MXPA97002011A - Microorganisms and methods for overproduction of dahp by clonedpps - Google Patents

Abstract

Genetic elements comprising expression vectors and a gene coding for phosphoenol pyruvate synthase is utilized to enhance diversion of carbon resources into the common aromatic pathway and pathways branching therefrom. The overexpression of phosphoenol pyruvate synthase increases DAHP production to near theorical yields.

Description

MICROORGANISMS AND METHODS FOR THE SUPERPRODUCTION OF DAHP THROUGH THE GENE PPS CLONADO DESCRIPTION OF THE INVENTION This work was supported in part by The National Science Foundation (Grant BCS-9257351) (National Science Foundation), The Welch Foundation (Grant A-1251) (Welch Foundation), and The Texas Higher Education Coordinating Board (Grant 999903-084 ) (Texas Higher Education Office). The Government of the United States may own the non-exclusive rights in and for the invention. The present invention relates to the biosynthetic production of organic chemical compounds. In particular, the present invention relates to methods for increasing the production of 3-deoxy-D-arabino-hetulosonate 7-phosphate (DAH P) in microorganisms through genetic alterations. The present invention also relates to methods for improving the production of cyclic and aromatic metabolites derived from DAH P in microorganisms through genetic alterations. For example, the biosynthesis of DAH P is the first step in the common aromatic pathway, from which tyrosine, tryptophan, phenylalanine and other aromatic metabolites are formed. Also, trajectories branching from the common aromatic path provide such useful chemicals as catechol and organic quinoids, such as quinic acid, benzoquinone, and hydroquinone. In addition, aspartame and Indigo can be produced from products derived from the common aromatic path. The production of chemicals from microorganisms has long been an important application of biotechnology. Typically, the steps involved in the development of a microorganism production strain include: (i) selecting an appropriate host microorganism, (ii) eliminating the metabolic pathways leading to byproducts, (iii) deregulating such trajectories at both the activity level of enzyme as in the transcriptional level; and (iv) overexpressing appropriate enzymes in the desired trajectories. The last three steps can now be achieved by using a variety of in vivo and in vitro methods. These methods are particularly condescending to the user in well-studied microorganisms such as Escherichia coli (E. coli). Therefore, many examples of microorganisms subjected to technical study for the physiological characterization and production of metabolites have been published. In many cases, the first objective for the process through engineering is the terminal trajectory that leads to the desired product, and the results are usually successful. However, further improvements in productivity (product formation regime) and yield (conversion rate) of desired products require the alteration of central metabolic trajectories, which provide the precursors and energy necessary for the desired biosynthesis of those products. Cyclic and aromatic metabolites such as tryptophan, phenylalanine, tyrosine, quinones, and the like, follow their biosynthesis towards the condensation reaction of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form DAH P. The biosynthesis of DAH P is the first obligatory step in the common aromatic trajectory. The biosynthesis of DAH P is mediated by three synthases or isoenzymes of DAH P. These isoenzymes are encoded by the aroF, aroG, and aroH genes, whose gene products are inhibited by feedback by tyrosine, phenylalanine and tryptophan, respectively. After the biosynthesis of DAH P, some DAH P is converted to corismato. Corismate is an intermediate in the biosynthetic trajectories that eventually leads to the production of aromatic compounds such as phenylalanine, tryptophan, tyrosine, folate, melanin, ubiquinone, menaquinone, prefénico acid (used in the production of bacilisina antibiotic) and enteroquelina. Due to the large number of biosynthetic trajectories that depend on corismato, the biosynthetic path used by organisms to produce corismato is usually known as the "common aromatic path". In addition to its use in the production of chorismate, DAH P can also be converted to quinic acid, hydroquinone, benzohydroquinone or catechol, as described by Draths et al.

(Draths, KM, Ward, TL, Frost, J .W., "Biocatalysis and Nineteenth Century Organic Chemistry: Conversion of D-Glucose into Quinoid Organics", J. Am. Chem. Soc, 1992, 1 14, 1925-26 ). These biosynthetic trajectories branch out from the common aromatic path before the shikimate is formed. The efficient production of DAH P by means of a microorganism is important for the production of aromatic metabolites, since DAH P is the precursor in the majority of the trajectories produced by the aromatic metabolites. The three aromatic amino acids, in addition to being essential building blocks for proteins, are useful precursor chemicals for other compounds such as aspartame, which requires phenylalanine. In addition, the trajectory of tryptophan can be genetically modified to produce indigo. The production of tryptophan and phenylalanine by E. coli has been well documented. For example, Aiba et al. (Aiba, S. H., Tsunekawa and T. Imanaka, "New Approach to Tryptophan Production by Escherichia coli: Genetic Manipulation of Composite Plasmids in vitro", (New Aspect for the Production of Tryptophan by Escherichia coli: Genetic Manipulation of Mixed Plasmids In Vitro), Appl. Env. Microbiol. 1982, 43: 289-297) have reported an overproduction of tryptophan containing genes overexpressed in the tryptophan operon in a host strain that is trpR and tna (encoding tryptophanase) negative. In addition, several enzymes, such as the product of the trpE gene, have been mutated to resist inhibition by feedback. Similar work has been reported for the production of phenylalanine. In the past, the improved compromise of cellular carbon sources entering and flowing through the common aromatic path has been achieved only with modest success (ie, such attempts have failed far below the theoretical yield). Typically, improvements were achieved by transferring to host cells, genetic elements encoding enzymes that direct the flow of carbon towards and / or through the common aromatic path. Said genetic elements may be in the form of plasmids, cosmids, extrachromosomal phages, or other replicas capable of transforming genetic elements to the host cell. The patent of E. U.A. No. 5, 168,056 to Frost, describes the use of a genetic element that contains an expression vector and a gene encoding transketolase (Tkt), the tkt gene. This genetic element can be integrated into the chromosome of microorganisms to provide overexpression of the Tkt enzyme. Additional examples include: Miller et al. (Miller, J. E., KC Backman, J. M. O'Connor, and TR Hatch, "Production of phenylalanine and organic acids by PEP carboxylase-deficient mutants of Escherichia coli" (Production of phenylalanine and organic acids by deficient mutants of PEP carboxylase of Escherichia coli), J. Ind. Microbiol .. 1987, 2: 143-149), who attempted to direct more carbon flux into the amino acid pathway by using a deficient phosphoenolpyruvate carboxylase mutant (encoded per ppc); Draths et al. (Draths, KM, DL Pompliano, DL Conley, J. W. Frost, A. Berry, GL Disbrow, RJ Staversky, and J.C. Lievense, "Biocatalytic synthesis of aromatics from D-glucose: The role of transketolase" (Biocatalytic synthesis of aromatics from D-glucose: The role of transketolase), J. Am. Chem. Soc, 1992, 1 14: 3956-3962), who reported that overexpression of transketolase (encoded by tkt A) and a feedback resistant DAH P synthase (encoded by aroGfbr) resulted in the improved production of DAH P from glucose. The overproduction of transketolase in tkt transformed cells has been found to provide an increased flow of carbon resources to the common aromatic trajectory in relation to the utilization of the carbon resource in whole cells that do not inhabit said genetic elements. Nevertheless, the increased carbon flow can be further improved by further manipulation of the host strain. In this way, it is desirable to develop genetically engineered strains of microorganisms that are capable of improving DAHP production almost at a theoretical yield. Said genetically engineered strains can then be used for the selective production of DAH P or in combination with another genetic material incorporated for the selective production of desired metabolites. The efficient and cost-effective biosynthetic production of corismate, quinic acid, hydroquinone, benzohydroquinone, catechol, or derivatives of these chemicals requires that carbon sources such as glucose, lactose, galactose, xylose, ribose, or other sugars be converted to desired product in high yields. Therefore, it is valuable from the point of view of the industrial biosynthetic production of metabolites to increase the influx of carbon sources for the biosynthesis of DAHP cells and their derivatives. The present invention provides genetically engineered strains of microorganisms that overexpress the pps gene to increase the production of DAHP very close to the theoretical yields. The present invention also provides genetically engineered strains of microorganisms, wherein at least one of the plasmids pPS341, pPSL706, pPS706, or derivatives thereof is transformed into a microorganism to increase the production of DAHP very close to the theoretical yields. The present invention further provides a method for increasing the carbon flux for the biosynthesis of DAHP in a host cell, comprising the steps of: transforming into the host cell recombinant DNA, comprising a pps gene, such that Pps is expressed at improved levels relative to wild-type host cells, concentrate the transformed cells through centrifugation, re-suspend the cells in a minimal, minimal nutrient medium, ferment the resuspended cells, and isolate DAHP from the medium. The present invention further provides methods for increasing the carbon flux in the common aromatic path of a host cell, comprising the step of transforming the host cell with recombinant DNA comprising a pps gene, so that Pps is expressed at an appropriate point in the metabolic trajectories at improved levels relative to wild-type host cells. The present invention also provides methods for improving a biosynthetic production of host cells of compounds derived from the common aromatic path in relation to the biosynthetic production of wild-type host cells of said compound, said method comprises the step of increasing the expression in a cell host of a protein that catalyzes the conversion of PEP to pyruvate. The present invention also provides methods for overexpressing Pps in strains of microorganisms, which utilize DAHP in the production of DAHP of metabolites. The present invention also provides a culture containing a microorganism characterized by overexpression of Pps, where the culture is capable of producing DAH P metabolites very close to the theoretical yields under fermentation in an aqueous resuspension, medium nutrient medium, minimum that It contains assimilable sources of carbon, nitrogen and inorganic substances. The present invention also provides a genetic element comprising a pps gene and one or more genes selected from the group consisting of an aroF gene, an aroG gene, aroH gene and an aroB gene. The present invention also provides a DNA molecule comprising a vector carrying a gene coding for Pps. Figure 1. Overexpression of Pps increases the production of DAHP. The host strain used was AB2847 and the plasmids used are as marked in the Figure. Note that pPSX1 and pUHE denote pPS341 X1 and pUHE23-2, respectively. These strains were first cultured in the medium YE (a rich medium) to delay the stationary phase, and then wash and resuspend in a minimal medium. (A) DAH P concentrations were measured at 10 and 27 hours after resuspension. (B) The synthase activities of DAHP (aroG) and Pps were measured 27 hours after the resuspension. Figure 2. Production of DAH P 10 and 27 hours after resuspension. (A) Strain AB2847 with plasmids labeled in the abscissa. pPSX1 and pU H E denote pP341 X1 and pU H E23-2, respectively. (B) Strains AB2847 (marked as AB), JCL1283ppc:: Km (marked as ppc), and JCL1362 pps :: MudK (marked as pps) with plasmids labeled in the abcisa. Figure 3. The reaction trajectories for the maximum conversion of glucose to DAH P for (A) strains without Pps, (B) strains overexpressing Pps. The numbers are the relative flows required to convert 7 moles of glucose to DAH P. The abbreviations are: G6P, glucose 6-P; F6P, fructose 6-P; 1 .6F DP, 1,6-fructose diphosphate; DAH P, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-P, R5P, ribose 5-P; X5P, xylulose 5-P; S7P, sedoheptulosa 7-P. Figure 4. The common aromatic trajectory is shown, so that E4P and PEP undergo a condensation reaction to initiate the common aromatic trajectory. Figure 5. The construction of pPSL706. Plasmids pPS706 and pGS103 were restricted with EcoRI and Seal. Fragments containing pPS of pPS706 and the fragment containing luxl were purified and ligated to generate pPSL706. Plasmid pPS706 was constructed by inserting a PCR fragment of pps into the cloning vector 9 pJ F1 18EH. Figure 6. Effects of Pps activity on the production of DAHP from glucose at different concentrations of I PTG and autoinducer. Strains are AB2847 / p PSL706 / pAT1 and AB2847 / pPSL706 / pRW5. Plasmid pGS 104 was used to replace pPSL706 as a control, and the data is the furthest left point on each graph. Many microorganisms synthesize aromatic precursors and aromatic compounds from the condensation reaction of PEP and E4P to produce DAH P. This condensation reaction to form DAH P, is the first step required in the biosynthetic path, known as the common aromatic path, From this path, the cells synthesize many cyclic metabolites, pre-aromatic metabolites, and aromatic metabolites, such as aromatic amino acids, quinone biomolecules, and aromatic and cyclic related molecules. The inventor has found that cell lines can be developed that increase the carbon flux towards the production of DAHP and obtain almost theoretical yields of DAH P by overexpression of phosphoenolpyruvate synthase (Pps) in the cell lines. Overexpression of Pps can increase the final concentration and the production of DAH P as much as twice, at a near theoretical maximum, compared to wild-type cell lines. Overexpression of Pps is achieved by transforming a cell line with recombinant DNA comprising a pps gene, so that Pps is expressed at an improved level relative to the wild-type cell line and, thus, the production of DAH P approaches its theoretical value. Generally, the present invention improves expression in a Pps host cell relative to a wild type host cell, either by transfer and stable incorporation of an extrachromosomal genetic element into the host cell, or by transfer of the genetic element. towards the genome of the host cell. The expressed gene products are enzymes configured to provide catalytic sites suitable for substrate conversion of compounds of common aromatic path. In addition to the use of the pps gene, the present invention also provides for the transfer of genetic elements comprising the tkt gene, the gene coding for DAH P synthase (aroF in E. coli), the gene encoding 3-dehydroquinate synthase (aroB). in E. coli), or other genes that encode enzymes that catalyze reactions in the common aromatic pathway. Said cell transformation can be achieved by transferring one or more plasmids containing genes coding for enzymes, which increase the carbon flux for the synthesis of DAHP and for the subsequent synthesis of other desired cyclic, pre-aromatic and aromatic metabolites. As a result of this transfer of genetic elements, more carbon enters and moves through the common aromatic pathway relative to wild-type host cells that do not contain the genetic elements of the present invention. In one embodiment, the present invention comprises a method for increasing the carbon flux towards the common aromatic path of a host cell, increasing the production of DAH P through overexpression of Pps at the appropriate point in the common aromatic path to provide Additional PEP at the point, where PEP is condensed with E4P. In order to increase the carbon flux, the step of transforming the host cell with recombinant DNA containing a pps gene is required, so that Pps is overexpressed at improved levels relative to the wild-type host cells. Then, DAH P is produced by fermenting the transformed cell in a nutrient medium, where DAH P can be extracted from the medium in an intermittent or continuous extraction procedure.

In another embodiment, the present invention involves the co-overexpression of a pps gene and other genes encoding enzymes of the common aromatic path, wherein the additional genetic material is transformed into the host cell. The transferred genes may include the tkt gene, the DAH P synthase gene, and the DHO synthase gene (preferably, the aroF or aroB genes, respectively). Although work so far has focused around the transformation of certain E. coli host cell strains, such as AB2847 aroB, this particular host cell may not be the preferred host cell of commercial production of DAH P or metabolites of DAH P through overexpression of Pps. Another embodiment of the present invention is a method for improving a biosynthetic production of host cells of compounds derived from the common aromatic path. This method involves the step of increasing the expression of Pps in the host cell relative to a wild-type host cell. The step of increasing the expression of Pps can include the transfer, towards the host cell, of a vector carrying the pps gene. The overexpression of Pps results in forcing the increased carbon flux towards the biosynthesis of DAH P. In another embodiment of the present invention, a method is provided for improving a host cell biosynthetic production of compounds derived from the common aromatic path in relation to to the host cell biosynthetic production of said compound. This method requires the step of increasing the expression in a host cell of a channel conversion from pyruvate protein to PEP. Expression of said protein may involve transfer to the host cell recombinant DNA including a pps gene. In another preferred embodiment, the present invention comprises a genetic element comprising the pps gene and a gene selected from the group consisting of an aroF gene, an aroB gene, and a tkt gene. Said genetic element may comprise the plasmid pPS341, a vector carrying a pps gene. To channel more carbon flux into the common aromatic path, it has been found that the production of PEP in a given cell line must be increased. This increase can be obtained by deactivating the trajectories that compete for PEP or by recirculating pyruvate back to PEP. In addition to being used in the biosynthesis of DAHP, PEP is used as a phosphate donor in the phosphotransferase system (PTS), which is responsible for the consumption of glucose. Additionally, PEP can be converted to pyruvate by pyruvate kinases and oxaloacetate by phosphoenolpyruvate. All these competition trajectories limit the availability of PEP for the biosynthesis of DAHP and all the metabolites derived from the common aromatic trajectory or trajectories that are derived from them. Once PEP is converted to pyruvate either by PTS or by pyruvate kinases, pyruvate is generally not recirculated back to PEP, due to a high energy cost. As a result, a large amount of carbon flux is channeled from PEP through pyruvate and finally into organic acids, carbon dioxide or cell mass. PEP is critical for the biosynthesis of DAH P and the metabolites of DAHP including all the metabolites of the common aromatic pathway. The first obligatory step of the common aromatic path involves the formation of DAH P from the condensation of E4P and PEP. This condensation involves a condensation of aldol between an intermediate carbanion of C-3 of PEP and the carbonyl C-1 of E4P. Most PEP molecules react stereospecifically with respect to the configuration on C-3. A key component of the methods of the present invention directed to the commitment of increased carbon flux towards DAH P and metabolites of DAH P, is the recirculation of pyruvate to PEP. Pyruvate is available in host cells as a final product of glycolysis. In glycolysis, the free energy of degradation of glucose to pyruvate is used to synthesize ATP. Generally speaking, this procedure involves an inversion of ATP to form a phosphoryl compound (FBP) of glucose, which is divided into two C3 units. The free energy of this reaction is used in the oxidation of GAP, which is then used to synthesize an acyl phosphate, a "high energy" intermediate (1, 3-BPG). 1, 3- BPG is used to phosphorylate ADP to ATP. The second "high energy" path compound, PEP, which is produced from 2 PG, also phosphorylates ADT to ATP. In this way, the degradation of glucose via the glycolytic pathway produces pyruvate. The total reaction of glycolysis is, therefore: Glucose + 2ADP + 2P + 2NAD + >; 2 pyruvate + 2ATP + 2NADH + 4H + 2H20 The first step of glycolysis is the transfer of a phosphoryl group from an ATP group to glucose to form glucose 6-phosphate (G6P) in a reaction catalyzed by hexokinase. Hexokinase is a relatively non-specific enzyme contained in all cells that catalyze the phosphorylation of hexoses, such as D-glucose, D-mannose and D-fructose. The second substrate for hexokinase, as with other kinases, is an Mg2 + - ATP complex. Actually, ATP without complex is a potent competitive inhibitor of hexokinase. Hexokinase has a Bi Bi random mechanism, in which the enzyme forms a ternary complex with glucose and Mg2 * - ATP, before the reaction occurs. With complex formation with phosphate-oxygen atoms, Mg2 + is thought to protect its negative charges, making the phosphorus atom more accessible to the nucleophilic attack of the C (6) -OH group of glucose. Then, G6P is converted to 6-fructose phosphate (F6P) by phosphoglucose isomerase (PG I). This reaction is an isomerization of an aldose to ketose. Since G6P and F6P both exist predominantly in their cyclic forms, the reaction requires ring opening, followed by isomerization, and subsequent ring closure. The entire reaction is believed to occur by catalysis based on general acid mediated by enzyme. Then, phosphofructokinase (PFK) phosphorylates F6P to produce 1,6-bisphosphate (FBP or F1, 6P). The PFK catalyzes the nucleophilic attack by the group C (1) -OH of F6P on the electrophilic atom? -phosphorus of the Mg2 + -ATP complex. PFK plays a central role in the control of glycolysis, since it catalyzes one of the path regime determination reactions. In many organisms, the activity of PFK is improved alloestéricamente by means of several substances, including AMP, and is inhibited alloestéricamente by several other substances, including ATP and citrate. The regulatory properties of PFK are intensely complex. Next, aldolase catalyzes the cleavage of FBP to form the two triglycerides glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). This reaction is a split of aldol (the opposite of a condensation of aldol). The cleavage of aldol between C (3) and C (4) of FBP requires a carbonyl in C (2) and a hydroxyl in C (4). Only one of the products of the aldol cleavage reaction, GAP, continues along the glycolic path. However, DHAP and GAP are ceosa-aldose isomers, such as F6P and G6P. Therefore, the interconversion of GAP and DHAP via an intermediate of arsenate analogous to the reaction of the phosphoglucomutase is possible. The triose isomerase phosphate (TIM) catalyzes this procedure. At this point, glucose, which has been transformed to two GAPs, has completed the preparatory stage of glycolysis. This procedure has required the expenditure of two ATPs. However, this investment has resulted in the conversion of one glucose to two C3 units, each of which has a phosphoryl group that, with a little chemical technology, can be converted to a "high energy" compound, whose Hydrolysis-free energy can be coupled to the synthesis of ATP. This double energy inversion will be repaired in the final stage of glycolysis, where the two phosphorylated units of C3 are transformed to two pyruvates with the coupled synthesis of four ATPs per glucose. The next step in glycolysis involves the oxidation and phosphorylation of GAP by NAD + and P, as they are catalyzed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In this reaction, the oxidation of the aldehyde, an exergonic reaction, directs the synthesis of 1,3-diphosphiglycerate acryl phosphate (1,3-BPG). The next reaction of the glycolytic path results in the first formation of ATP together with 3-phosphoglycerate (3PG) in a reaction catalyzed by phosphoglycerate kinase (PGK). Then, 3PG is converted to 2-phosphoglycerate (2PG) by phosphoglycerate (PGM) mutase. This reaction is necessary for the preparation of the next reaction in glycolysis, which generates a "high energy" phosphoryl compound for use in the synthesis of ATP. Subsequently, 2PG is dehydrated to phosphoenolpyruvate (PEP) in a reaction catalyzed by enolase. The enzyme forms a complex with a divalent cation, such as Mg2 + before the substrate binds. The fluoride ion inhibits glycolysis with the accumulation of 2 PG and 3PG. It does this by strongly inhibiting enolase in the presence of Pi. The inhibiting species is the fluorophosphate ion (FPO33"), which probably forms complexes with Mg2 + bound to the enzyme, thus inactivating the enzyme.The 2PG enolase substrate, therefore, develops and as it does so, it is balanced with 3PG by PGM Finally, pyruvate kinase (PK) couples the free energy of PEP hydrolysis for the synthesis of ATP to form pyruvate, at this time, glycolysis has produced PEP, one of the precursors for the production of DAHP and the path of entry to the common aromatic path In addition, the biosynthesis of PEP, DAHP, as well as other products derived from the common aromatic trajectory and trajectories that branch off from it, depend on the biosynthesis of E4P. Figure 4, E4P is a biosynthetic intermediate of the pentose phosphate path The path of pentose phosphate is located between glycolysis and a variety of different biosynthetic cascades This trajectory produces E4P via a non-oxidizing branch of the trajectory. The non-oxidizing pentose phosphate path converts D-fructose 6-phosphate to variable equivalents of D-ribose 5-phosphate, D-sedoheptulose 7-phosphate, and E4P. The first two end products are associated with the biosynthesis of gram-negative bacterial lipopolysaccharide nucleotides, respectively, while E4P is a precursor of the aromatic amino acids: phenylalanine, tyrosine and tryptophan. The initial siphoning of intermediates from glycolysis via the pentose phosphate pathway involves the catalyzed transfer of transketolase from a cetol group from 6-phosphate of D-fructose to 3-phosphate of D-glyceraldehyde to form E4P and -D-xylulose phosphate. Next, the pentose phosphate epimerase converts D-xylulose 5-phosphate to D-ribulose 5-phosphate folloby pentose phosphate isomerase-mediated transformation of D-ribulose 5-phosphate to 5-phosphate. D-ribose. At this stage, D-ribose 5-phosphate can be exploited by transketolase as an acceptor of a cetol group derived from another D-fructose 6-phosphate molecule forming a second molecule of E4P and 7-phosphate of D-sedohetulose . Finally, the enzyme transaldolase catalyzes the transfer of a dihydroacetone group from 7-phosphate D-sedoheptulose to 3-phosphate D-glyceraldehyde producing the third molecule of E4P and 6-phosphate D-fructose. In this way, the non-oxidizing pentose phosphate path achieves a net conversion of two D-fructose 6-phosphate molecules to three E4P molecules. The condensation of PEP and E4P is catalyzed by the enzyme DAH P synthase. Many microorganisms, including E. coli, produce three isozymes of DAH P synthase: DAH P synthase sensitive to phenylalanine (phe), DAH P synthase sensitive to tyrosine (tyr), and DAH P synthase sensitive to tryptophan ( trp). The tetrameric DAHP (phe) synthase has a subunit molecular weight of 35,000. The dimeric DAHP synthase (tyr) and the DAHP synthase (trp) have subunit molecular weights approaching 40,000. The native forms of the enzymes are probably protein-PEP adducts. In E. coli, the structural genes for the DAH P synthase (tyr), DAH P synthase (phe) and DAH P synthase (trp) are aroF, aroG, and aroH, respectively. These genes are located at 56, 17 and 37 minutes, respectively, on the binding map of E. coli. In wild-type E. coli, 80% of the total DAH P synthase activity is contributed by the phenylalanine-sensitive isoenzyme, while 20% is contributed by the tyrosine-sensitive isoenzyme. There are only traces of the DAH P (trp) synthase in E. coli. After the condensation of PEP and E4P, the next reaction of the common aromatic path is an intramolecular exchange of oxygen from the DAH P ring with C-7, accompanied by an oxidation at C-6 and a reduction at C-2. The cleavage of the phosphoester provides the im pulsing force to form 3-dehydroquinate (DAH). This reaction is catalyzed by the dehydroquinate synthase (DAH synthase). The pure DAH synthase is a single polypeptide chain having a molecular weight of 40,000-44,000. The enzyme requires Co and NAD for the activity, the latter in catalytic quantities. The formation of 3-dehydroquinate from DAH P is stereospecific and proceeds with inversion in C-7 of DAH P without exchange of hydrogen with the growth medium. Organic quinoids are formed from the trajectories that branch off from the common aromatic and use DAH. A stereospecific non-dehydration of 3-dehydroquinate introduces the first double bond of the aromatic ring system to produce 3-dehydrosicimate. The reaction is catalyzed by 3-dehydroquinate dehydratase. The Schiff base formation between the enzyme and the substrate causes a conformational change in the substrate (twisted capsule) that leads to the stereospecific course of the reaction. The biosynthesis of shikimate from 3-dehydroshikimate is catalyzed by shikimate dehydrogenase. This NADP-specific enzyme facilitates the transfer of hydrogen from the A side of NADPH. The shikimate is phosphorylated to shikimate 3-phosphate by shikimate kinase. The shikimate kinase is a 10,000 dalton polypeptide that forms complexes with the DAH P-mutase synthase of bifunctional chorismate. The kinase, only active in the complex, has been purified to homogeneity. Since the enzyme is inhibited by corismate, prefenate, ADP, and 5-enolpyruilsyl-waste-3-phosphate are derepressed by growth on limiting tyrosine, it is believed that the shikimate kinase represents a key allosteric control point of the trajectory in some types of host cells. The shikimate-3-phosphate reacts with PEP to form 5-enolpiruvoyl-3-phosphate and inorganic phosphate. The reaction catalyzed by the reversible enzyme is a transfer of a portion of enolpiruvoil without change of PEP. The protonation of C-3 of PEP combined with a nucleophilic attack of the 5-hydroxyl of shikimate leads to a presumed intermediate from which is obtained shikimate-3-phosphate of 5-enolpiruvoil in a 1, 2-elimination of orthophosphate. The reaction is catalyzed by the 5-enolpiruvoyl siquimate-3-phosphate synthase. The second double bond in the aromatic ring system is introduced through a trans-1,4-elimination of the orthophosphate from 5-enolpiruvoyl-3-phosphate to produce chorismate. The reaction is catalyzed by corismate synthase. From the chorismate, the end point of the common aromatic path, the biosynthesis of a different number of aromatic compounds is possible. For example, as indicated in Figure 4, the aromatic amino acids tryptophan, tyrosine, and phenylalanine can be synthesized from corismat along their respective biosynthetic trajectories. As previously noted, other commercially important aromatic compounds can also be produced from corismate and include folates, aspartame, melanin, pregenic and Indigo acid. In addition to the common aromatic path, other pathways that use DAHP produce other aromatic metabolites. For example, catechol and organic quinoids, such as quinic acid, benzoquinone and hydroquinone, can be produced from pathways branching off the common aromatic path. According to the theoretical analyzes, the inventor believes that the maximum production of DAHP and aromatic amino acids from glucose can be doubled if pyruvate is recirculated back to PEP. The maximum production can be calculated assuming that the branched trajectories are blocked and that the carbon flux is directed by the most efficient trajectories with minimal loss for carbon dioxide and other metabolites. Under these conditions and under steady state conditions, the relative flow through each step can be calculated by balancing the inflows and outflows of each metabolite deposit. As shown in Figure 3A, for maximum yield of DAHP production by strains without the overproduction of Pps, 7 moles of glucose are needed to produce 3 moles of DAHP (43 mole% yield) and 7 moles of pyruvate, which is additionally metabolized. The relative flow through each intermediate step is also shown in Figure 3A. The formation of pyruvate is necessary due to the stoichiometry of the phosphotransferase system for glucose consumption. In the presence of glucose, pyruvate is not recirculated back to PEP, efficiently, since the Pps enzyme is not induced. It has been found that pyruvate is effectively recirculated to PEP via overexpressed Pps, even in the presence of glucose, resulting in a double increase in DAH P, which is close, if not obtained, at the theoretical levels for the synthesis of DAH P. As shown in Figure 3B, at the theoretical maximum, 6 moles of DAH P can be produced from 7 moles of glucose (yield 86% molar). The non-oxidizing part of the pentose path provides E4P, while the overexpression of Pps recirculates pyruvate back to PEP. The data described above and shown in the Figures 3A and 3B go according to this flow distribution model. The controls with inactive Pps and without Pps show that the improved activity, through the overexpression of Pps, is required to obtain high yields of DAH P and, of course, metabolites of DAH P, including metabolites of the common aromatic path.

In previous studies, it was demonstrated that overexpression of Pps in host cells cultured in a nutrient-rich glucose-containing medium led to growth inhibition, increased glucose consumption and excretion of pyruvate and acetate. This previous study also showed that the effects of the overexpression of Pps in the production of DAH P, in actively growing cultures, are not so significant, and that the adverse effects of the overexpression of Pps on cell growth, denied any effects beneficial in the production of DAH P. The stimulation of glucose consumption, in the previous work, was attributed to the altered relationship of PEP / pyruvate. It was hypothesized that the increased PEP / pyruvate ratio stimulates the phosphotransferase system for increased glucose consumption, which, in turn, results in the excretion of pyruvate. The inventor discovered that the problem of growth deterioration can be overcome through the use of high-density resuspension crops that grow on glucose media, lacking in nutrients. Said resuspension cultures achieve a high metabolic activity with low growth rates. This discovery led to productions of DAH P that approach the theoretical values. In the present invention, PEP was redirected to the aromatic path, and, thus, the ratio of PEP / pyruvate was decreased. This flow redirection explains the insensitivity of the specific glucose consumption regime for the overexpression of Pps in the experimental system of the present invention. The increased production of DAH P from glucose, caused by the overexpression of Pps, also suggests that Pps actually functions in its physiological direction (from pyruvirate to PEP) in vivo, even under glycolytic conditions. PEP is also a precursor for the trajectories that use the Ppc enzyme encoded by the ppc gene. It has been reported that the elimination of ppc increased the production of phenylalanine and acetate. In addition, overexpression of Ppc in a wild type host has been shown to reduce acetate production. Both results may indicate that the flow through Ppc (from PEP to OAA) is reasonably significant under those conditions, and therefore, the modulation of the level of Ppc expression may affect the use of PEP. However, in the present invention, the elimination of the chromosomal ppc gene did not have a positive effect on the production of DAH P, suggesting that the flow through Ppc is not important in the methods of the present invention. A preferred embodiment of the present invention encompasses the modification of a host cell to cause overexpression of an enzyme having the catalytic properties of naturally derived Pps, and, thereby minimizing the production of DAH P at quasi-theoretical yields. . Enzymes that have catalytic activity of Pps include, but are not limited to, Pps produced by the expression in whole cells of a naturally derived pps gene, enzymes produced by the expression in whole cells of a naturally derived pps gene, modified by elimination or the addition of the sequence, such that the expressed enzyme has an amino acid sequence varying from unmodified Pps, produced abzymes having catalytic sites with steric and electronic properties that correspond to catalytic Pps sites, or other produced proteins that have the ability to catalyze the conversion of pyruvate to PEP by any other means recognized in the art. In another preferred modality, the inventor has observed that the effect of Pps on the production of DAH P is improved by the simultaneous overexpression of Tkt. Such simultaneous overexpression ensures that both precursors, necessary for the biosynthesis of DAH P, are overproduced. Although overexpression of Pps and Tkt may be required to obtain almost theoretical yields of DAH P, overexpression of Pps only in the methods of the present invention significantly improved the production of DAH P over overexpression of Tkt alone (Figure 2A). This result may suggest that without a Pps-mediated pyruvate recirculation (Figure 3A), a sufficient flow of PEP can not be obtained for the synthesis of DAH P. In addition, the transformation of DNA, including the pps gene, to microorganisms processed by Engineering for overexpression of other substrates, and / or overexpression or derepression of enzymes in pentose phosphate or common aromatic path, can be used to design the microorganism to obtain almost theoretical yields of said DAH P metabolites, such as tyrosine, tryptophan, phenylalanine and other aromatic metabolites such as Indigo, catechol and organic quinoids such as quinic acid, benzoquinone and hydroquinone. Enzymes that catalyze reactions in pentose phosphate or in the common aromatic pathway include those enzymes produced by expression in whole cells of naturally derived pentose phosphate or genes of common aromatic pathway, the enzymes produced by the expression of pentose phosphate derivative naturally or genes of the common aromatic pathway that have been modified by the elimination or addition of sequence, so that the expressed enzyme has an amino acid sequence that differs from the natural enzyme, or abzymes that have catalytic sites with steric and electronic properties that correspond to catalytic sites of a natural enzyme in the common aromatic pathway or pentose phosphate. Pps or enzymes having catalytic activity similar to Pps can be overexpressed in relation to the production of Pps in wild type cells (as measured by normal PEP synthase activity assays, known in the art and described in Example 1) along with any number of other enzymes in the common aromatic path or paths that branch off from it. For example, overexpression of Pps, DAH P synthase, and transketolase; Pps, DHQ synthase and transketolase; Pps, DAH P synthase, DHQ synthase and transketolase; Pps, transketolase and shikimate kinase; Pps, transketolase (Tkt), and corismato mutase; or any other enzymes of common aromatic path together with Pps, overproduction can improve the carbon source entry to and / or through the entire common aromatic path. The improved expression of genes encoding proteins capable of making or controlling pentose phosphate and the enzymatic functions of the common aromatic pathway is mediated by genetic elements transferable to a host cell. Genetic elements, as defined herein, include nucleic acids (generally DNA or RNA) that have coding sequences expressible for products such as proteins, apoproteins or antisense RNA, which can perform or control pentose phosphate or enzymatic functions of the common aromatic path. The expressed proteins can function as enzymes, as repressor or depressant agents, or to control the expression of enzymes. The nucleic acids encoding these expressible sequences can be either chromosomal (e.g., integrated to a host cell chromosome by homologous recombination) or extrachromosomal (e.g., carried by plasmids, cosmids, etc.). In addition, genetic elements are defined to include optional expression control sequences, including promoters, repressors and enhancers that act to control the expression or derepression of sequences encoding proteins, apoproteins, or antisense RNA. For example, such control sequences can be inserted into wild type host cells to promote overexpressions of selected enzymes already encoded in the host cell genome, or alternatively they can be used to control the synthesis of extrachromosomally encoded enzymes. The genetic elements of the present invention can be introduced into a host cell by a genetic agent that includes, but is not limited to, plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that can mediate the transfer of genetic elements. to a host cell, or mixtures thereof. These vectors can include an origin of replication together with cis-acting control elements, which control the replication of the vector and of the genetic elements carried by the vector. Selectable markers may be present in the vector to aid in the identification of host cells, into which the genetic elements have been introduced. For example, selectable markers can be genes that confer resistance to particular antibiotics such as tetracycline, ampicillin, chloramphenicol, kanamycin or neomycin. Means are used to introduce the genetic elements to a host cell of a multi-copy, extrachromosomal plasmid vector, to which the genetic elements have been inserted, according to the present invention. The plasmid carrying the introduction of the genetic element to the host cells involves an initial cleavage of a plasmid with a restriction enzyme, followed by the ligation of the plasmid and the genetic elements according to the present invention. Under the recirculation of the ligated recombinant plasmid, transformation or other mechanisms are used for plasmid transfer (e.g., electroporation, microinjection, etc.) to transfer the plasmid to the host cell. Suitable plasmids for the insertion of genetic elements into the host cell include, but are not limited to, pBR322 and its derivatives such as pAT153, pXf3, pBR325, and PBR327, pUC vectors, pACYC and its derivatives, pSC101 and its derivatives, and ColEi. The patent of E.U.A. No. 5,168,056, incorporated herein by reference, teaches the incorporation of the tkt gene, which codes for the Tkt enzyme to the host cell. Tkt catalyzes the conversion of D-fructose 6-phosphate from the carbon source to E4P, a precursor of DAHP. Suitable host cells for use in the present invention are members of those genera capable of being used for the industrial biosynthetic production of desired aromatics. Accordingly, host cells can include prokaryotes belonging to the genus Escherichia, Corynebacterium, Arthrobacter, Bacillus, Pseudomonas, Streptomyces, Staphylococcus, or Serratia. Eukaryotic host cells can also be used, with yeasts of the genus Saccharomyces or Schizosaccharomyces being preferred. More specifically, prokaryotic host cells suitable for use in the present invention include, but are not limited to, Escherichia coli, Corybacterium glutamicum, Corybacterium herculis, Brevibacterium divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus. coagulans, Bacillus lichenformis, Bacillusmegaterium, Bacillus mesentericus, Bacillus pumilis, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas angulata, Pseudomonas fluorescens, Pseudomonas tabaci, Streptomyces aureofaciens, Streptomyces to vermitilis, Streptomyces coelicolor, Streptomyces griseus, Streptomyces kasugensis, Streptomyces vendulae, Streptomyces lipmanii , Streptomyces lividans, Staphylococcus epidermis, Staphylococcus saprophyticus, or Serratia marcescens and their genetically engineered strains by engineering or mixtures thereof. Preferred eukaryotic host cells include Saccharomyces cerevisiae or Saccharomyces carlsbergensis and their genetically engineered strains or mixtures thereof. For the industrial production of primary metabolites derived from corismato (such as aromatic amino acids), strains of deregulated mutant of the aforementioned species that lack the inhibition of feedback of one or more enzymes in the biosynthetic metabolic path are preferred. Said strains can be created by random or directed mutagenesis, or are commercially available. Examples of E. coli strains having DAH P synthase, prephenate dehydratase or feedback inhibition of removed corismal mutase, are described in the patent of E. U.A. No. 4,681, 852 of Tribe and patent of E.U.A. No. 4, 753, 883 to Backman et al. , incorporated herein by reference. To overcome the stoichiometric limitations in the condensation of E4P and PEP, the present invention overexpresses Pps in the presence of glucose and directs more carbon flux towards the production of DAHP. The following list of abbreviations for compounds commonly observed in the specification and Examples is presented as follows: DHQ 3-dehydroquinate DAH 3-deoxy-D-arabino-heptulosonic acid DAHP 7-deoxy-D-arabino-heptulosonic acid phosphate TSP sodium salt 2, 2, 3, 3-d-sub 4 3- (trimethylsilyl) propionic acid PEP phosphoenol pyruvate NADH beta-nicotinamide adenine dinucleotide phosphate, reduced form Kan kanamycin Ap ampicillin Te tetracycline Cm chloramphenicol Strains and plasmids Escherichia coli AB2847 aroB mal T6r, obtained from the Genetic Supply Center of E. coli (E. coli Genetic Stock Center), Yale University, was used as the preferred host strain for the production of DAH P. For identification from the tkt clone was used BJ502 tkt-2 fhuA22 garB IO ompF627 fadL 701 rela I pit10 spo T1 mcrB1 phoM510, also from the Genetic Supply Center of E. coli. As described above, ppc:: Km was constructed by inserting a cassette of kanamycin into a cloned ppc gene, which was then integrated into the chromosome by homologous recombination. Plasmid pPS341 (available from the Department of Chemical Engineering, University of Texas A &M, College Station, Texas, E.U.A.) was constructed by cloning a chromosomal DNA fragment of E. coli containing the pps gene to the expression vector induced by IPTG, pUHE23-2 (a derivative of pBR322) as taught by Patnaik et al. , and the contents of which are incorporated herein by reference. Plasmid pPS341 X1, which contains the inactive gene product of pps was constructed by codon insertion mutagenesis, the details of which are fully described in Patmaick et al. The pps gene on pPS341 was inserted with a Mu dlll 1734 lac * Kmr (Mud K), according to the published protocol of Castiho et al. , the contents of which are incorporated herein by reference. In summary, a Mu lisate was made from a donor strain POII 1734 / pPS341, which was lysogenized by the mini-Mu element and a Mu cts. Lysate was used to infect a Mu lisogen of HG4 pps pck, and colonies were selected for Apr and Kmr simultaneously to ensure that the mini-Mu element carried to the plasmid. The colonies were also classified for the Pps' phenotype (inability to grow pyruvate). Restriction analysis of the plasmid DNA further confirmed the insertion of the MudK element to the pps gene on the plasmid pPS341. 20% of these selected colonies showed IPTG-dependent expression of β-galactoside, indicating an insertion in the frame. The plasmid from one of said colonies was named pPS1734, which was then linearized at the Seal site, and then transformed to strain JC7632 recB21 recC22 sbcB15. Transformants were selected for Kmr and were classified for Ap sensitivity. Said colonies presumably contained pps :: MudK on the chromosome. By using the transduction of P1, this site was moved to AB2847 and the Kmr transducers were further classified for the inability of pyruvate growth. One of these colonies was designated JCL1362 and was used for further studies. The insertion of MudK to the chromosomal pps was also confirmed by the cotransduction frequency (89%) with the Tef marker of strain CAG12151 zd7-925 :: Tn70. Plasmid pRW5, Genencor International, South San Francisco, CA, is derived pACYC and contains aroGfbr This plasmid also contains a lacl gene, and the aroGrbr is expressed from a lac promoter. To construct plasmid pAT1 containing both aroGrbr and tktA, a 5-Kb BamH1 fragment of E. coli DNA was cut from phage 473 of the Kohara minigroup (National Institute of Genetics, Japan), and inserted into the -3amH 1 site of pRW5. It was reported that this fragment contains the tktA gene, and it was confirmed, by its ability, that it complements a tkt strain (BJ502) for growth on ribose, and also of the migration distance of the gene product as judged on 12% SDS -PAG E (molecular weight of approximately 72,500).

Construction of pPS706 and control Plasmid pPS706 was constructed by inserting a 2.4 kb PCR fragment containing the pps minus promoter gene into the pJ F1 18EH vector. The primers were designed from the published pps sequence and contained an EcoRI site and a 5 'end of the? 10 ribosome binding site of the pps sequence and a 3' end of the BamK l site of the sequence. The PCR product was then cloned to the EcoRI or Bam sites of pJF1 18EH. Positive clones were selected based on the complementation of HG4 pps for growth on pyruvate. The expression of pps, of this construction, was controlled by means of the inducible tac promoter by IPTG. Plasmid pPSL706 was constructed after pPS706, as shown in Figure 5. Briefly, a ScalEcoR fragment, containing the pps gene, was cut from pPS706 and purified from the restriction pH regulator. This fragment was then cloned into a purified Scal-EcoR1 fragment, containing the luxl promoter of pGS 103, amicably given to the inventor by Tom Baldwin. Department of Biochemistry and Biophysics, University of Texas A & M. The expression, which uses this system, was controlled by the autoinducer (Al) in the culture media. pPSL706 is ampicillin resistant and compatible with other pACY184 derivatives, such as pRW5 and pAT1. The strains and plasmids used are summarized in Table I and Table I I.

TABLE I Bacterial strains TABLE II Plasmids Growth Media and Conditions All the cloning procedures were performed in a Luria-Bertani medium. Medium YE contained K2H PO4 (14 g / L), KH2PO4 (16 g / L), (N H4) 2SO4 (5 g / L), MgSO4 (1 g / L), yeast extract (15 g / L) , and D-glucose (15 g / L). The minimum medium used for high density cell resuspension cultures contains per liter, K2HPO4 (14 g), KH2PO4 (16 g), (N H4) 2 SO4 (5 g), MgSO4 (1 g), D-glucose (15 g). g) and also supplement with thiamin (1 mg), sialic acid (50 mg), L-tyrosine (8 mg), L-phenylalanine (8 mg), and L-tryptophan (4 mg). The minimal medium was supplemented with succinate (0.1 g / L), when the ppc mutant and its control grew. For the stable maintenance of the plasmids, ampicillin (100 mg / ml), chloramphenicol (50 mg / ml) was added to the culture medium. The concentration of antibiotics was reduced by half when the minimum medium was used. Cultures were grown overnight in the YE medium at 37 ° C in a roller drum and then subcultured in the same medium with appropriate training. Cultures were grown in 250 ml shake flasks, at 37 ° C in a rotating water bath, shaken at 200 rpm. After four hours of incubation (ODS50: 2-3) the cultures were induced with isopropyl-β-D-thiogalacto-pyranoside, IPTG (1 M). Cells were harvested from the last stationary phase by centrifugation at 6000 x G, and washed twice with minimal medium before resuspension in the same minimal medium supplemented with appropriate training of I PTG (1 mM). The OD5so of the high density resuspension cultures were approximately 4.0. The optical density can be greater than 4.0. In other words, the media can contain at least 5 x 109 cells / mL. Samples from the resuspension cultures were periodically removed to analyze the concentration of DAH (P) and glucose in the medium.

Determination of glucose and DAHP Cells were removed from samples by centrifugation and the supernatants were stored at 4 ° C until the samples were collected. The residual glucose in the culture supernatant was determined by the dinitrosalicylic acid test for the total reducing sugars. For additional information on this test, see Miller (Millet, G. L. 1958. Use of dinitrosalicylic acid reagent for the determination of reducing sugars, Anal. Chem. 31: 426-428) and Patnaik et al. (Patnaik, R., WD Roof, RF Young, and JC Liao, 1992. Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle, J. Bacteriol 174: 7527-7532), the contents of which are incorporated here for reference. The concentration of DAH (P) in the supernatant was determined by the thiobarbiturate assay. For additional information on this trial, see Draths et al. , and Gollub et al. (Gollub, E., H. Zaikin, and DB Springson, 1971. Essay for Synthase 7-phosphate of 3-deoxy-D-arabino-hetulosonic acid, Methods in Enzymology 17A: 349-350), the contents of which are incorporated herein by reference, this assay does not distinguish between DAH and DAH P.

Enzyme assays Cells were harvested by centrifugation at 6000 x G and washed and resuspended in a pH buffer of potassium phosphate (50 mM) pH, 7 or 5 mM Tris-CI-1 mM MgCl 2 (pH 7.4) , for the DAHP synthase assay or PEP synthase, respectively. Extracts of cells were prepared by disrupting the cells through a French pressure cell (S LM Aminco, Urbana, Y1 1) at 1248 kg / cm 2. The DAH P synthase activity was analyzed by the Schoner method, as described more fully in Schoner et al. (Schoner, R. and K. M. Herrmann. 1976. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. J. Biol. Che. 251: 5440-5447), the contents of which are incorporated herein by reference. The activity of Pps was analyzed as previously described. The total protein was determined with the Bio-Rad dry reagent (Bradford assay) with bovine serum albumin as normal.

Effects on Pps in the production of DAHP from glucose The purpose of constructing pPS706 was to express Pps with an inducible promoter unaffected by I PTG. This plasmid, together with pRW5, provided a means to vary the activities of the enzymes, Pps and AroG, independently under the control of two different promoters. The third enzyme, TktA, was under the control of its natural promoter and thus variable only in the on / off mode (presence or absence of the gene), this system then allowed the examination of the effect of Pps on a wide variety of terms. Furthermore, it is possible that this system could show an optimal point, where the activities of the enzyme were high enough to provide a maximum production of DAH P, but so high that it exerts a load of protein on the system, reducing the production of DAH P as a result. Therefore, the production of DAHP was measured by AB2847 / pRW5 / pPS706 and AB2847 / pAT 1 pPS706 in a glucose medium at varying concentrations of ITPG and autoinducer (N- (3-xox-hexanoyl) -humoserin lactone).

Figure 6 shows the effect of Pps at various concentrations of Al of IPTG in a glucose medium. At low concentrations of IPTG (low AroG activities), Pps has little or no effect. When the concentration of I PTG exceeded 50 mM, the effect of Pps began to show. As a control plasmid pGS104 was used, isogenic with pPS706, except for the pps site, and no effect was shown with or without the addition of Al. The effect of Pps was more significant in the strain that overexpressed TktA, and not it was due to a variation in the activities of AroG or TktA, since the activities of AroG and TktA showed to be constant with or without overexpression of Pps. From the measurement of residual glucose (data not shown), the productions of DAH P from glucose reached 100%, which corresponds to 70-80% after adjusting the overestimation of DAH P. The last value is consistent with that predicted by the stoichiometric analysis, which indicates a maximum theoretical yield of 86% from glucose, when pyruvate was recycled to PEP by Pps. Although increases in DAH P levels and productions with Pps were observed, a fall with higher Pps activity was not evident, it could have provided a peak. The levels of DAHP rather appear to reach saturation with additional induction of Pps.

Formation of by-products To obtain a discernment in the distribution of metabolic flux, the culture broth was analyzed for the fermentation of by-products, through the use of H PLC. Samples of the cultures were taken in glucose media with variable activities of Pps, AroG and TktA. The results indicate that host strain AB2847 produced acetate, succinate and formate, as the main byproducts, when neither AroG nor Pps were overexpressed. The production of these acids was generally reduced with the increase in the concentration of I PTG, except formate. This reduction correlates with the increase in the production of DAHP. When AB2847 / pAT1 / pPS706 was grown in glucose with an IPTG concentration beyond 50 mM, the broth showed unacceptable levels of these acids (data not shown). While the levels of formic acid and acetic acid were reduced with the increase in the activity of Pps, the succinic acid either remained constant (OμM IPTG) or was increased (10.50μM IPTG) with an increase in the activity of Pps. This increase can be contributed to the increase induced by Pps in the level of PEP, which is poured through PEP carboxylase and finally succinate.

EXAMPLE 1 Production of DAHP This example demonstrates that E. coli AB2847 is incapable of using DAH P, and accumulates DAH P in the medium, if the DAHP synthase is overexpressed. This strain was used as a host to detect the deposited flow to the aromatic trajectories. Since Draths et al. (Draths, K. M., DL Pompliano, DL Conley, J. W. Frost, A. Berry, GL Disbrow, RJ Staversky, and J.C. Lievense, "Biocatalytic synthesis of aromatics from D-glucose: role of Transketolase. "J. Am. Chem. Soc. 1992, 1 14, 3956-3962) have shown a possible limitation in the production of DAHP by E4P, pAT1 (containing both aroGfrb and tktA) was transformed to AB2847 to eliminate the limitation of E4P. To test whether the PEP supply limits the production of DAH P, the PEP synthase (Pps) was overexpressed in AB2847 / pAT1 by transforming the plasmid pP341 to this strain. 20-70 copies of the pps gene were expressed in the host cells. As a control, pPS341 was replaced by pPS341 X1, which encodes an inactive but stable pps gene product. The use of inactive Pps allowed the discrimination between the effect of the activity of Pps and that of overexpression of the protein. A B2847 / pAT1 / pUH E23-2 and AB2847 / pAT1 were also used without any other plasmid, as additional controls to identify the effect of the cloning vector, pUH E23-2, on the production of DAHP. As described above, the strains were grown in an enriched medium (YE) with I PTG and resuspended in a minimal medium. Since overexpression of Pps under Neolithic g conditions may cause growth inhibition, resuspension cultures were used to minimize the effect of cell growth on the icatic biocatalyst conversion. After resuspension, excreted DAH P and residual glucose were measured periodically. At 27 hours after resuspension, samples were taken for the Pps and AroG assays. Figure 1A shows the strain that overexpresses active Pps increased the production of DAHP almost twice. Strains containing pPS341 X1 or pU HE23-2 produced the same amount of DAH P, as the only one containing only pAT1. Figure 1B shows that, as expected, the Pps activity was overexpressed 10-fold in the strain containing pPS341, while the aroG activity in all strains remained almost constant. These data strongly suggest that the activity of Pps is responsible for the increase in the production of DAHP, while the inactive Pps or the cloning vector had no observable effect on the production of DAH P. The specific glucose consumption regimes of these strains were not influenced by the presence of active or inactive Pps, nor by the cloning vector (data not shown). Therefore, the strain that overexpresses Pps showed an increase, almost double, in the total production of DAH P (approximately 90% molar), compared with the controls (approximately 52% molar), suggesting that Pps improves both the productivity as the yield of DAHP production. The maximum theoretical yield of glucose at DAH P is 86%, which is slightly lower than the measured yield of the strain that overexpresses Pps. Because the measurements of both glucose and DAHP were reasonably reproducible, the discrepancy can be attributed to the inaccuracy of the extinction coefficient used to calculate the concentration of DAH P. However, the extinction coefficient has been calibrated by biosynthesized DAHP to from the cell extract and from known amounts of E4P and PEP. The results show that the extinction coefficient is absolutely within 30% accuracy. Therefore, the yield of DAH P is reasonably close to the theoretical maximum, although it can be and probably is less than the theoretical value. To determine whether the effect of Pps also requires transketolase overexpressed (Tkt), plasmid pRW5, which contains only aroGfbr, was used instead of pAT1 in the previous experiments. It was found that the overproduction of Pps did not increase the production of DAHP (Figure 2A) without the high activity of Tkt. Therefore, as regards the limitation of small molecules in the biosynthesis of DAH P, the first limitation arises from the supply of E4P. This hurdle changes to the PEP supply when Tkt is overexpressed, which is believed to increase the supply of E4P.

EXAMPLE 2 As noted above, overexpression of Pps improved the production of DAH P from glucose. There is great interest in knowing if the basal level of Pps expression, in the glucose medium, contributed to the production of DAH P. Therefore, the chromosomal pps gene in strain AB2847 was suppressed. The resulting strain (JCL1362) was used as the host to repeat the previous experiments. The results show that the inactivation of chromosomal pps did not significantly affect the production of DAH P in strains containing pRW5 or pAT1 (Figure 2B). Thus, the basal level of pps expression in the glucose medium did not contribute to the production of DAH P. Since PEP was also converted to OAA by Ppc, the elimination of this enzyme can increase the supply of PEP . Thus, the ppc gene on chromosome AB2847 was inactivated to determine if the production of DAH P can be increased without overexpression of Pps. This was done by transducing AB2847 with a P 1 lysate grown on JCL1242ppc:: Km. The resulting transducer, JCL1283 aroB ppc:: Km was then transformed with pAT1 or pRW5 and tested for the production of DAH P in the resuspension culture, as described above. To avoid the limitation of OAA in the ppc strain, the culture medium was supplemented with succinate, which showed no effect on the production of DAH P (data not shown). Contrary to what was expected, the ppc mutation did not increase the production of DAH P (Figure 2B), suggesting that the metabolic flux of PEP to OAA was not significant under the experimental conditions tested here. In fact, the ppc mutation actually reduced the production of DAH P for unknown reasons.

EXAMPLE 3 Production of Tryptophan The existing technologies for the production of tryptophan use either naturally occurring microorganisms, mutated microorganisms or genetically engineered microorganisms. These microorganisms include, but are not limited to, Escherichia coli, Brevibacteria, Corynebacteria, and yeast. The altered trajectories may include: (1) the elimination of trajectories that branch to phenylalanine and tyrosine; (2) the elimination of pyruvate kinases (pyk); (3) elimination of PEP carboxylase (ppc); (4) the elimination of phosphoglucose isomerase (pgi); (5) desensitization of the inhibition of enzyme feedback in the corismatous path and the trp operon; (6) elimination of the repressor, trpR, and the attenuation sequence in the trp operon; (7) removal of tryptophan degradation enzymes; (8) overexpression of trp operon enzymes; (9) the overexpression of wild-type or feedback-resistant AroF, AroG or AroH, or any enzyme in the corismatous pathway; (10) overexpression of SerA; and (1 1) overexpression of TktA or TktB. To produce tryptophan, strain ATCC31743, which contains chromosomal markers, such as trpRα (trAE) tna, can be used as a host. This strain also contains a plasmid pSC 102trp, which inhabits the trpE operon. Plasmids pAT1 and pPS341 (or pPS706 or pPSL706) can be transformed to this strain. The SerA gene can be cloned to any of the plasmids. Alternatively, these cloned genes (trpAE, aroG, tktt, pps, or serA) can be bound to one or two plasmids. The resulting strain was grown in an MT medium, which contains, per liter: KH2PO4, 3g; K2HPO4, 3g; K2H PO4, 7 g; N H4CL, 3g; MgSO 4, 0.2 g; FeSO4 7H2O, 10 mg, glucose 0 to 30 g. The Pps technology is compatible with all previous alterations in metabolism. Alterations that favor the delivery of E4P, such as the elimination of phosphoglucose isomerase, can eliminate the need for overexpression of Tkt associated with Pps in the preferred embodiment. Higher levels of AroG can also eliminate the need for overexpression of Tkt. Pps technology can be used in microorganisms, such as Brevibacteria and Corybacteria.

EXAMPLE 4 Production of Phenylalanine The path alterations for the production of phenylalanine are similar to the previous ones, except in the terminal trajectories that lead to phenylalanine. These include (1) the overexpression of corismate to phenylalanine enzymes; (2) trajectory operon elimination; and (3) elimination of phenylalanine degradation enzymes, and (4) destabilization of all enzymes from DAHP to phenylalanine, so that they are not inhibited by the latter. To produce phenylalanine, an E. coli mutant (W3110 Atrp Atyr Aphe) can be used as a host (ref: Forberg, Eliaeson and Haggstrom, 1988). Plasmids pAT1 and pPS341 (or pS706, pPSL706) can then be transformed to this strain. In addition, the phefbr gene of plasmid pJN6 (same ref) can be cut and ligated to either pPS341 or pAT1. The resulting strain can be grown in the following medium containing, per liter, NH4CL, 5g; K SO4, 0.8g; KH2PO4, 0.5 G; NaHPO4, 1 G; Na citrate, 2.5 g; FeCL36H2O, 0.01 g; CaCl 2 2 H 2 O, 0.20; MgCl 2 6H 2 O, 0.8 g; tyrosine, 0.05 g; tryptophan, 0.025 g, glucose 10-30 g.

EXAMPLE 5 Production of Tyrosine The trajectory alterations for tyrosine production are similar to the previous ones, except in the terminal trajectories that lead to tyrosine. These include: (1) the overexpression of the enzymes of chorismate to tyrosine; (2) elimination of the trp operon and the phenylalanine branching; (3) the elimination of tyrosine degradation enzymes; and (4) the desensitization of all the enzymes from DAHP to tyrosine, so that they are not inhibited by the latter.

EXAMPLE 6 Production of indigo Indigo production can be achieved by converting tryptophan or an intermediate from the tpr to indigo pathway, either in vitro or in vivo. Since the Pps technology increases the production of DAH P, it will also increase the supply of any metabolite that serves as the precursor for indigo. To produce indigo, the tryptophan producing strain, described above, can be used as a host. However, the strain needs to be made tna + and overexpressing naphthalene dioxygenase from Pseudomonas putida. In this strain, the tryptophan produced will be generated by the tryptophanase to indole, which is then converted to cis-indole-2,3-dihydrodiol by the cloned naphthalene dioxygenase. This cis-indole-2, 3-dihydrodiol produced is spontaneously converted to indigo in the presence of oxygen. ATCC31743 is the strain used in the conversion of DAHP to tryptophan.

EXAMPLE 7 Production of organic quinoids Organic quinoids can be derived from dehydroquinate, which is a 3 'end metabolite of DAHP. To produce quinic acid, can be used as a host E. coli AB2848 aroD housing pTW8090A, which contains the qad gene (dehydrogenase quinic acid Klebsiella pneumoniae) (ref: Draths, Ward, and Frost, 1992, JACS, 1 14, 9725-9726), and pKD136 (ref: same as above), which contains tkt, aroF, and aroB genes. The pps gene can be cloned to one of these plasmids and can be simultaneously overexpressed. It has been reported that at least 80 mM of D-glucose can be converted to 25 mM of quinic acid. After removal of cell, quinic acid in the supernatant it can be converted to benzoquinone after addition of sulfuric acid and manganese dioxide (IV) technical grade, and heating at 100 ° C for 1 hour. In the absence of acidification, the aqueous solutions of purified quinic acid were converted to hydroquinone in a 10% yield under heating at 100 ° C for 18 hours, with technical grade manganese dioxide.

EXAMPLE 8 Production of Catecol Catechol production can be achieved by transforming pps to E. coli expressing pKD 136. Since Pps technology increases the production of DAH P, it will also increase the supply of any metabolite that serves as the precursor for catechol.

EXAMPLE 9 Characterization of the expression of pps directed by luxl ' To characterize the expression of ppl directed by luxl ', pPS706 was transformed to AB2847 / pRW5 and AB2847 / pAT1, and Pps activity was measured when the strains were cultured in either glucose or xylose medium. The activity of Pps was increased with the concentration of the autoinducer (N- (3-xox-hexanoil) -hemoserin lactone) and saturation was reached when 1 μM of autoinducer was used. The activity of Pps, in the same strain that grew in a medium of xylose, was equal to that in glucose. The Pps activity was independent of the concentration of I PTG (data not shown). Therefore, the inventor achieved the independent modulation of three key enzymes in the production of aromatics: AroG (I PTG-inducible), TktA (on / off or presence / absence) and Pps (autoinductor-inducible). Although according to the patent statutes, the best mode and preferred embodiments of the invention have been described, it should be understood that the invention is not limited thereto, but rather will be measured by the scope and the scope of the invention. spirit of the appended claims.

Claims (6)

1. CLAIMS 1 .- A method to increase the flow of carbon towards the common aromatic trajectory of a microorganism, said method comprises modifying a microorganism to eliminate the step of limiting the production of PEP.
2. 2. A method according to claim 1, wherein the modification of the microorganism is such that it increases the expression of a protein that catalyzes the conversion of pyruvate PEP.
3. 3. A method according to claim 2, wherein the protein is Pps.
4. 4. A method according to any of claims 1, 2, or 3, wherein the microorganism is modified by transformation with a vector comprising a Pps gene.
5. 5. A method according to any of claims 1 to 4, further comprising the step of culturing the modified microorganism in a medium.
6. 6. A method according to any of the preceding claims, wherein the host cell is transformed with a plasmid, which is pPS341, PSL706, pPS706. 7. - A method according to any of claims 1 to 6, wherein said method is for improving the host cell biosynthetic production of compounds derived from the common aromatic path. 8. - A method according to any of claims 1 to 7, wherein said method is for improving a host cell biosynthetic production of compounds derived from DAH P. 9. A method according to any of the preceding claims, wherein said method is a method for the production of an aromatic metabolite, which is tryptophan, tyrosine, phenylalanine, catechol, indigo, quinic acid, benzoquinone or hydroquinone. 10. A method according to any of the preceding claims, wherein said method is a method for the production of a cyclic metabolite, wherein the modified microorganism is a genetically engineered strain adapted to produce said cyclic metabolite. 1. A method according to any of claims 1 to 6, wherein said method is a method for producing DAMP. 12. A method according to claim 1, wherein said method further comprises the step of isolating DAMP. 13. A method according to any of the preceding claims, which further comprises the step of modifying the microorganism in order to increase the expression of a second enzyme that catalyzes an additional reaction in the common aromatic path. 14. A method according to claim 13, wherein the enzyme is DAM P synthase, DHQ synthase, PEP synthase or Tkt. 15. A method according to claim 12, 13 or 14 wherein the microorganism is modified by transforming the microorganism with a vector comprising a gene encoding said enzyme. 16. A method according to claim 15, wherein the gene is aroF or aroB. 17. A method according to claim 15 or 16, wherein the vector is the PAT1 plasmid. 18. A method according to any of the preceding claims, wherein the microorganism is an E. coli strain. 19. A method according to claim 18, wherein the E. coli strain is AB2847AROB. 20. The product of a method according to any of claims 1 to 19. 21 .- A vector comprising a gene coding for Pps. 22. A vector according to claim 21, further comprising an aroF gene or an aroB gene. 23. A vector according to claim 21 or 22, which is the plasmid pPSL706, pPS706 or pPS341. 24. A DNA molecule comprising a plasmid according to any of claims 20 to 23. - A DNA molecule according to claim 24, further comprising a gene encoding DAHP or Tkt synthase. 26.- A method to produce a microorganism that has increased levels of DAH P, said method comprises the step of modifying a microorganism in order to eliminate the step of limiting the production of PEP. 27. A method according to claim 26, wherein the elimination of the step of limiting the production of PEP is effected by increasing the expression of a protein that catalyzes the conversion of pyruvate to PEP. 28. The method according to claim 27, wherein the protein that catalyzes the conversion of pyruvate to PEP is Pps. 29. A method according to claim 18, which comprises transforming the microorganism with a vector according to any of claims 20 to 23. 30.- A method according to any of claims 26 to 28, wherein the The microorganism is a strain genetically engineered to use increased levels of DAH P to produce a desired product. 31.- A method according to any of claims 26 to 29, wherein it further comprises the step of modifying the microorganism in order to increase the expression of one or more enzymes to use the increased levels of DAH P to produce a desired product. 32 - A method according to any of claims 30 to 31, wherein the desired product is tryptophan, tyrosine, phenylalanine, catechol, indigo, quinic acid, benzoquinone or hydroquinone. 33. A method according to any of claims 26 to 31, wherein it further comprises the step of modifying the microorganism in order to increase the expression of Tkt. 34.- A method according to claim 32, wherein the microorganism is transformed with pAT1. 35.- A microorganism that can be produced by the method of any of claims 26 to 34. 36.- A culture comprising a microorganism according to claim 35. 37.- A method for producing cyclic metabolites comprising the steps of: (a) transforming a microorganism to overexpress Pps and Tkt; and (b) include glucose to cultivate the transformed microorganism in a medium. 38.- The method according to claim 37, further comprising the step of isolating the DAH P. 39.- The method according to claim 37, wherein Pps is overexpressed by coupling the Pps gene with a promoter sequence. 40. The method according to claim 37, wherein the microorganism is Escherichia coli AB2847. 41.- The product of the method of claim 37. 42.- The method according to claim 37, wherein the step of overexpressing the Pps comprises transforming a plasmid selected from the group consisting of pPS341, pPSL706 and pPS706 to the microorganism. 43. The method according to claim 37, wherein Tkt is provided by the overexpression of the Tkt gene by transforming the plasmid pAT1 to the microorganism. 44.- A culture containing a microorganism modified with DNA, comprising a pps gene and a tkt gene, said culture is capable of producing increased levels of DAHP by fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances. 45. The method according to claim 44, wherein the microorganism is adapted to use DAHP to produce cyclic metabolites. 46. The method according to claim 37, wherein the metabolites are selected from the group consisting of tryptophan, tyrosine, phenylalanine, catechol, indigo, quinic acid, benzoquinone and hydroquinone. 47.- A genetic element comprising a Pps gene and a gene selected from the group consisting of an aroF gene and an aroB gene. 48. A vector carrying the genetic element of claim 47. 49. The DNA of claim 48, wherein the vector is a plasmid selected from the group consisting of pPSL706 and pPS706. 50. - The DNA of claim 49, further comprising a gene encoding DAH P. synthase 51. The DNA of claim 50, further comprising a gene encoding Tkt. 52.- A method for improving a host cell biosynthetic production of compounds derived from DAH P in relation to the wild-type host cell biosynthetic production of said compounds, said method comprises the step of increasing the expression in a host cell of a protein that catalyzes the conversion of pyruvate to PEP. 53. The method according to claim 52, further comprising the step of increasing the expression in the host cell of a protein that catalyzes reactions in the common aromatic path. 54. The method according to claim 53, wherein the protein exhibits DAH P synthase activity or DHQ synthase activity. 55. The method according to claim 53, wherein the protein exhibits PEP synthase activity.