and lac operon regulated yiaX2 gene to K. oxytoca strain MGK002. DKG uptake assay under +/- IPTG induction using MGK002 and Tester strain confirmed that yiaX2 encoded a poly peptide having 2,5-DKG transport activities (Fig 9). Example V Example V provides the selection methodology for screening 2,5-DKG permeases form microorganisms. With the information that yiaX2 gene encoded a transporter protein having 2,5-DKG transport activities, it became possible to design a selection host for finding 2,5-DKG transporter protein of P. citrea and other biological sources. K. oxytoca with yiaX2 gene deletion and addition of genes to express enzymes involved in the catabolism of 2,5-DKG to gluconic acid, which is assessable to the central metabolism of an organism. Enzymes capable of catabolizing 2,5-DKG to gluconic acid are encoded by tkr and idno genes of the tkr idnD idnO operon present in some Gram negative organisms (Bausch, O, et al, J. Bacteriol., 1998, 180:3704-3710). The resulting tester strain of K. oxytoca was yiaX2[tkr idno] and had a all the components needed for growth on 2,5-DKG as a sole carbon source except its inability to import DKG into the cytoplasm. Therefore, a nucleic acid molecule that encodes a 2,5-DKG permease, upon expression in the tester strain, should confer the ability of the tester strain to grow on 2,5-DKG. This selection methodology is shown in figure 9. The cloning vector used for constructing the P. citrea genomic libraries is plasmid pCI1920 (Lerner et al., Nucleic Acid Res., 1994, 18:4621 ), a low-copy number expression vector which carries a spectinomycin/streptomycin resistance determinant. Expression is driven by the lacPO promoter/operator region which is repressed by laclq gene product when provided by the host. Genomic DNA from P. citrea (ATCC 39140) was isolated using standard protocol and genomic library was created (Sambrook, et al, Molecular Cloning: A laboratory manual, Cold Sprint Harbor Laboratory, New York (1992)) . The amplified libraries were stored in the form of Plasmid DNA for further use to find 2,5-DKG permease of P. citrea. Example VI Example VI provides the proof that by overexpressing DKG transporter in the host cell, one can enhance the DKG import rate into the cell. Genomic library was introduced into tester strain K. oxytoca yiaX2[tkr idno] strain. Clones that grew on 2,5-DKG using M9-agar plates with 2.5% 2,5-DKg and 0.1 mM IPTG were tested for DKG uptake using radiolabeled 14C (U) DKG. Various clones were found to have improved DKG uptake than the control tester strain (Fig. 10) Genomic library DNA from these positive clones was transformed into P. citrea (139-2A) and DKG uptake assay was performed to measure the improvement in DKG uptake over the P. citrea 139-2A strain. Three to five fold improvement in DKG uptake rate was seen in the transformants having additional copies of plasmid encoded DKG permeases found through genomic library screening and selection methodology (Fig 11). Example VII Example VII provides the proof that by overexperssing DKG transporter in the host cell one is able to improve the production of 2KLG. Nucleic acid molecule encoding Pantoea citrea DKG permease PermA (seq ID no2) when subcloned using low copy vector pBCL1920 into a Pantoea citrea strain 139-2A suitable for biosynthesis of 2KLG from glucose (US Patent 5,032514), the production rate of 2KLG production improved form 2.5 g/l/hr 13.2 g/l/hr and yield on the sugar improved form 45 % to 53% (fig 12) Example VIII Example VIII describes the characteristics of 2,5-DKG permease PermA from P. citrea. This example describes the membrane topology of PermA of P. citrea. PFAM analysis (Hirokawa, T., et al., Bioinformatics, 1998, 4(4): 378) predicts that the PermA has 11 transmembrane spanning domains, with 8 primary domains and 3 secondary spanning domains (Fig. 13). The amino terminal is in the periplasm and caboxy terminal being localized in the cytoplasm. Two major and two minor loops exist and both periplasm and cytoplasm have one majoe and one minor loop. The PermA is a membrane protein with hydrophobicity of 0.62 and has molecular weight of 48 Dalton. It is a one H+ associated symporter of 2,5-DKG based on the accumulation ratio analysis (Lolkema, J.S., et al. 1996, Handbook of Biological Physics, Chapter 11 , 229-260). It belongs to ACS family of MFS based on the consensus sequence analysis (Pao, S.S., et al., Microbiol. Molecular Biol. Rev., 1998, 62:1-34) (Fig. 14). Various other examples and modifications of the foregoing description and examples will be apparent to a person skilled in the art after reading the disclosure without departing from the spirit and scope of the invention, and it is intended that all such examples or modifications be included within the scope of the appended claims. All publications and patents referenced herein are hereby incorporated in their entirety.

Claims: CLAIMS: 1. A method for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway of said host cell, the method comprising: a) selecting a host cell that has an at least partially intracellular synthetic pathway which utilizes an extracelluar substrate; b) increasing the transport of said extracellular substrate into said host cell while maintaining the integrity of the host cell; c) culturing the host cell to produce said extracellular substrate; and d) producing the desired compound. 2. The method of claim 1 , wherein said increasing the transport of said extracellular substrate into said host cell includes the step of transforming into said host cell DNA encoding for one or more polypeptides that increase the transport of said substrate into said host cell. 3. The method of claim 2, wherein said one or more polypeptide is a transport promoter 4. The method of claim 2, wherein said one or more polypeptides is a major facilitator superfamily transporter. 5. The method of claim 4, wherein said one or more polypeptides is a anion/cation H+ symporter. 6. A method for enhancing a host cell's biosynthetic production of a desired compound derived from an at least partially intracellular pathway of said host cell, the method comprising: a) selecting a host cell that has an at least partially intracellular synthetic pathway which utilizes an extracelluar substrate; b) increasing the transport of said extracellular substrate into said host cell while maintaining the integrity of the host cell wherein the transport of said substrate into said host cell is a rate limiting step in said host cell's biosynthetic production; c) culturing the host cell to produce said extracellular substrate; and d) producing the desired compound. 10. The method for enhancing the transport of a substrate into the cytosol across the inner cell membrane, the method comprising: Selecting a host cell; and Transforming into said host cell DNA encoding for one or more enzymes transporting of said substrate into said host cell. 11. The method of claim 10, wherein said host cell is selected from the group consisting of bacteria and yeast. 12. The method of claim 11 , wherein said host cell is pantoea. 13. A method of overexpressing an annion/cation symporter comprising the steps: Selecting a host cell; and Transforming into said host cell, DNA encoding for one or more anion/cation symporters.

Inventors: KUMAR MANOJ, VALLE FERNANDO, KUMAR, MANOJ, VALLE, FERNANDO
Applicants: GENENCOR INT, KUMAR MANOJ, VALLE FERNANDO, GENENCOR INTERNATIONAL, INC., KUMAR, MANOJ, VALLE, FERNANDO
Application year: 2001
Main classification: C12N15
IPC codes: C07K14, C07K16, C12N1, C12N9, C12N15, C12P7