MXPA97008363A - Application of mutants that transport glucose for the production of compounds of la via aromat - Google Patents

Abstract

The present invention relates to methods for increasing the flow of carbon towards a pathway of a host cell to increase the biosynthetic production of compounds thereof, the host cells are selected based on the Pts / glucose phenotype. Such host cells are capable of transporting glucose without consuming PEP, resulting in conservation of the PEP, which can be redirected to the pathway to increase the production of the desired compounds along the pathway. Pts / glucose mutants have been shown to be advantageous for increasing the production of aromatic amino acids

Description

APPLICATION OF MUTANTS THAT TRANSPORT GLUCOSE FOR THE PRODUCTION OF COMPOUNDS OF THE VIA AROM TICA FIELD OF THE INVENTION This invention relates to the increase of glucose transport in host strains, which are normally used by the Transport Systems of phosphoenolpiruvirate: Phosphotransferase (PTS) for such transport, by reducing the consumption of phosphoenolpyruvirate (PEP) and redirecting such PEP towards the desired metabolic pathway, such as an amino acid pathway, in the host strain.

BACKGROUND OF THE INVENTION The biosynthetic pathway known as the shikimate pathway or "common aromatic pathway" leads to the production of many aromatic compounds, including aromatic amino acids and other compounds such as folate, melanin, indole, catechol, enteroqueline, shikimate, dehydroshikimate and L-DOPA. In addition, by introducing specific cloned genes into an organism possessing the shikimate pathway, the range of compounds that can be produced expands enormously. Indigo production via, the way of the REF: 25993 aromatic amino acids is an example of the metabolic potential of this route. The low cost of efficient biosynthetic production of the compounds or derivatives thereof throughout the common aromatic route requires that carbon sources such as glucose, lactose and galactose be converted into the desired product with high yield percentages. Thus, from the point of view of industrial biosynthetic production of aromatic compounds or other biosynthetic derivatives along the common aromatic pathway, it could be valuable to increase the influx of carbon sources into and through the common aromatic pathway. , thereby allowing the biosynthetic production of the desired compound. Phosphoenolpyruvirate (PEP) is one of the main building blocks that these cells use in their biosynthetic pathways, particularly in amino acid biosynthesis (see Figure 1). For example, the synthesis of a molecule of corismato (the common precursor of all aromatic amino acids) requires two molecules of PEP. To date, the methods taken to increase the influx of carbon sources into and through the common aromatic pathway are typically related to the increase of PEP supply in the cells by the elimination of pyruvate kinase (pyk mutants) [1] and / or the elimination of the PEP carboxylase (ppc mutants) [2]. A third method for increasing the supply of PEP in the cells is to amplify the expression of the pps gene (which codes for the PEP synthase, which converts pyruvate PEP) (USSN 08 / 307,371, the description of which is incorporated herein by reference). ). Additional methods to increase the flow of carbon into and through the common aromatic pathway are related to the increased intracellular supply of D-erythrose 4-phosphate (E4P), the other necessary precursor (with PEP) for aromatic biosynthesis. This method can use overexpression of a transketolase gene (tktA or tktB), the product of the. which (trancetolase) catalyzes the conversion of 6-phosphate from D-fructose to E4P (US Patent No. 5,168,056, the disclosure of which is incorporated herein by reference). Another method to increase the availability of E4P can use the overexpression of the transaldolase (talA) gene, which codes for the enzyme transaldolase [3], which catalyzes the conversion of 7-phosphate D-sedoheptulase plus 3-phosphate glyceraldehyde to E4P plus 6-fructose phosphate. Contrary to the methods described above, the present invention addresses the aspect of increasing the availability of PEP, and thus the flow of carbon towards a given pathway, generating strains capable of transporting glucose without consuming PEP during the process. In this way, the conserved PEP is then redirected to a given metabolic pathway to increase the production of the desired product. These sepals were generated by inactivating the transport system of the phosphotransferase that depends on the PEP (PTS) used by such strains to transport glucose, and then selecting mutants that are capable of transporting glucose efficiently by a non-PTS mechanism (independent of the PEP). Using the strategy of inactivating PTS, the inventors have found that PEP is not consumed in glucose transport and, therefore, can be redirected to other metabolic pathways. These strains (Pts ~ / glucose ') have been successfully used to increase the production of tryptophan, phenylalanine, tyrosine and other compounds and are considered useful in the production of other aromatic as well as non-aromatic compounds along the metabolic pathway in the biological systems. For example, oxaloacetate (OAA) is synthesized by at least two routes: (i) through the tricarboxylic acid (TCA) cycle site; and (ii) through an anaplerotic route; the latter is catalyzed by the PEP carboxylase (PPC), which converts the PEP to C02 to OAA. Removal of the PTS could increase the level of PEP available to the PPC enzyme, thereby increasing the production of OAA. Since OAA is the precursor of aspartate, lysine, methionine, isoleucine and threonine (see Figure 1), the production of any of the latter compounds could be increased in a Pts' / glucose * strain.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, what is provided by the present invention is a method for increasing the flow of carbon to the common aromatic pathway, or any other biosynthetic or metabolic pathway that uses PEP as an intermediate precursor, of a host cell capable of using a host system. transport of phosphotransferase for the transport of carbohydrates, the method comprises increasing the availability of PEP for such a pathway by selecting a host cell, which is phenotypically PtsVglucose * and culturing the host cell with the appropriate carbon source. In a preferred embodiment the selected host cell is modified to suppress or inactivate all or substantially all of one or more of the ptslf ptsH and crr genes encoding the PTS, HPr and IIAGXc proteins of PTS [6], respectively. In another embodiment of the invention, the host cell (which is phenotypically Pts "/ glucose +) can be transformed with recombinant DNA containing genes encoding enzymes such as transketolase (tktA or tktB genes), transaldolase (talA gene) and / or phosphoenolpiruvirate synthase (pps gene) so that the products thereof are expressed at high levels in relation to the natural host cells In another embodiment of the invention the phenotypically host PtsVglucose 'may contain mutations in the pykA and / or pykF genes, which code for pyruvate kinase. Similarly, the host may contain a mutation in the ppc gene, which codes for the PEP carboxylase, and it could be expected that the pykA, pykF or ppc mutations would further increase the availability of PEP in the cell, as compared to a Pts strain. "/ glucose + alone. In yet another embodiment of the invention, the phenotypically host cell Pts "/ glucose + may further comprise additional recombinant DNA comprising one or more genes coding for the enzymes that catalyze the reactions in the common aromatic pathway of the host cell. The host cell can be transformed with DNA containing one or more of the aroB, aroD, aroE, aroL, aroA, and aroC genes.These genes code for the DHQ synthase, DHQ dehydratase, shikimate dehydrogenase, shikimate kinase, EPSP synthase and corismato synthase. , respectively (see Figure 1) In addition, the host cells can be transformed with a wide variety of genes of inherent viscosities, depending on the product desired to be produced by the cells by fermentation. increase biosynthetic cell production of compounds derived from the common aromatic pathway of the host cell, the The method comprises the step of cultivating, under suitable conditions, a phenotypically host cell Pts "/ glucose +. The host cell can preferably be transformed with recombinant DNA containing the tktA, tktB, talA or pps genes, so that the products of those genes are expressed at high levels relative to natural host cells. Alternatively, increased levels of such gene products can be achieved by chromosomal mutation or chromosomal integration by methods available to those skilled in the art. Chromosomal mutations include mutations in the tktA, tktB, talA or pps genes themselves, or in the promoters or regulatory genes that control their expression. In yet another embodiment of the invention which relates to the overproduction of the desired compounds, the host cell may further comprise additional recombinant DNA containing one or more genes coding for enzymes that catalyze reactions in the common aromatic pathway of the host cell. As for tktA, tktB, talA or pps, the increased expression of the genes encoding the enzymes of the common aromatic pathway can be reflected by the mutation of the genes themselves, or the promoters or regulatory genes that govern their expression. The host cells can be transformed with a wide variety of genes from a given pathway, depending on the product that is desired to be produced by the cells by fermentation. In another aspect of the present invention there is provided a method for obtaining PtsVglucose mutant cells, the method comprising: a) selecting a host cell which normally uses the phosphotransferase transport system; b) causing the host cell to mutate by inactivating the phosphotransferase transport system; c) culturing the mutant host cell using glucose as a carbon source; and d) selecting mutant cells growing on glucose having a specific growth rate of at least about 0.4 h "1. In a preferred embodiment the host cell is modified to inactivate the phosphotransferase transport system by deleting one or more genes selected from ptsl, ptsH and crr, which code for the El, HPr and IIAGlc proteins of the PTS, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the pathway of central carbon metabolism in E. coli, which shows the derivation of carbon skeletons for aromatic amino acid biosynthesis. From the figure it can be seen that the PTS is the main consumer of PEP, the percentages shown in the. L. figure represent the amount of PEP channeled to the competent pathways shown, as described by Holms [4]. The thick lines in Figure 1 indicate the steps of the tricarboxylic acid (TCA) cycle, dotted lines indicate the glyoxylate shuttle. Abbreviations PEP, phosphoenolpyruvate; DAHP, 3-deoxy-D-ara-ino-heptulosonate 7-phosphate; FHQ, 3-dehydroquinate; DHS, 3- dehydroshikimate; SHK, shikimate; S3P, 3-shikimate phosphate; EPSP, 3-phosphate of 5-enolpiruvil shikimate; PHE, phenylalanine; TYR, tyrosine, TRP, tryptophan; EtOH ethanol; 2-KG, 2-ketoglutarate; pgi, phosphoglucose iso erase; pyk, pyruvate kinase; pps, PEP synthase; ppc, PEP carboxylase. The aroG, aroF and aroH genes that code for the three isozymes of DAHP synthase in E. coli. The products of the other aro genes of the common aromatic pathway were defined in the text. Figure 2 shows the plasmid map of pRW5. Only the cloned gene relevant to the restriction site is shown. PlacUV5 represents the lacUVS cascade promoters that control the expression of the aroG. Figure 3 shows the plasmid map of pRW5tkt. Plasmid pRW5tkt was constructed by cloning a 5 kb fragment of E. coli DNA containing the tktA gene [5] into the unique pIAW5 site of pIAm5. The precise location of the tktA gene in the 5 kb fragment, or the orientation of the 5 kb fragment in relation to the -gen aroG gene, is unknown. Figure 4 shows the increased production of DAH (P) in Pts "strains (NF9) compared to Pts + strains (PB103) expressing the aroG gene (plasmid pRW5) or the aroG + tktA genes (plasmid RW5tkt). shows the plasmid map of the pCLlOlEA Only the relevant cloned genes are shown Ptac represents the tac promoter that controls the expression of the aroE and aroACB genes Figure 6 shows the increased production of phenylalanine and tyrosine production in Pts hosts "(p. NF9) and PTS + hosts (PB103) expressing the aroG gene (plasmid pR 5) or the genes aroG + tktA (plasmid pRW5tkt), in the presence of the plasmid pCHOlEA (which expresses the aroACBLE genes). Strain 1, PB103 / pR 5, pCLlOlEA; Strain 2, PB103 / pRW5t'kt, pCLlOlEA; Strain 3, NF9 / pRW5, pCLlOlEA; Strain 4, NF9 / pRW5tkt, pCLlOlEA. Figure 7 shows the plasmid map of pBE7.

Only the relevant cloned genes are shown. PlacUV5 represents the lacUVS cascade promoters that control the expression of the aroG and trp genes. Figure 8. shows the plasmid map of pBE6tkt. Only the relevant cloned genes are shown. The orientation of the tktA gene relevant to the other genes cloned in the plasmid pBE6tkt is unknown. PlacUVS represents the cascaded acUVS promoters that control the expression of the aroG and trp genes. Figure 9a shows the increased production of tryptophan, the production of anthranilate and indole in PTS " (NF9) and PTS 'hosts (PB103 and JB102) expressing the aroG + trp genes (plasmid pBE7) in the presence or absence of the plasmid pCLlOlEA (expressing the aroACBLE genes), or expressing the genes aroG + trp + tktA ( Plasmid pBEdtkt) in the presence of pCLlOlEA. Strain 1, PB103 / pBE7; Strain 2, NF9 / pBE7; Strain 3, NF9 / pBE7, pCLlOlEA; Strain 4, JB102 / pBE6tkt, pCLlOlEA. Figure 9b shows the total potential tryptophan produced in the host Pts "(NF9) and PTS + (PB103 and JB102) expressing the aroG + trp genes (plasmid pBE7) in the presence or absence of the plasmid pCLlOlEA (expressing the aroACBLE genes), or expressing genes aroG + trp + tktA (plasmid pBEdtkt) in the presence of pCLlOlEA strain 1, PB103 / pBE7, strain 2, NF9 / pBE7, strain 3, NF9 / pBE7, pCLlOlEA, strain 4, JB102 / pBE6tkt, pCLlOlEA. Figure 9c shows the specific tryptophan productivity (tryptophan g / dry cell weight / hour) Pts "(NF9) and Fts + (PB103 and JB102) expressing the aroG + trp genes (plasmid pBE7) in the presence or absence of the plasmid pCLlOlEA (which expresses aroACBLE genes), or expressing genes aroG + trp + tktA (plasmid pBE6tkt) in the presence of pCLlOlEA. Strain 1, PB103 / pBE7; Strain 2, NF9 / pBE7; Strain 3, NF9 / pBE7, pCLlOlEA; Strain 4, JB102 / pBE6tkt, pCLlOlEA.

DETAILED DESCRIPTION OF THE INVENTION One of the goals of metabolic engineering is the improvement of cellular activities by manipulating the enzymatic, transport, and regulatory functions of cells with the use of recombinant DNA techniques. To date, most of the mesophilic bacteria that metabolize glucose through the glycolytic pathway have been shown to possess a PTS for glucose transport [6,7]. The PTS uses PEP to phosphorylate glucose during its internalization, providing a strong link between the transport of sugar and its subsequent metabolism. Also, since the PEP is twice as energetic as the ATP, it can theoretically direct the consumption of its sugar substrates to a much greater extent than is allowed by the use of other sources of biological energy [8]. Obviously, the PTS system is advantageous in natural environments where carbon sources are scarce. However, under laboratory or industrial conditions this is not the case and, depending on the product to be biosynthesized, the consumption of PEP for glucose transport may decrease the availability of PEP for other biosynthetic reactions. PEP is one of the main precursor metabolites used by the cell in many biosynthetic reactions. Recently Varma, et al. [9] demonstrated the importance of the PEP-pyruvate node for the distribution of the optimal catabolic flux for maximum biochemical productions. Many bio-molecules can now be produced by the fermentation of genetically modified microorganisms, for example, catechol (US Patent 5,272,073), indigo (US Patent 4,520,103 and USSN 07 / 956,697), chemical acid (USSN 07 / 954,623), melanin, tryptophan, phenylalanine, etc. In general, the method for producing these products using recombinant techniques and fermentation processes has been the amplification of the genes that code for the speed-limiting enzyme (see, for example, USSN 08 / 257,354). However, in addition to the amplification of certain genes in the pathway, another important factor to consider is the flow of carbon through the central metabolic pathways of given organisms, which is the focus of the present invention. Host cells or strains useful for the present invention include any organism capable of using a PTS system for the transport of carbohydrates. These include prokaryotes belonging to the genus Escherichia, Corynebacterium, Brevibacterium, Bacillus, Pseudomonas, Streptomyces or Staphylococcus. A list of suitable organisms is shown in Table 1. Removal of the transport system allows PTS in any of those organisms could potentially increase the availability of PEP in the cell for alternative metabolic pathways and consequently increase the production of the desired compounds (eg, aromatics) of such cells.

Table 1 Reference Escherichia coli (6) Salmonella typhimuri um (6) Continuation of Table 1 Reference KJeiDsielJa pneumoniae (6) Bacill us subtilis (6) Mycoplasma capricolum (6) Acholeplasma florum (10) Staphylococcus aureus (6) Staphylococcus camosus (6) Staphylococcus xylosus (11) Rhodobacter capsulatus (6) Rhodopseudomonas sphaeroides (12) Streptococcus (Enterococcus) ) faecalis (6) Streptococcus mutans (6) Streptococcus salivarius (6) Streptococcus sanguis (6) Streptococcus sobrinus (13) Erwinia chrysanthemi (6) Xanthmonas campestri s (6) Corynebacteri um gl utamicum (14) Brevibacteri um lactofermentum (15) Bifidiobacterium breve (16) Azospirill um brasiliense (17) Listeria monocytogenes (18) Spirocheta aurant-ia (12) Continuation of Table 1 Reference Lactojaciiius jrevis (12) Lactobacillus buchneri (12) Lactobacillus casei (6) Lactococcus cremori s (19) Lactococcus lactis (6) Pseudomonas aeruginosa (12) Vibrio alginolyticus (6) Vibrio fumissii (20) Vibrio parahae olytica (12) Preferred strains are those that are known to be useful for producing aromatic compounds, including selected cells of the genera Escherichia, Corynebacterium um, Brevibacterium um and Bacill us. All the bacterial strains and plasmids used in this work are listed in Tables 2a and 2b. The selection of Pts mutants "capable of efficiently transporting glucose can be achieved using techniques available to those skilled in the art." For the case of Pts mutant selection "/ gucose + E. coli as exemplified herein, a chemostat was used to select glucose mutants * (from an initial population of Pts' / glucose cells) that have certain rates of specific growths with glucose as the sole carbon source.The spontaneous E. coli mutants that were selected were able to transport glucose efficiently through A non-PTS transport system Those mutants were selected for their ability to reach again rapid growth rates (meaning that they have a specific growth rate of at least about 0.4h-1) in the chemostat, having glucose as the only source of carbon, the mutants characterized in this paper apparently contain more than one and they need the activity of the galactose permease (gaJP) to give the phenotypes described. It is known that a better substrate for gJaP is D-glucose [21]. Under normal conditions (ie, with a functional PTS) this is not physiologically relevant, especially since the PTS is responsible for the 'exclusion of the inducer and the galactose regution is not induced, even if the galactose is present in the medium [22]. However, deletion of the ptsHIcrr operon creates a new situation, the preferred glucose transport system is absent and the exclusion effect of the inducer is lost [23]. Under those circumstances any mutation that is made about the galP gene (or any other transporter gene whose product could transport glucose) will produce cells that can use glucose. However, the degree of glucose utilization will depend on the specificity, level, efficiency, etc., of the transporter. The use of the chemostat as described herein, allowed the isolation of a collection of spontaneous mutants that can grow on glucose with different growth rates. Presumably these differences are due to variations in glucose transport speeds. The mutants described so far are distinguished from those reported by Biville, et al., [24]. This was surprising since the undifferentiated strain contains the same deletion of the PTS genes. Without intending to be limited to a particular theory, it is proposed that the difference lies in the fact that in all current experiments the level of dissolved oxygen was controlled and the cells were never under limited oxygen conditions. The behavior of the Pts strains was affected by the oxygen levels in the medium.Now, without intending to limit itself to that, a tentative hypothesis based on these mutants could be that the cells do not consume PEP during glucose transport and intracellular levels. However, considering that in E. coli the PEP is an allosteric regulator of several enzymes similar to phosphofructokinase and the ethylglioxal bridge, it is difficult to believe that by altering the carbon flux in the PEP-node pyruvate (that is, by interrupting the pyruvate kinase genes and / or by using mutants that use a non-PTS transport mechanism) there could be an accumulation of PEP in the cells. , that in E. coli this situation is avoided by several mechanisms and, to redirect the flow of carbon to some other pathways, concomitantly with the removal of competitive pathways, the metabolic pathways The desired ones need to be deregulated or amplified. In one embodiment of the present invention the strains Pts "/ glucose + were transformed asynchronously with recombinant DNA that codes for one or more genes that direct the flow of carbon into and through the common aromatic pathway, one such gene is transketolase (tktA or tktB). an enzyme of the pentose phosphate pathway that catalyzes two separate reactions each of which produces E4P as a product The amplification of the tktA gene increases the intracellular concentration of the aromatic precursor E4P (US Patent 5,168,056, incorporated herein by reference). Consequently, the amplification of the tktA gene (ie, the increase of intracellular E4P levels) in strains that also contain high levels of DAHP synthase (for example, strains that have amplified expression of the aroG gene) gives or results in an increase significant in the carbon committed in the aromatic pathway compared to strains containing high DAHP synthase activity only (U.S. Patent 5,168,056, which is incorporated herein by reference). Thus, having a host cell which creates an increase in carbon flux due to the amplification of transketolase in addition to a host cell which retains inactivation of the PTS pathway of the PTS (Pts), is a preferred embodiment since the effects can be additive as shown in the examples here, it should be noted that the host cell was cultured under conditions that create an increase in the carbon flux towards the aromatic pathway, it may be necessary to identify and overcome the limiting steps of the reaction On the way, this methodology is available to those skilled in the art, see, for example, USSN 08 / 257,354 (incorporated herein by reference.) As an example, in the next conversion DHQ DAHP Sintasa Sintasa. (aroB) E4P + PEP > DAHP > DHQ under conditions that create an increase in carbon flux towards the pathway (ie, Pts and tkt strains amplified), the activity level of the DHQ synthase is insufficient to consume the DAHP as soon as it is formed as a result of this limiting step of the natural reaction in the aroB, the DAHP accumulates and is excreted into the culture supernatant.This allows the accumulation of DAHP to be used as a means to test the increased intracellular PEP levels resulting from the Pts "/ glucose + mutations channeled towards the aromatic route. Similar methodologies are available with respect to the PEP synthase (pps) (USSN 08 / 307,371), and transaldolase (talA) [3], both of which are incorporated herein by reference. In addition to the amplification of enzymes such as transketolase to increase the flow of carbon to the common aromatic pathway, any genes encoding enzymes that catalyze reactions within the common aromatic pathway can be amplified (eg, DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroL, aroK), EPSP synthase (aroA) and corismato synthase (aroC)) in the Pts mutants "/ glucose + of the present invention Of course, as is readily apparent to those skilled in the art, it would be desirable to amplify a variety of different genes depending on the desired product.For example, if the desired product is tryptophan, any of the genes in the specific segment can be amplified. for the tryptophan of the aromatic pathway including the genes encoding tryptophan synthase (tr A and trpB), phosphoribosyl anthranilate isomerase-indoleglycerol phosph ato synthase (trpC), anthranilate phosphoribosyl transferase (trpD) and anthranilate synthase (trpE), while other genes can be deleted, such as tryptophanase (tnaA). If, for example, the desired compound is catechol, it is possible, in addition to using a mutant Pts "/ glucose *, to further transform this mutant with DNA encoding one or more of the following enzymes: DAHP synthase (aroF, aroG, aroH); 3-dehydrocinate (DHQ) synthase (aroB); transketolase (tktA or tktB); 3-dehydroshikimate (DHS) dehydratase (aroZ) or protocatequate (PCA) decarboxylase (aroY) (see USSN 08 / 122,919 and US Patent 5,272,073, the disclosure of which is incorporated herein by reference). In addition, by way of example, if the desired product is adipic acid, one or more of the following enzymes can be overexpressed (by amplification of the corresponding gene): 3-dehydroshikimate (DHS) dehydratase (aroZ); protecatechuato (PCA) decarboxylase (aroY) or catechol 1,2-dioxygenase (ca tA); and, optionally, transketolase (tktA or tktB); DAHP synthase (aroF, aroG, aroH) or DHQ synthase (aroD). (See USSN 08 / 122,920, the description of any patent issued thereon is incorporated herein by reference).

Similarly, if the desired product is indigo, the host strain Pts "/ glucose can be further transformed with DNA encoding a polypeptide analogous to a beta subunit of tryptophan synthase and DNA encoding an aromatic enzyme dioxygenase. USSN 07 / 956,697, the description of any patent issued thereon is incorporated herein by reference.) Examples of amplification of several genes (depending on the desired compound) in a Pts mutant strain "are provided in the following Examples. In this way, having provided a Pts / glucose + host cell which retains the PEP and thereby increases the flux of carbon towards the pathway (redirecting the PEP towards the desired pathway), the inventors have provided a host system which can be used for the production of virtually any compound or derivative along the common aromatic pathway, as well as other pathways such as non-aromatic pathways for lysine and threonine, etc. Daier, et al., [25] have reported that a Salmonella typhimuri um strain deleted from some phosphotransferase genes (so that it does not grow on minimal medium plus glucose) give rise to mutants that can use glucose as the sole carbon source.These mutants were found to have a mutation in the galR gene, and thus they had a constitutively expressed galactose permease gene (galP) which resulted in a glucose transport to obtain Pts mutants of E. coli that could In using glucose efficiently, strains PB11 and NF6 were used which contain a deletion of the ptsH, ptsl and ccr genes (the ptsHIcrr operon) in the experiments herein. The introduction of the pts deletion was done by the methods detailed in the Experimental Procedures section below. In general, the methodology used was as follows. Strains PB11 and NF6 are derived from strains JM101 [30] or PB103 (Trp + derivative of C534 [33]), respectively, in which the ptsHIcrr operon has been deleted. Although this deletion can be carried out using many different methodologies, in the present invention we use the generalized translation according to that described by Silhavy, et al. [26], using phage Pl vir to effect translation and strain TP2811 [27] as donor of the ptsHIcrr deletion. This process was carried out in two stages. First, a cell-free phage suspension was prepared by growing the bacteriophage Plvir in strain TP2811. In strain TP2811 most of the ptsHIcrr operon had been deleted and the kanamycin resistance marker inserted in the same region of the DNA [27]. -. The Pl vir lysate is able to transduce the suppression of ptsHIcrr and the kanamycin resistance marker simultaneously. Second, that phage was used to infect a genetically different recipient cell (JM101 or PB103) and the genetic (transducing) recombinants were selected by plating the infected cells on MacConkey-glucose plates containing kanamycin. After incubating the plates for 16 hours at 37 ° C, several white colonies appeared. It is important to note that the recipient strains (JM101 and PB103) are sensitive to kanamycin and form red colonies on the MacConkey-glucose plates. The color of the colony is an important factor to consider in this experiment. MacConkey-glucose plates contain an indicated dye that, depending on the pH, can vary from white to dark red. If the cells can transport glucose at a rapid rate, they will normally secrete organic acids and produce red colonies. On the other hand, if the transport of glucose decreases or is absent, the cells will not produce organic acids and the colonies will be white. The fact that after transduction all colonies resistant to the resulting kanamycin were white indicated that the ability of cells to assimilate glucose was affected, probably due to the transfer of the suppression of the ptsHIcrr operon. To corroborate this hypothesis, some transductants were selected and inoculated in minimal medium containing glucose as the sole carbon source. As expected, 12 hours after incubating the cultures at 37 ° C, no cell growth was detected. Under the same conditions, strains JM101 and PB103 grew very well (data not shown). Another proof of the absence of the PTS system was based on the fact that the Pts' strains become more resistant to the antibiotic fosfomycin [28], This phenotype was also tested in our transductants and was found to be resistant to such an antibiotic (no show the data). Based on these results, it was concluded that we transferred the suppression of the ptsHIcrr operon to the recipient strains (JM101 or PB103). The derivative Pts "of JM101 was designated PB11, while the derivative Pts" of PB103 was designated as NF6. The suppression of ptsHIcrr caused a very pleiotropic phenotype, which affects the utilization of PTS carbohydrates and not PTS. In addition, it affects the assimilation of tricarboxylic acid intermediaries and certain amino acids [23]. Biville et al. [24] demonstrated that an E. coli strain containing the same deletion was able to grow very slowly on glucose as the sole carbon source. 2-3 days after incubation, that strain gave rise to cells that grow rapidly. It was found that these mutants were able to produce pyrroloquinolin quinone (PQQ) and had glucose dehydrogenase activity. Presumably this strain absorbed glucose via the Entner-Duodoroff route, converting glucose into gluconate [24]. To select the spontaneous glucose reversals of the Pts strain (PB11), the selection was made with a chemostat [29]. The experiment was designed to isolate mutants with specific growth rate of at least 50% of the strain not Differentiated (Pts') (JM101) (see Experimental Procedures section below) Increasing the feed flow rate in the chemostat, mutants with different specific growth rates were selected.These growth rates were confirmed in the experiments independent for each strain With this method to select glucose mutants of PB11 having a specific growth rate of at least 0.4h "', a collection of mutants was obtained. Initially, 8 colonies were purified to a single colony by irradiating several times on MacConkey-glucose agar plates. Some of these isolates showed a normal E. coli colony morphology with a homogeneous red color. Others, however, presented an unstable phenotype. Still others were mucoid or produced small colonies. There were also differences in the degree of red color of the colony. One of the non-mucoid isolates, stable (designated PB12) that had a normal colony morphology and homogeneous red color was further characterized (see below). After increasing the feed flow rate to the chemostat to select PtsVglucose 'mutants having a specific growth rate of 0.8h ", the cells were again cultured out of the chemostat.All the colonies obtained now had a colony morphology. E.coli, stable, normal, and were not mucoid and had a homogeneous red color, one of such mutants, designated as PB13, was further characterized as described below.In addition to the PtsVglucose mutants' (PB12 having a specific growth of 0.4h_1, and PB13 having a specific growth rate of O.dh "1) derived from host JM101 of E. coli, another mutant PtsVglucose 'designated as NF9 was derived in the same manner but from the strain of E. col i PB103. NF9, like mutant PB13, had a specific growth rate in the chemistry of O.dh "1, and had the normal colony morphology of E. coli described above for PB13 Some of the phenotypic characteristics described above For mutants PB12 and PB13 were also carried out for the mutant NF9, mutants PB13 and NF9 gave similar results in those tests (data not shown). Strains JM101 (undifferentiated), PB11 (Pts "), PB12 (PtsVglucose ', specific growth rate of 0. 4h_1) and PB13 (PtsVglucose ', specific growth rate of 0.8h "1) were compared for their ability to oxidize a variety of carbon sources using the Biolog microplate assay system as described in the Experimental Procedures section below. It was found that the ability or inability of strains to oxidize a carbon source was very reproducible It was also noted that the oxidation of a particular carbon source does not always indicate that the test organism can use that carbon source to grow. The introduction of pts deletion in strain JM101 had a strong effect on the ability of cells to oxidize several carbon sources (strain PB11) (Table 3), however, after selecting mutants that could grow on glucose with different growth rates (strains PB12 and PB13), some of the phenotypes changed and some remained the same.In general, the reversion to a phenotype of glucose * does not produce a new phenotype not present in the original or undifferentiated strain (Pts *) JM101. Also, the two PtsVglucosa * strains analyzed showed a very similar carbon use pattern. Based on previous information in the literature, [24, 25], it was thought that the PtsVglucose * mutants selected in this work used a constitutive galactose permease to transport glucose. To confirm this notion of interrupted the galP gene in strains PB11, PB12 and PB13 with a TnlO transposon. This was done using a Pl vir phage lysate prepared on the E. coli strain CGSC6902 (see Table 2a) to transduce the galP:: TnlO insert into strains PB11, PB12 and PB13, creating strains PB11P, PB12P, and PB13P-, respectively. The strains in which the gaJP was interrupted lost their ability to use glucose as a carbon source (judging by their color on the MacConkey-glucose plates). These results indicate that PtsVglucose 'need the galactose permease to produce the glucose * phenotype. Saier et al. [25] reported that a history of Pts "in the introduction of the galR mutation is sufficient to produce a glucose phenotype. * More recently it has been shown that E. coli exists in two repressors, galR and galS, involved in the control of the galactose regu- lation [22] Based on this, PB11 derivatives containing the galR, galS or galR, gal S mutations were constructed by transduction with Plvir using the AG701 gal5 :: TnlO strain and / or the JT247 gaJP strain: : CmR as the source of the inactivated galS and galR genes The resulting strains were designated PB111 (PtsVglucose ", galR:: Cmk), PB114 (PtsVglucose ", galS:: TnlO) and PB115 (Pts" / glucose ", gaiP:: CmR, galS:: TnlO) After culturing these mutants on MacConkey-glucose plates, the color of the colonies was recorded 24 h later, none of the mutants was enough to give a red phenotype on MacConkey-glucose plates, however, the introduction of the galR mutation in the PB11 strain produced pink colonies, suggesting that this mutation restored In this way, it is believed that the PtsVglucosa * mutants isolated in this work may contain more than one mutation.This belief is supported by the fact that the use of Plvir phage lysates prepared from strains PB12 and PB13, they were unable to transduce strain PB11 back into the M9-glucose medium again, these experiments were repeated several times, using different amounts of phage, and the ability of these phage lysates to tr ansducing an unrelated genetic marker was verified on the same with "joint experiments (data not shown). The fact that the PtsVglucose mutants of the present invention need ga 1 P to grow on glucose distinguishes them from the mutants reported by Biville [24] that were able to sustain high growth rates on glucose in the absence of a functional gaiP gene. In addition, the mutants of the present do not produce gluconate and use glucose in MacConkey-glucose plates under anaerobic conditions (the data are not shown), although the mutants isolated by Biville [24] were oxygen dependent for the oxidation of glucose in gluconate .

Experimental procedures Bacterial Strains and Growth Conditions The bacterial strains are listed in Table 2a, while the plasmids used are listed in Table 2b. Strains Pts "PB11 and NF6 were obtained by transduction with phage Pl vir using TP2811 [27] as donor according to what was described by Silhavy [26] Several of the phenotypic characteristics of the Pts mutation" were confirmed using plates based on MacConkey-agar agar supplemented with different carbohydrates. Also, resistance to the antibiotic fosfomycin was used as another indication of the phenotype pts ~ [28]. Minimum M9 medium supplemented were used thiamine and glucose [32] for the determination of growth characteristics in liquid medium.

Use of Different Sources of Carbon To characterize the catabolic properties of the strains, they used ES and GP Microplates (Biolog, Inc.). Equal numbof cells were inoculated into the 96-well microplate, incubated for 24 hours at 37 ° C and the results were analyzed with a microplate reader and the distributor's computer program (Biolog, Inc.).

Table 2a Genotype and Characteristics Source or Relevant Strains Reference JM101 supE, thi, (? Lac-proAB) F '[30] [traD36, lacl '', lacZ? M15, ProAB] TR2811 F ", xyl, argHl, lacX74, aroB, il vA [27] A (ptsH, pstl, crr), Km" CGSC6902 FI, hiS / jeu, üvA,? Lac, mglP, E. coli galP :; TnlO Genetic Stock Center AG701 galS:: Tnl O t31] Table 2a (continued) Genotype and Characteristics Source or Relevant Strains Reference JT247 galR:: Cm [31] PBll JM101,? (pstH, pstl, crr), KmR [This Work] PB12 the same as NF6, but glucose '[This Work] with a specific growth rate of 0.4h "' PB13 the same as PBll, but glucose 'l [This Work! With a specific growth rate of O.ßh -1 PB11P the same as PBll, but gaiP :: TnlO [This Work] PB12P the same as PB12, but gaiP:: TnlO [This Work] PB13P the same as PB13, but gaiP :: TnlO [This Work] PBl 11 the same as PBll, but gaiP:: CmR [This Job] PB114 the same as PBll, but gaiS:: TnlO [This Job] PB115 the same as PBll, but gaJP :: CmR [This Job] PB103? lacU169 trpR tnaA2 [This Job] anthranilate (TrpR 'derived from strain C534 [33]) JB102 the same as PB103, but it will be [This Job] NF6 the same as PB103, but? (pstH, [This Job] pstl, crr), KmH Table 2a (continued) Genotype and Characteristics Source or Relevant Strains Reference NF9 the same as PBll, but the glucose '[This Work] with a specific growth rate of O.dh "1 NF6P the same as NF6, but gaiP :: TnlO [This Work] NF9P the same as NF9, but gaIP: : TnlO [This Job] Table 2b Source or Plasmids Relevant Cloned Genes Reference pRW5ktk the same as for pRW5, but [This Job] also contains tktA PCLlOlEA P, l?: - aroACB, aroL, P, "..- aro £ [This Job] pBE7 P / cw5- ar Gfbr, Py Wi * -trpE'brDCBA, [This Job] will be pBEdtkt P / fJ,. "v ', - a oGfbr, P," tw < J-trpEtbrDCBA, [This Job] will be, tktA Example 1 Transduction of the Suppression of the ptsHIcrr Operon to Strains JM101 and PBl03 Phase I: Preparation of the phage lysate Plvir To prepare the phage lysate Pl vir of strain TP2811 [27], 0.5 ml of a culture of this strain was inoculated overnight in 5 ml of LB culture medium (1% Bacto-tryptone, 0.5% extract of Bacto-yeast, 1% sodium chloride, pH 7.'4) with a content of 0.2% glucose and 5mM CaCl2. The culture was incubated for 30 minutes at 37 ° C with aeration. A volume of 0.1 ml of a Plvir lysate (approximately 5 x 10"phage / ml) was added and the mixture was shaken at 37 ° G for 2-3 hours until the cells were used, 0.1 ml of chloroform was added and the mixture was stirred vortexically, the resulting sample was centrifuged at 4500 g for 10 minutes to pellet the remains, the supernatant was transferred to a sterile tube, 0.1 ml of chloroform was added to the tube, mixed and stored at 4 ° C.

Phase 2: Genetic Transduction To effect transduction, a single colony of the recipient strain (JM101 or PB103, see Table 2a) was inoculated in 5 ml of LB culture medium and incubated with shaking at 37 ° C overnight. The overnight culture was centrifuged at 1500 g for 10 minutes and the pelleted cells were resuspended in 2.5 ml of 10 mM MgSO4 with a content of 5 mM CaCl2. In a sterile tube, 0.1 ml of cell suspension and 0.1 ml of phage lysate were combined and incubated for 30 minutes at 30 ° C without agitation. Phages that lacked control were also included. 0.1 ml of 1 M sodium citrate was added to the tubes and mixed. Then 1 ml of LB medium was added and the mixture was incubated at 37 ° C for 1 hr and cultured on medium containing MacConkey-agar containing 50 micrograms / ml kanamycin and 1% glucose. Several white colonies appeared on the plates after 12 hours of incubation. Those colonies were further purified by growing on fresh plates containing medium containing MacConkey-agar, 50 micrograms / ml kanamycin and 1% glucose. Those white colonies, as indicated above, were unable to transport glucose, and thus are believed to comprise the suppression of the ptsHIcrr operon. One of the white colonies derived from each of the undifferentiated or original strains (JM101 or PB103) was selected for further work. These PtsVglucose mutants were designated PBll (derived from JM101) and NF6 (derived from PB103) (see Table 2a).

Example 2 Method of Manufacturing the Selection in a Chemostat Strain PBll or strain NF6 were inoculated in a 1 liter chemostat containing M9 medium supplemented with 0.2% glucose and incubated at 37 ° C. The dissolved oxygen remained above 20% controlling the speed of the agitator. The pH of the medium was maintained at 7.0 by the addition of base. After the culture reached a DOeoo of about 2.5, the washing of the fermenter was started by feeding fresh M9 medium at a rate of 0.52 liters / hour. This flow rate could wash all the cells that grow with a specific growth rate of less than 0.4 h "1 (under the same conditions the specific growth rate of the undifferentiated strain or of origin Pts * was 0.8h-1 After at least 3 residence times, the feed flow rate was increased to wash the cells with a growth rate of less than 0.5h-1. This procedure was repeated (i.e., in increments of 0.1) until the strains were selected with a double time of at least 0.8h_1 No attempts were made to isolate the strains with faster growth rate Before each increase in the glucose feed flow rate, the samples were removed from the chemostat, were diluted and cultured on MacConkey-glucose plates, after incubation of the plates for 24 hours at 37 ° C, the total number of plate colonies, total red colonies, morphology of the colony was recorded. , 'etc., as a measure of the tracking of the appearance of PtsVglucose * cells that had a normal colony morphology of E. coli and a homogeneous red color. As stated above, only strains that had a non-mucoid, normal colony morphology and a homogeneous red color were further studied. All PstVglucose * mutants isolated from the chemostat that were further characterized were listed in Table 2a. PB12 and PB13 were derived from strain PBll, while NF9 was derived from strain NF6.

Example 3 Phenotypic Characterization of Mutants Pts' / glusosa * To characterize the catabolic properties of strains JM101, PBll, PB12 and PB13 (see Table 2a), ES and GP microplates were used as discussed in the Experimental Procedures section. After performing several experiments with this system it was found that the quantitative values varied. However, the ability or inability to oxidize a carbon source was very reproducible. In the data shown in Table 3 provide the qualitative results of this experiment. These data reflect the capacity (+) or inability (-) of a given strain to oxidize a certain carbon source. The pleiotropic nature of the suppression of ptsHIcrr is evident from these results.

Table 3 Strain Cepa Cepa Cepa Carbon Source M101 PBll PB12 PB13 Glucose L-Asn L-Gln +/- Continuation of Table 3 Cepa Cepa Cepa Cepa Carbon Source JM101 PBll PB12 PB13 L-Pro + - L-Asp + - L-Glu + - L-Thr + - D-Ala + - + Glicil-L-Asp + +/- + Glicil-L-Glu + +/- + N-Acetyl-D-Glucosamine + - D-galactonic acid? -Lactone - - Glycerol + + Sugar acid + - D-Glucuronic + + + + D-malic acid + - +/- + Fumaric Acid + - D-Sorbitol + - Lactose - - Fructose + - D-Mannose + - D-Galactose + + L-Ramnosa + - + D-Gluconic Acid + + + a-Methyl Galactoside Continuation of Table 3 Cepa Cepa Strain Strain Carbon Source JM101 PBl l PB12 PB13 L-Galactonic acid? -Lactone + - + + Mucic acid + - + + Example 4 Interruption of the galP To determine whether glucose transport in the PtsVglucose * strains occurs via the galactose permease (encoded by the gaiP gene), the galP gene was disrupted in strains JM101, PBll, PB12, PB13, PB103 and NF9 (see Table 2a) . Example 1 was repeated with the following modifications: To prepare the phage lysate Pl vir, strain CGSC6902 (Table 2a) was used as the donor of the galP :: TnlO mutation. After performing genetic transduction using strains JM101, PB103, .PB11 (Example 1), NF6 (Example 1), PB12 (Example 2), PB13 (Example 2) or NF9 (Example 2) as receptors, the cells were cultured in agar-MacConkey medium containing 50 micrograms / ml kanamycin, 10 micrograms / ml tetracycline and 1% glucose. After 12 hours of incubation at 37 ° C, the phenotypes were determined. The results are shown in Table 4.

After transferring the gaiP :: TnlO insert to all the PtsVglucose * strains selected in this invention as for Examples 1 and 2, they had a white phenotype. This strongly supports the hypothesis that glucose transport in the PtsVglucose * strains occurs via the galactose permease (encoded by gal P).

Table 4 Strain Phenotype JM101 Red PBll White PB12 Red PB13 Red PBllP (gaJP ') White PB12P (gai p- White PB13P (gaiP ~) White PB103 Red NF6 White NF9 Red NF6P (galP') White NF9P (galP White Example 5 Effect of mutations ga IR:: CmR, gaiS :: TnlO The results presented in Example 4 strongly suggest that in the PtsVglucose * strain, glucose transport occurs via the galactose permease encoded by galP. The galR and galS genes code for the repressor and isorrepressor, respectively of the gal operon [31], and it is known that galR represses the expression of the gaJP gene [25]. Thus, inactivation of the gaiP gene (and possibly the galS) at the Pts origin "could lead to anti-repression of the galactose permease and a glucose * phenotype." This hypothesis was tested as follows: To transfer the mutations galR:: CmR and / or galS :: TnlO to strain PBll, Example PBl was repeated with the following modifications: To prepare phage lysate Pl vir, the strains AG701 (gaiS:: TnlO, Table 2a) or JT247 (galR: : CmR, Table 2a) as donors The lysates prepared on this strain were used to transduce the strain PtsVglucosa 'PBll, selecting the transductants that had the resistance to the appropriate antibiotic Three derived strains were obtained: PBl 11 (galR:: CmR); PBl14 (galS:: TnlO) and PB115 (galR:: CmR, galS:: Tnl0). Those strains, together with the strains of origin or undifferentiated JM101 and PBll, were analyzed for their ability to use glucose by scratching on agar from MacConkey with a content of 1% glucose The results shown in Table 5 indicate that none of the introduced mutations were sufficient to completely restore the utilization of glucose (ie, production of red colonies). However, the introduction of the galR:: CmR mutation to strain PBll (strains PB111 and PB115) generated pink colonies, indicating that the ability to transport glucose and secrete organic acids had been partially restored.

Table 5 Strain Phenotype JM101 Red PBll White PBl 11 Pink PB114 White PB115 Pink The results presented in Examples 1-5 show that PTS can be effectively abolished by the deletion of the ptsHIcrr operon, resulting in cells that are incapable of using glucose (glucose). (Example 1) The spontaneous glucose mutants of those strains can be obtained using the present novel method of selecting the glucose derivatives' in a chemostat (Example 2) The pleiotropic effect of the suppression of ptsHIcrr is evident from the large number of phenotypic differences between the Pts strain "(of origin) and Pts "or PtsVglucose" derivatives (Example 3) Inactivation of the galP gene coding for galactose permease, in PtsVglucosa * mutants ablated its ability to use glucose, strongly suggesting that strains PtsVglucosa * uses galactose permease for transport of glucose (Example 4) The additional support of this hypothesis comes from the fact that the inactivation of the repressor galR, which suppresses the expression of the galP, in the origin PtsVglucosa ", partially restores the capacity of the strain to use glucose (Example 5). The remaining three Examples that follow (Examples 6, 7 and 8 relate specifically to the detection of the increase of PEP introduced by PtsVglucose mutations in a specific biosynthetic pathway, in this case the pathway of the aromatic amino acids, although the present invention does not is limited to this route only.

Example 6 The method described above for the selection of PtsVglucose * mutants that import glucose via a mechanism not dependent on PEP could result in an increased intracellular availability of phosphoenolpyruvate (PEP). One method to test this hypothesis is to compare the carbon committed in the biosynthetic pathway of the aromatic amino acids (a pathway in which the PEP is an initial precursor) in the mutants PtsVglucosa 'and their respective origin or undifferentiated (Pts *) strains . The PEP that is exchanged during glucose transport in the PtsVglucose * mutants could be available to be directed to the aromatic pathway. To test whether PtsVglucose * strains direct more PEPs to aromatic biosynthesis, strain PB103 (a Trp 'derivative of strain C534 [33]) and its PtsVglucose' NF9 derivative (refer to Table 2a for the description of the strains) was used. strains). In minimal glucose medium, the mutant PtsVglucose 'NF9 exhibits a growth rate identical to that of its parent strain or non-differentiated PB103 (data not shown). To measure the carbon committed in the aromatic pathway, strains PB103 and NF9 were each transformed with the designated plasmids, pRW5 and pR 5tkt (Figures 2 and 3). The plasmid? RW5 contains the aroG gene of E. coli cloned under the control of the lacUVS promoters [34]. Thus, the expression level of aroG is controlled by the addition of isopropyl β-D-thiogalactopyranoside (IPTG) inducer to bacterial cultures. The aroG gene that codes for the DAHP synthase enzyme, which catalyzes the initial reaction of the aromatic amino acid pathway (refer to the legend in Figure 1 for the abbreviations). (aroG) DAHP synthase - E4P + PEP > DAHP Strains containing ΔR 5, when grown in medium containing IPTG, had amplified DAHP synthase activity (unpublished). This generated DAHP synthase activity serves to pull the E4P and PEP pathway from the central metabolism and direct it towards the aromatic amino acid pathway (Figure 1). In fact, high levels of DAHP synthase are a prerequisite for the production of compounds derived from the aromatic pathway. Plasmid pR 5tkt is identical to tRW5 but also contains the cloned E. coli tktA gene, which codes for the enzyme transketolase (US Patent No. ,168,056). Transketolase is an enzyme of the pentose phosphate pathway that catalyzes two separate reactions, each of which produces E4P as a product. In this way, the amplification of a tkt gene (tktA or tktB) increases the intracellular concentrations of the precursor of the aromatic pathway E4P. In consecuense, the amplification of a tkt gene (ie, the increase in intracellular E4P level) in strains that also contain high levels of DAHP synthase (eg, strains containing amplified aroG) results in the significant increase in carbon compromised in the aromatic pathway compared to strains containing high DAHP synthase activity only. The second enzymatic step of the aromatic amino acid pathway is catalyzed by the enzyme dehydroquinate (DHQ) synthase. This enzyme, encoded by the aroB gene, catalyzes the conversion of DAHP to DHQ.

DHQ DAHP synthase synthase (araS) E4P + PEP > DAHP > DHQ Under conditions that create an increase in carbon flux into the aromatic pathway, for example, in strains containing pR 5 or pRW5tkt, the activity level of the DHQ synthase is sufficient to consume DAHP as soon as it is formed. As a result of this limiting step of the natural reaction in the aroB, DAHP accumulates and is excreted into the culture supernatant. This allows the accumulation of DAHP to be used as a means to test the hypothesis that the increased intracellular PEP levels resulting from PtsVglucose * mutations can be channeled into the aromatic amino acid pathway. Four strains were compared for the production of DAHP: Cloned genes Phenotype strain Pts on the plasmid PB103 / pRW5 Pts7 aroG PB103 / pRW5tkt Pts' aroG + tk tA NF9 / pRW5 PtsVglucose 'aroG NF9 / pRW5tkt PtsVglucose' aroG + tktA The strains were grown with shaking in cultures in 30 ml flasks at 37 ° C. The medium used was medium YE, which contains (per liter of distilled water); 15 g of yeast extract, 14 g of K2HP04, 16 g of KH2HPO4, 5 g of (NH4)? S0, 15 g of glucose, 1 g of MgSO4-7H0 and 1 drop of antifoam P-2000. The cultures were inoculated with cells from cultures planted overnight. The initial OD of the cultures was 0.2. IPTG was added to the cultures (to induce high expression levels of the aroG gene in the plasmids RW5 or pRW5tkt) when the D066 reached 2.0. The pH of the cultures was maintained at 6.5 through the experiment by periodic additions of 45% KOH. Culture samples were removed at specific intervals, the cells were removed by centrifugation, and the supernatant (cell-free culture broth) was assayed for DAHP using the standard thiobarbituric acid assay [35]. The results shown in Figure 4 show that the strain PtsVglucose ', NF9 containing the plasmid pRW5 accumulates more than 2X more than DAHP than the isogenic control strain PB103 / pRW5. This level of increase in the production of DAHP is similar to that observed for the PTS strain 'PB103 / pRW5tkt, which contains aroG + tk tA amplified. The highest level of production of DAHP was observed in the PtsVglucose strain 'NF9 containing pRW5tkt (approximately 2X of DAHP on PB103 / pRW5tkt or NF9 / pRW5, and approximately 4X of DAHP on PB103 / pRW5). These results show that in the PtsVglucosa * strains the carbon committed in the aromatic amino acid pathway was doubled compared to the isogenic control strain. In addition, the individual positive effects of the PtsVglucose 'and tk tA amplified mutations act in an additive manner, resulting in a 4-fold increase in the carbon committed to the aromatic amino acid pathway relative to the control strain.

Example 7 In Example 6, it was shown that the carbon committed to the aromatic amino acid pathway was doubled in the PtsVglucose strains 'NF9 / pRW5 and NF9 / pRW5tkt in relation to their strains (PTS') isogenic control, PB103 / pRW5 and PB103 / pRW5tkt, respectively. To illustrate this better phenomenon, each of the strains mentioned above was transformed with plasmid pCLlOlEA (Figure 5). The last plasmid is compatible with the plasmids pRW5 and pRW5tkt and contains the genes aroA, aroC, aroB, aroL and aroE of cloned E. coli (referred to collectively as aACACBLE). The presence of pCLlOlEA results in high levels of the aroACBLE gene products, which catalyze five of the steps within the common trunk of the aromatic amino acid pathway that leads to the intermediate branch point of the corismato (described schematically below) . The abbreviations used in the diagram are given in the legend of Figure 1. aroB aroD aroE aroL aroA aroC DAHP > DHQ > DHS > SHIK > S3P > EPSP > CO ISMATO In strains containing pCLlOlEA, the limitation of natural speed in the passage of the aroB (and any other step within the common trunk of the aromatic amino acid pathway) is mitigated, allowing the unimpeded carbon flow from the DAHP to the corismat. The corium formed in this way is converted by the products of the genes pheA, tyrA and tyrB of E. coli (encoded ^ chromosomally) to phenylalanine and tyrosine. This allows the carbon committed to the pathway of the aromatic amino acids to be measured as the total production of phenylalanine and tyrosine. Four strains were compared for the production of phenylalanine and tyrosine; Clone genes Phenotype strain Pts on Plasmid PB103 / pRW5, pCLlOlEA Pts • aroG, aroACble PB103 / pRW5tkt, pCLlOlEA Pts * aroG + tktA, aroACBLE NF9 / pRW5, pCLlOlEA PtsVglucose 'aroG, aroACble NF9 / pRW5tkt, pCLlOlEA PtsVglucose' aroG + tktA, aroACBLE The strains were grown with shaking in cultures in 30 ml flasks at 37 ° C. The medium used was medium YE, which contains (per liter of distilled water); 15 g of yeast extract, 14 g of K, > HP04, 16 g of KH2P04, 5 g of (NH) 2S04, 15 g of glucose, 1 g of MgSO4-7H20 and 1 drop of antifoam P-2000. The cultures were inoculated with cells from cultures planted overnight. The initial crop D06feo was 0.2. IPTG was added to the cultures (to induce high expression levels of the aroG and aroACBLE genes) when the DO ^ o reached 2.0. The pH of the cultures was maintained at 6.5 through the experiment by periodic additions of 45% KOH. Culture samples were removed at specific intervals, the cells were removed by centrifugation, and the supernatant (cell-free culture broth) was analyzed by high performance liquid chromatography to detect the presence of phenylalanine, tyrosine and the common pathway intermediates. of the aromatic amino acids. The results shown in Figure 6 show that PtsVglucose 'NF9 host-based strains accumulated 2-3X more phenylalanine and 1.6X more tyrosine than the relevant control strains than those based on the host Pts * PB103. In addition, in all cases, the presence of amplified transketolase activity (ie, plasmid pRW5tkt) gave an improvement of 27-47% in the production of phenylalanine and tyrosine. None of the strains accumulated detectable levels of the amino acid common pathway intermediates, illustrating the unblocking effect of plasmid pCLlOlEA. These results show that the aromatic amino acids, measured as the total production of phenylalanine and tyrosine, increased significantly in the Pts strains. " / glucose 'compared to its isogenic control strains (Pts').

Example 8 In Examples 6 and 7 it was demonstrated that the increase of the intracellular PEP level in the strain PtsVglucose 'NF9 (in relation to its strain of origin or natural Pts' PB103) can be translated into a compromised carbon increase of the aromatic amino acids. This increased committed carbon was demonstrated as the increase of the first intermediary of the aromatic amino acid pathway, DAHP, and as the increased production of two of the aromatic amino acids themselves, phenylalanine and tyrosine. In this example, it was shown that the carbon concomitant with the increased aromatics can also result in an increase in the production of the third aromatic amino acid, tryptophan. Three host strains were used in this experiment. PB103 (Pts1) and its derivative PtsVglucose 'NF9 were described in Example 6. Strain JB102 (see Table 2a) is a mutant serA derived from PB103 and was included in this example because in separate experiments it had shown a functioning of improved tryptophan compared to that of PB103 containing the same plasmids (unpublished). Plasmid pBE7 (Figure 7) confers resistance to tetracycline and contains six cloned genes required for the production of tryptophan, aroG (coding for the first enzyme of the aromatic pathway, DAHP synthase) and the trpEDCBA genes (coding for the five enzymes of the tryptophan branch of the aromatic pathway). The trpE gene on pBE7 has been altered so that its product, anthranilate synthase, has become resistant to the inhibition of feedback by tryptophan (trp £ lbr). Plasmid pBE6tkt (Figure 8) is identical to plasmid pBE7, except that it specifies resistance to chloramphenicol instead of resistance to tetracycline, and also contains the transketolase gene (tktA) cloned, the function of the last gene is to increase the carbon committed to the aromatics increasing the intracellular E4P supply (see Example 6). Plasmid pCLlOlEA, which confers resistance to spectomycin and is compatible with pBE7 or pBE6tkt, contains the aroACBLE genes (see Example 7) and functions to attenuate the steps that limit the reaction in the common trunk of the aromatic pathway .

Four strains were compared for the production of tryptophan: Phenotype Strain Pts Relevant Cloned Genes on Plasmids _______ pts' aroG trpEtbrDCBA NF9 / pBE7 PtsVglucose 'aroG trpE "DCBA NF9 / pBE7 pCLlOlEA PtsVglucose 'aroG trpEtb'DCBA aroACble JB102 / pBE6tkt, Pts' aroG tk tA pCLl OlEA trpE brDCBA aroACBLE Strains were grown with shaking in cultures in 30 ml flasks at 37 ° C. The medium used was Amisoy medium, which contained (per liter of distilled water): 7 g of Amisoy soy hydrolyzate, 14 g of K ^ HP04, 16 g of KH? P04, 5 g of (NH4) > S0, 15 g of glucose, 1 g of MgSO4"7H20, 0.27 g of FeClt and 1 drop of antifoam P-2000. The cultures were inoculated with culture cells seeded overnight growing in the seeded medium (identical to the medium Amisoy except that 15 g of yeast extract was replaced by 7 g of Amisoy and FeCl3 was omitted.) The initial D066o of the cultures was 0.2, IPTG was added to the cultures at time zero to induce high expron levels of the aroG genes. , trpEDCBA and aroACBLE on the different plasmids (the tktA gene in the plasmid pBE6tkt is under the control of its native promoter) .The pH of the cultures was maintained at 6.5 through the experiment by periodic additions of 45% KOH. samples of the cultures at specific intervals, and were mixed 1: 1 with 95% ethanol.The cells and debris were removed by centrifugation, and the supernatant was analyzed for tryptophan, two specific intermediates for the branch of the tryptophan from the aromatic pathway (anthranilate and indole), and intermediates from the common trunk of the aromatic pathway (refer to Example 7), by high-performance liquid chromatography. The experimental results are shown in Figures 9a, 9b and 9c. Three important comparisons were made. First, the PtsVglucose strain 'NF9 / pBE7 produced 4. 4X more tryptophan than its isogenic control strain PB103 / pBE7 (Kifir; »'). I,;? N 'VpHK' produced an i Crimen t o I *, r i pto fano, ns say, that did not accumulate significant levels of any of the intermediaries of the common pathway or the specific intermediaries of the chain of tryptophan anthranilate and indole. K to r (\ your II rui;; <? II; ¡i .st in l. (With ls * eu II,? Two preadolested ri h I Of »I '| < -il \ l | > I ofi (* 'and /, Second, the strain NF9 / pBE7, pCLlOlEA produced 1.3X more tryptophan than NF9 / pBE7 (which lacks the aroACBLE unblocking genes on pCLlOlEA) and 5.9X plus tryptophan that PB103 / pBE7 (which lacks both PtsVglucosa * mutations and the aroACBLE genes) (Figure 9a) The NF9 / pBE7 layer, pCLlOlEA also produced almost exclusively tryptophan, only a small accumulation of anthranilate being observed, and these results are also consistent with those presented in Examples 6 and 7. Third, in terms of the total potential tryptophan produced, strain NF9 / pBE7, pCLlOlEA functioned almost identically to strain JB102 / pBE6tkt, pCLlOlEA (Figure 9a) The last strain had been found to have a flow markedly improved carbon through the pathway of aromatic amino acids compared to cep as appropriate control, due to the presence of the combination of tktA and aroACBLE. However, the carbon flux increased in JB102 / pBE6tkt, pCLlOlEA does not fully convert to tryptophan; 55% accumulated as anthranilate and indole (Figure 9a). Since NF9 / pBE7, pCLlOlEA almost exclusively produced tryptophan, this strain is actually superior in this respect .. to the best previous strain JBl02 / pBE6tkt, pCLlOlEA. Because NF9 / pBE7, pCLlOlEA produced almost exclusively tryptophan, whereas JB102 / pBE6tkt, pCLlOlEA coaccumulated tryptophan, anthranilate and indole, the specific tryptophan production rates for the two strains are only equivalent if the tryptophan, anthranilate is considered and indole for strains JB102 / pBE6tkt, pCLlOlEA (Figure 9c). Like the results presented in Examples 6 and 7, the results presented in this example clearly show that the increased intracellular availability of PEP in the PtsVglucose 'strains increases the carbon committed to the aromatic amino acid pathway compared to the control strains Isogenic Pts.

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) P.R. Srinivasan and D.B. Sprinson (1959) "2-keto-3- deoxy-D-ara_? O-heptonic acid 7-phosphate synthetase" J. Biol. Chem. 234: 716-722. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (22)

1. A method for increasing the flow of carbon towards a metabolic pathway of a host cell capable of using a phosphotransferase transport system to transport carbohydrates, the method is characterized in that it comprises increasing the availability of PEP to the metabolic pathway by selecting a cell host that is phenotypically PtsVglucose ', and cultivate the host cell with an appropriate carbon source.

2. The method according to claim 1, characterized in that the selected host cell was modified to suppress all or substantially all of one or more genes selected from the group consisting of ptsl, ptsH and crr.

3. The method according to claim 1, characterized in that it further comprises transforming the host cell with recombinant DNA encoding transketolase so that the transketolase is expressed at increased levels relative to natural host cells.

4. The method according to claim 1, characterized in that it further comprises transforming the host cell with recombinant DNA encoding transaldolase, so that the transaldolase is expressed at increased levels relative to natural host cells.

5. The method according to claim 1, characterized in that it further comprises transforming the host cell with recombinant DNA encoding phosphoenolpyruvate synthase, so that the phosphoenolpyruvate synthase is expressed at increased levels relative to natural host cells.

6. The method in accordance with the claim 1, characterized in that it further comprises mutating the host cells to reduce or eliminate the activity of pyruvate kinase.

7. The method in accordance with the claim 6, characterized in that the activity of pyruvate kinase is reduced or eliminated in the host cell by introducing a mutation in one or more of the DNAs encoding pyruvate kinase, or in the promoter or other regulatory DNA that controls the expression of pyruvate kinase

8. A method for increasing the flow of carbon towards a metabolic pathway of a host cell capable of using a phosphotransferase transport system for the transport of carbohydrates, the method is characterized in that it comprises: a) selecting a host cell which is phenotypically PtsVglucose *; b) modifying the host cell of step a) to increase the expression of one or more enzymes selected from the group consisting of transketolase, transaldolase and phosphoenolpyruvate synthase; and c) culturing the host cell with an appropriate carbon tributary.

9. The method in accordance with the claim 1, characterized in that it further comprises the step of transferring to the host the DNA encoding one or more enzymes that catalyze reactions in the common aromatic pathway of the host cell.

10. The method according to claim 9, characterized in that the DNA codes for one or more enzymes selected from the group consisting of DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase (aroD), shikimate dehydrogenase ( aroE), shikimate kinase (aroL, aroK), EPSP synthase (aroA) and choris ato synthase (aroC).

11. The method according to claim 8, characterized in that it further comprises the step of transferring to the host cell the DNA encoding one or more enzymes that catalyze reactions in the common aromatic pathway of the host cell.

12. The method in accordance with the claim 8, characterized in that the DNA codes for one or more enzymes selected from the group consisting of DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroL) , aroK), EPSP synthase (aroA) and chorismate synthase (aroC).

13. A method for increasing the biosynthetic production of the host cell of a desired compound derived from an aromatic pathway of the host cell, the method is characterized in that it comprises: a) using a phenotypically selected host cell PtsVglucose *; b) transforming into said host cell the DNA encoding one or more enzymes that catalyze reactions in the aromatic pathway of the host cell; c) culturing the host cell with an appropriate carbon source; and d) producing the desired compound.

14. The method according to claim 13, characterized in that the phenotypically selected host cell PtsVglucose * is prepared by modifying a precursor host cell to suppress all or substantially all of one or more genes selected from the group consisting of ptsl, ptsH and crr.

15. The method in accordance with the claim 13, characterized in that the DNA encodes one or more enzymes selected from the group consisting of DAHP synthase (aroF, aroG, aroH), DHQ synthase (aroB), DHQ dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroL) , aroK), EPSP synthase (aroA) and chorismate synthase (aroC).

16. The method in accordance with the claim 13, characterized in that it further comprises transforming the host cell with recombinant DNA encoding one or more enzymes selected from the group consisting of transketolase, transaldolase and phosphoenolpyruvate synthase, so that the enzyme is expressed at increased levels relative to host cells natural

17. A method for obtaining PtsVglucosa * mutant cells, the method is characterized in that it comprises: a) selecting a host cell which utilizes a phosphotransferase transport system; b) causing the host cell to mutate by inactivating the phosphotransferase transport system by suppressing or inactivating selected genes; c) culturing the mutant host cells using glucose as a carbon source; and d) selecting mutant cells growing on glucose that have a specific growth rate of at least about 0.4 h "1.

18. The method according to claim 17, characterized in that the phosphotransferase transport system is inactivated by suppressing / activating one or more genes selected from the group consisting of the genes ptsl, ptsH and crr.

19. The method according to claim 17, characterized in that the mutant cells are selected in a continuous culture.

20. The method in accordance with the claim 13, characterized in that the desired compound is selected from the group consisting of tryptophan, tyrosine and phenylalanine.

21. The method in accordance with the claim 20, characterized in that the desired compound is tryptophan and the host cell PtsVglucose * is transformed with DNA encoding one or more genes selected from the group consisting of aroG, aroA, aroC, aroB, aroL, aroE, trpE, trpD, trpC, trpB, trpA, and tktA or tktB.

22. The method according to claim 1, characterized in that it further comprises increasing the expression of an enzyme selected from the group consisting of transketolase, transaldolase and phosphoenolpyruvate synthase in the host cell in relation to the expression of the enzymes in natural host cells, the expression The increase in such enzymes results in the introduction of one or more mutations in the DNA encoding such enzymes, or in the promoter or regulatory DNA that controls the expression of the genes encoding such enzymes.