MX2011008256A - Novel xylose tranporters and their applications thereof. - Google Patents

Abstract

Described is a novel transporter for five-carbon sugar best known as xylose, which corresponds to a protein obtained from the Escherichia coli bacterium, also referring to the DNA encoding for said protein (gene gatC, SEQ. ID NO: 13) and mutant protein with a better transport capacity (gen gatCS184L, SEQ. ID NO: 14). The present invention shows the capacity of the protein GatC (SEQ. ID NO: 15) for transporting xylose. Present is a mutant protein which transforms a Serine into a Leucine, a polar amino acid into a non polar amino acid, in the amino acid 184 (GatCS184L, SEQ. ID NO: 16) the improved capacity thereof to transport xylose is shown. As a consequence of the need of xylose transporting proteins that are energetically favourable, the present invention suggests the use of the product resulting from the genes gatC (SEQ. ID NO: 13) and gatCS184L (SEQ. ID NO: 14) as transporters in organisms searching the property of xylose transport or the improvement thereof for the utilization of said organism in the conversion of xylose-rich syrups, such as those obtained in the lignocelluloses hydrolysis as a raw material.

Description

NEW XILOSA TRANSPORTERS AND THEIR APPLICATIONS TECHNICAL FIELD The present invention relates to a novel sugar carrier of five carbones called xylose, corresponding to a protein found in the bacterium Escherichia coli and a mutant of said protein; likewise, it refers to the DNA encoding said protein and its mutant; to its expression in microorganisms transformed with said DNA and its growth from xylose using the expression of said transporter proteins; and the application of said transport proteins and the recombinant microorganisms that contain them, for the use of xylose in fermentation processes aimed at obtaining products of commercial interest from different sources of sugars containing xylose as the wide variety of hydrolysates of agroindustrial residues that exist worldwide.

BACKGROUND OF THE INVENTION In recent years the use of recombinant DNA technology and the systematic analysis of biological data have increased considerably, giving rise to the Metabolic Pathway Engineering (IVM), which is defined as the modification and / or introduction of new biochemical reactions for the direct improvement of cellular properties by recombinant DNA technology (Stephanopoulos, 2002). In particular, the development of new strains has been initiated by means of IVM, with the property of using the sugars present in hydrolysates of a wide variety of lignocellulosic materials and capable of producing metabolites with high productivity and yield (Clomburg and González, 2010; Trejo et al., 2010) The future depletion of oil reserves and various environmental aspects raise the need to develop sustainable processes to replace products that are obtained from it. Research at the international level focuses on reducing production costs of metabolites of interest, through: the use of new substrates; new fermentation and separation technologies: as well as new microorganisms capable of reaching high concentrations of product; high yields; and high productivities. Agroindustrial waste is a renewable source of great abundance and low cost, which can potentially be used as raw material for the manufacture of high volume chemical products. These materials are highly heterogeneous and are composed mainly of hemicellulose, cellulose, lignin and pectins, which for their bioconversion need to be hydrolyzed in soluble sugars, using thermochemical methods (hydrolysis with mineral acids, alkalis and mechanically) and / or enzymatic. The hemicellulosic fraction is composed mainly of pentoses, mainly the five-carbon sugar known as xylose and small fractions of arabinose (another sugar of five carbons) and hexoses (such as glucose, galactose and mannose, Martinez et al., 2001). After glucose, xylose is the most abundant monosaccharide in nature and is generally polymerized in the hemicellulosic fraction of plant material. Several of the microorganisms most commonly used in industrial fermentations, such as Saccharomyces cerevisiae, Bacillus subtilis, Lactococcus lactis, etc., lack the ability to use xylose (Hahn-Hagerdal, Karhumaa, Fonseca, Spencer-Martins, &Gorwa-Grauslund, 2007). The variety of microorganisms that have the capacity to metabolize both pentoses and hexoses is reduced, furthermore, there are no known wild microorganisms that can efficiently catabolize under anaerobic conditions xylose or arabinose, mixtures of glucose-xylose, xylose-glucose-arabinose or glucose- cellobiose in fermentation products (Hernández-Montalvo et al., 2001). Although the use of these wastes is attractive due to its high carbohydrate content, its current commercial use to obtain fermentation products is null, limited mainly by the scarce or no use of sugars present in the hemicellulosic fraction that many of the Used microorganisms do not efficiently convert into products.

Among the microorganisms that consume xylose is the Gram negative bacterium Escherichia coli, which has been genetically modified using metabolic pathway engineering techniques to produce metabolites with high productivity and yield. Compared with other organisms used at industrial level in fermentative processes E. coli has several advantages, such as: rapid growth in aerobiosis and anaerobiosis; ability to ferment pentoses; simple nutritional requirements; there is also a great knowledge of their physiology and ease of genetic manipulation. Due to the above, the development of new strains has started with IVM, with the capacity to grow in mineral media and produce alcohols (ethanol and butanol among others); diols (1,2 propanediol, 1,3 propanediol, 1,4-butanediol, 2,3-butanediol); organic acids (lactate, succinate, propionate, acetate, pyruvate and fumarate among others), polyhydroxyalkanoates, lipids and waxes among a wide variety of products (Steen et al., 2010; Orencio-Trejo et al., 2010; Zeng and Sabrá, 201 1; Steinbüchel, 2003) The transport of xylose in E. coli is carried out by two different systems reported: an ABC type system encoded by the xylF, xylG and xylH genes; and a simporte system encoded by the xylE gene (Sumiya et al., 1995). In a study for the production of xylitol, it was found that even with the elimination of both xylose transport systems, a basal transport probably carried out by non-specific or promiscuous transporters can be found (Khankal et al., 2008). According to the functional metabolic network of E. coli under fermentation conditions, for each mole of glucose (Glc) catabolized to pyruvate, which is a precursor metabolite of the fermentation products mentioned above, two moles of the ATP energy carrier are obtained, If half of the pyruvate generated is converted to acetic acid the yield is increased to 3 molATp / molGic- In the case of xylose (Xil) the yield is only 0.67 molATp / molxii, when E. coli catabolizes this sugar to pyruvate . In such a way that the functionality of the enzymes pyruvate format lyase (Pfl), fostoacil transferase (Pta) and acetate kinase (Ack) are essential in the growth of E. coli with xylose, this because the conversion of one mol of pyruvate in acetyl-CoA and in turn in acetate it generates an extra mole of ATP, being able to increase its yield up to 1.5 molATp / molxü. Consequently, the strains of homologous E. coli W31 10 interrupted in pflB can not grow in said pentose, because only 0.67 molATp / molx¡i are obtained. The ATP insufficiency was confirmed by inactivating the acetate kinase (ack) gene in W31 10 (with functional Pfl), the mutant was unable to grow in minimal medium supplemented with xylose under anaerobic conditions (Hasona et al., 2004), verifying the importance of ATP yield for growth in xylose as a carbon source. In the case of glucose, transport and phosphorylation is carried out by the phosphotransferase system (PTS) with the expenditure of one equivalent of ATP, while for xylose the cell spends two molecules of ATP, one for transport ( high affinity transporter type ABC encoded by xylF, xylG, xylH) and the second for the phosphorylation of xylose to generate xylose-5-phosphate, which can be metabolized by the pentose phosphate pathway and glycolysis to pyruvate (Lin, nineteen ninety six). In the case of the arabinose pentose, the internalization to the cell is carried out by a mechanism of simporte (arabinose / H +) by means of AraE, a transporter of low affinity and high capacity; this conserves an ATP molecule, which would be spent in the transport of pentoses by the ABC type transporter and both mutants (pfl and ack) grow in arabinose (Hasona et al., 2004). The ability to grow in arabinose, unlike xylose, is attributed to the transporter that is used for the internalization of that pentose is not dependent on ATP to energize it. In addition to their importance in growth, transporters are also important in other factors such as tolerance to fermentation products. Many of the product tolerance mechanisms involve the use of ATP, to export the product causing the toxicity from the cell (Russell, 1992).

These data reflect the importance of finding and / or developing novel transport systems of pentoses and other sugars, mainly xylose, whose internalization and phosphorylation imply a consumption of less than two ATPs per molecule of xylose.

As mentioned above, many of the organisms mostly used in industrial production are not able to use xylose as a carbon source. In many cases, this limitation is due to the lack of transport systems of xylose. Although it can transport xylose by low affinity systems, S. cerevisiae lacks an efficient transport system for xylose, which limits its application for the fermentation of hydrolysates of the hemicellulosic fraction of lignocellulosic residues. (Leandro et al., 2006; Fernandes and Murray, 2010). The use of a xylose transporter is of particular importance in this case to achieve that S. cerevisiae is modified for the use of xylose (Fernandes &Murray, 2010). In yeast, the production of organic acids is dependent on the yield of ATP by the mechanism of export of them (Maris et al., 2004), which highlights the importance of having xylose transporters and phosphorylation systems that only consume one mole of ATP to activate one mole of xylose.

Another product of industrial importance are polyhydroxyalkanoates (PHA's). In a study to find bacteria that could produce it from xylose, it was found that only 22% of 3, 152 bacterial isolates studied showed significant growth in xylose (Lopes et al., 2009). When studying the production of PHA's from xylose with these strains, it was found that in general they have a productivity 40% lower than with glucose. This was related to the theoretical ATP yield for one mole of 3-hydroxybutyrate monomer (3-HB): for xylose it is 3 moles of ATP per mole of 3-HB, while for glucose it is 7 moles of ATP per mole of 3-HB (Lopes et al., 2009). The above is another example of how useful it would be to be able to use a system of transport and phosphorylation of xylose that does not use two moles of ATP, if it were available.

B. subtilis is an organism of industrial importance widely used for the production of enzymes and other compounds by fermentation. B. subtilis has the enzymes necessary for the metabolism of xylose, however it lacks a gene that codes for a transport protein of xylose (Schmiedel &Hillen, 1996).

Through the expression of genes that allow the transport of xylose, it has been possible to generate strains capable of consuming it (Pariente T., 2007 Licenciatura Thesis).

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Graphs showing growth kinetics during the adaptive evolution of strain JU01 in mineral medium (AM2) supplemented with 120 g / L of xylose (A) and a bar chart of organic acid productivity at 48 h fermentation. P10, P1 1, P12 ... P15 are the passes made through the process of evolution (B).

Figure 2. Image showing a gel with the PCR products in colony of possible AgafC mutants. Lanes 1, 2, 3 negative control of wild strain. Lanes 5 and 6; 8 and 9; 1 1 and 12 products of 433 and 516 bp corresponding to the wild, JU01 and JU15 strains with the gatC gene (SEQ ID NO: 13) (inactivated).

Figure. 3 Bar graph showing the specific growth rate (μ) in AM2-xylose (40 g / L) of the strains JU15, JU15 LgatCS184L, JU01 and JU01 AgafC; and growth kinetics of strains JIM 5, JU01 gatC and JU 15 AgatCS184L.

Figure. 4. Image showing a gel with PCR products in colony of possible xyIE mutants. Lanes 1 -4 negative product of 1, 600 bp, lane 6 and 9: product of 1, 315 bp that correspond to the strains JU01 LxylE and JU15 LxyiE respectively.

Figure 5. Graph showing the specific growth rate (μ) in AM2-xylose (40 g / L) of the strains JU15, JU15 Axy / E, JU01 and JU01 Axy / E; and Graph showing kinetics of growth of strains JU 5, JU01 LxylE and JU15 xyIE.

Figure 6. Image showing a gel with the 5.4 kbp PCR products corresponding to the amplified gatYZABCD transcriptional unit of the JU15 strain.

Lanes 1-5 and 7-1 1 PCR products. Lane 6 molecular weight marker.

Figure 7. Image showing the enzymatic restriction products of the plasmid pGatC and pGaíCS184L with the enzymes Ncol and Mlul. Lanes 2 and 4 show the product of 9.3 kbp, which corresponds to the linearized vector and lanes 3 and 5 show the products of 5.4 and 3.9 kbp, which correspond to the empty vector and the cloned product respectively.

Figure 8. Image showing the vector obtained pGatC, through the ligation of the transcriptional unit gatYZABCD in the vector pCR2.1 TOPO.

Figure 9. Image showing the vector obtained pGaíCS184L through the ligation of the transcriptional unit gatYZABCD containing the mutation S184L in the vector pCR2.1 TOPO.

Figure 10. Graph showing the specific growth rate (μ) in AM2-xylose (40 g / L) of the strains JU 15 gatCS184L, JU15 AgarCS184L transformed with the plasmid pGafCS184L (JU15 AgafCS184L / pGaíCS184L) and the strain JU15 AgafCS184L transformed with plasmid pCR 2.1 as control.

Figure 11.- Image showing the obtaining of the plasmid pHCMgaíC through the ligation of the gatC gene (SEQ ID NO: 13) in the vector pHCMC05.

Figure 12.- Image showing the obtaining of the plasmid pHCMa / afCS184L through the ligation of the o / afCS184L gene (SEQ ID NO: 14) in the vector pHCMC05. Figure 13. Image showing a gel with the digestion of the vector obtained with the enzymes Xbal and Smal, the empty vector 8.3 Kpb and the release of the cloned fragment of 1.3 Kpb are shown.

Figure 14. Graph showing the kinetics of growth and consumption of xylose in mineral medium, anaerobic conditions and with xylose as the sole carbon source of the fí strain. subtilis 168 trp + transformed with the plasmid pHCMgafCS184L and graph showing the specific growth rate of the strain transformed with the pHCMgafC184L and the wild strain.

DETAILED DESCRIPTION OF THE INVENTION.

As can be seen from the background, the use of xylose as a carbon source is of great importance so that the use of lignocellulosic materials can be carried out as a carbon source in fermentative processes. Some of the microorganisms most used in industrial fermentations, such as S. cerevisiae, B. subtilis, Lactococcus lactis, etc., lack the ability to use xylose. As mentioned in the background, Escherichia coli is a Gram negative bacterium widely used in fermentative processes and has the ability to use xylose; however, the main transport system of xylose is dependent on ATP (Sumiya et al., 1995, Hasona et al., 2004). The ability of E. coli and other organisms to utilize xylose is largely dependent on the ATP yield that is obtained by metabolizing this sugar (Hasona et al., 2004); The above is particularly important under fermentation conditions (not aerobic). In the same way, the ATP yield is of particular importance for the mechanisms of tolerance to the same products that in many bioprocesses limit their productivity (Foster, 2004).

One of the limitations for the generation of microorganisms modified by genetic engineering to grant them the ability to use xylose is the existence of transport proteins of the same available for their use. In the present invention, by means of an original design and the use of metabolic engineering techniques, adaptive evolution and genomic sequencing, a new transporter capable of transporting xylose was identified and used, corresponding to a protein that, as far as is known, has not previously reported with the ability to transport xylose. This protein has been previously studied (Saier, Hvorup, and Barabote, 2005, Barabote and Saier, 2005) and was described as the membrane component (IIC) of the PTS system for galactitol (Gal).; polyalcohol of the six-carbon monosaccharide called galactose, also known as dulcitol) from E. coli. In a study on the evolution of bacterial PTS systems, it is mentioned that the IIC components of the GAT family seem to be able to function as secondary transporters or as group translocators dependent on phosphorylation by PTS systems (Saier, Hvorup, and Barabote, 2005 Barabote and Saier, 2005). It is also mentioned that it is possible that GatC or any protein of the IIC-Gat family works by both mechanisms depending on the availability of complementary proteins. This is based on the fact that the IIC proteins of the ASC-Gat superfamily (ASC: Ascorbate) have 12 transmembrane sections, unlike the 6 transmembrane sections that have other PTS systems (Saier et al., 2005). It has been proposed that PTS transporters with 12 transmembrane sections arose from permeases of 12 transmembrane sections of the superfamily of major facilitators (MFS, for its acronym in English: Major Facilitator Superfamily). The previous evidences pointed out that the IIC component of the PTS-Gat system could have a secondary conveyor function, however, this function had not been verified; nor was there evidence that he was able to transport xylose.

In a previous invention of the same working group of the inventors of the present invention, by means of an adaptive evolution protocol carried out by a process of subsequent transfers in mineral medium with xylose (Example 1), a new mutant strain was found whose phenotype it is faster for the consumption of xylose and its conversion into fermentation products (D and L lactate and ethanol) (Martínez et al, 2010. PCT WO201 1016706). In the present invention said mutant was studied by sequencing its genome and was found to have a mutation in the gatC gene (Example 2). Said mutation causes the change of a Serine by a Leucine in amino acid 184, a polar amino acid for a non-polar amino acid. To evaluate their contribution, the inactivation of the gatC gene (SEQ ID NO: 13) was carried out in strains JU01 (strain of E. coli genetically modified to produce only lactate D, Martínez et al, 2010. PCT WO201 1016706) and JU15 (strain derived from JU01, evolved to ferment faster xylose to D lactate, Martínez et al, 2010. PCT WO201 1016706), it was found that: a) the growth rate for strain JU15 was reduced 69% when inactivating gatC (SEQ ID NO: 13); b) for the JU01 strain the growth rate was reduced only 14%, and c) the base consumption used to neutralize the organic acids in the fermentation at 48 hours was reduced 71% and 31% respectively (see Example 3 and Example 4, and also figure 3). The above showed the participation of gatC (SEQ ID NO: 13) in the transport of xylose in the strains evaluated.

Recently, in a genomic study to study and increase the tolerance to isobutanol in evolved strains of E. coli, a mutation was found in the gatC gene (SEQ ID NO: 13) when the strains were subjected to evolution in the presence of isobutanol and xylose. This mutation only appeared in the populations subjected to adaptation in xylose and isobutanol and did not appear in the populations subject to adaptation in glucose and isobutanol (Minty et al., 201 1). The authors of the previous work do not explain how gatC (SEQ ID NO: 13) may be involved in the mechanism of tolerance to isobutanol, nor give gatC (SEQ ID NO: 13) the property of transporting xylose as demonstrated in the present invention. However, the occurrence of the mutation in gatC (SEQ ID NO: 13) in another study, independent of the one proposed in the present invention, demonstrates the importance of the participation of gatC (SEQ ID NO: 13) and the role that has in the adaptation to the consumption of xylose in evolved strains.

In Example 6 the difference in growth rate is shown in strains to which the two reported transport systems of xylose (XylE and XylFGH) have been inactivated (see Example 5). It was found that the strain having the wild version of the gatC gene (SEQ ID NO: 13) reduced its growth rate with respect to the control strain by 65%, the strain having the mutated version of gatC (gafCS184L, SEC. ID NO: 14) reduced only 26% in its growth rate. In this way it is demonstrated that the nonspecific transport of xylose that has been previously reported is largely carried out by gatC (SEQ ID NO: 13) and that the mutation therein, isolated in the present invention, increases its capacity to transport the xylose. Furthermore, it is demonstrated that cloning the gene in an expression vector completes an inactivated strain in gatC in its transport of xylose (See examples 7 and 8).

In such a way that in the present invention the use of gatC (SEQ ID NO: 13) or garCS184L (SEQ ID NO: 14) is proposed as transporters in organisms where it is sought to grant the transport property of xylose (Examples 9 and 10) or its improvement for the use of said organisms in the conversion of syrups rich in xylose, such as those obtained from the hydrolysis of lignocellulose as a raw material.

MATERIALS AND METHODS The microorganisms and plasmids used in the present invention are presented in Tables 1 and 2.

Table 1. Ceas of E. coli em leadas in this work Nomenclature: ? interrupt, inactivate pflB pyruvate gene lyase format AdhE alcohol dehydrogenase gene from E. coli frdA fumarate reductase gene xylFGH xylose transport system dependent on ATP E15 adaptive evolution strain 15 Table 2. Plasmids read in this work Both the PCR product and the plasmids used in this work analyzed on 1.0 -1.2% agarose gels by restriction patterns.

Culture and inoculum conditions They were carried out anaerobic cultures in fleakers (mini-fermentors) (Beall, Ohta, &L. O. Ingram, 1991) with a work volume of 200 mL. The temperature control at 37 ° C was maintained with a thermoswitch and a water bath. The pH was controlled in the range of 6.6 - 7.0 with the automatic addition of KOH 2 or 4N, while the stirring speed was maintained at 100 rpm using a cross-shaped magnet with a diameter of 3.81 cm. The experiments were carried out at least in duplicate and most of the cases in triplicate. The fleakers system used in the present work consists of the following elements: a) 6 mini-fermenters (300 mL nominal volume) with magnetic stirrer; b) temperature control, integrated by a thermal cycler and the water bath; c) pH control, integrated by six automatic controllers with valves for the release of the base and six pH electrodes; and d) stirring control, integrated by a magnetic plate (100 - 850 rpm).

The inoculum was incubated for 24 h until it reached an approx. from 1.5 to 2. The cultures were inoculated by centrifugation (4,000 rpm, 10 minutes at room temperature), conditions necessary to have an initial D06oo of approximately 0.1 (0.037 gDcw L) in the culture. Subsequently, the cells of the cell pack were transferred to each culture by suspending them in the medium.

Culture medium The composition of the mineral medium AM2 (Martínez et al., 2007) for the cultivation of Escherichia coli in fleakers (mini-fermenters) was: 2.63 g / L (NH4) 2HP04, 0.87 g / L NH4H2P04, 1.0 mL / L MgSO447H2O (1 M), 1.5 mL / L trace elements, 1.0 mL / L KCI (2M), 1.0 mL / L Betaine HCl (1 M), 100 mg / L citric acid. The medium was supplemented with different concentrations of xylose. The trace elements contain (in g / L): 1 .6 of FeCI3, 0.2 of CoCI26H2O, 0.1 of CuCI2, 0.2 of ZnCI24H20, 0.2 of Na2Mo04, 0.05 of H3BO3 and 0.33 of MnCI2.4H202.

Minimum medium for S. subtilis whose composition was (per liter) 4 g of (NH4) 2SO4; 5.32 g of K2HPO4; 6.4 g of KH2P0; 10 mg of Citric Acid; 0.4 g of MgSO4-7H2O; 5 mg of MnCl2; 40 mg of CaCl2; 30 g FeS04-7H20; supplemented with 2 or 10 g / L of sugar. The ammonium sulfate and the phosphate salts were sterilized together (by heat) and standard solutions of the other components of the medium were prepared, which were sterilized by filtration (0.22 μ) and added to the base medium before inoculating the crops.

Analytical methods Determination of cell concentration by spectrophotometry The optical density was measured at 600 nm (D060o) in a Beckman spectrophotometer (DU-70) (Beckman instrument, Inc. Fullerton, CA, USA) and converted to dry cell weight (DCW: dry cellular weigth). English), according to a calibration curve: 1 unit of ?? ß ?? equals 0.37 gDcw / l for E. coli and 0.35 for ß. subtilis. All samples were centrifuged (5,000 rpm at room temperature); the cell packet was discarded and the supernatant was frozen for further analysis. The specific rate of growth was determined during the exponential decrease phase of the microorganisms.

Determination of organic acids and sugars Calculation based on KOH base consumption (Organic acids) The consumption of base, by the control of pH, in a culture to obtain a kinetic of growth provides an approximate data of the amount of organic acids (lactic acid) present in the culture medium. The calculation for the determination of the concentration of organic acids (CA) is made based on the following known data: concentration of the base (CB); consumed base volume (VBA); initial work volume in the mini-fermentor (VT); and with Equation 1.

Equation 1 where: CA and CB, is given in molar concentration (mol / L) VBA and VT, is given in mL Quantification of organic acids and sugars by high performance liquid chromatography (HPLC) The determination of organic acids and sugars by HPLC was carried out by hydrochromatographic analysis with a 5 mM H2SO4 solution as a mobile phase at a flow of 0.5 mL / min on an Aminex HPX-87H (Biorad) column at 50 ° C. . The detection of the separated compounds was carried out simultaneously with a diode array detector (Waters 996) and a refractive index detector (Waters 410). The analysis and data processing was done with the "Millenium" system (Version 3.01 Waters). The internal and external temperatures of the column were adjusted to 45 and 50 ° C respectively. The supernatants of the samples to be analyzed were filtered with membranes of 0.45 pm and injected automatically with the help of the autoinjector (Waters 717). For the confirmation of the sugars and the products analyzed by HPLC, standards of xylose, glucose, arabinose, sodium acetate and organic acids were injected. The data obtained from the concentrations of each of the measured compounds were calculated using a calibration method-Interpolated from the same software.

EXAMPLES In the following examples, the present invention is better illustrated, but without restricting its scope.

The plasmid microorganisms and oligos used in the present invention are presented in the MATERIALS AND METHODS section or in the LIST OF SEQUENCES annex.

Example 1. - Adaptive evolution of strain JU01.

Strain JU01 (Martínez A., et al 2010 PCT WO201 1016706) underwent a process of adaptive evolution in xylose (40 g / L) seeking to improve its consumption capacity. The process was carried out in the following manner: 9 transfers were made and in these the growth rate and the production of organic acids were not significantly increased, evaluated by means of the base consumption used to control the pH. It was decided to use a concentration of 120 g / L of xylose, to increase the selection pressure and thus obtain mutants with better capacity to grow in xylose, 6 transfers were made and in the present invention it was possible to improve the growth and production capacities of organic acids of strain JU01 (Figure 1), a new strain of E. coli named JU15 was isolated (Martínez A et al., 2010 PCT WO201 1016706).

Example 2.- genome sequencing of strain JIM 5.

In the present invention, with the purpose of discovering which genomic changes were granted to the strain JU15 (Martínez A et al., 2010 PCT WO201 1016706) the ability to consume the xylose with greater speed was carried out comparative genomic sequencing of the Complete genome of the strain (CGS: comparative genome sequencing, Roche-NimbleGen). Through this sequencing, point mutations or SNP's (Single nucleotide polymorphisms) can be detected; deleted chromosomal regions, as well as gene duplications. Mutation in the gatC gene was found in the mutation map reported, to confirm this mutation, a PCR product was amplified using the gatCfor oligos (SEQ ID NO: 1) and gatCrev (SEQ ID NO: 2) and it was confirmed said mutation by Sanger type sequencing in the sequencing unit of the Biotechnology Institute of the UNAM. The mutation found is located in the PTSIIC domain of the GatC protein (SEQ ID NO: 15), in a transmembrane region, causes the change of a Serin by a Leucine in amino acid 184 of the protein, this is a polar amino acid by one not polar. The new gene was called gatCS184L (SEQ ID NO: 14).

Example 3.- Inactivation of the gatC gene.

To carry out the inactivation of the gatC gene (SEQ ID NO: 13), the PCR product was obtained with the regions of homology to said gene using the oligos gatCF (SEQ ID NO: 7) and gatCR (SEQ ID NO. : 8), using the pKD3 plasmid as a template (Datsenko, 2000). The product was electroporated into cells induced with arabinose and carrying the helper plasmid (pKD46). Several chloramphenicol-resistant colonies were obtained and analyzed by PCR with the oligo pairs a) gatC-CF forward (SEQ ID NO: 9) and C2 (SEQ ID NO: 12) and b) gatC-CR (SEQ ID NO: 10) and C1 (SEQ ID NO: 11), which amplify products corresponding to regions of 50 bp upstream of the gene and part of the sequence of the chloramphenicol resistance gene. When the iruption has been carried out, products of 433 bp are obtained with the pairs gatC-CR (SEQ ID NO: 10) and C1 (SEQ ID NO: 1 1), corresponding to the chromosomal region of gatC (SEQ ID NO: 13) plus the sequence of the chloramphenicol resistance gene, in the same way the gatC-CF (SEQ ID NO: 9) and C2 (SEQ ID. NO: 12) to check for the presence of the chloramphenicol resistance cassette at the gatC site, said pair of oligos amplifies a 516 bp product as shown in figure 2. The above indicates that the gatC gene (SEQ. NO: 13) was interrupted with the chloramphenicol resistance gene and its function has been inactivated. The strains JU01 gatC and JU15 gatCS184L were obtained using the previously detailed methodology Example 4.- Growth and consumption of xylose of strains lacking gatC.

As mentioned above, the gatC gene (SEQ ID NO: 13) is part of the PTSGat system whose function has been attributed to the transport of galactitol by a phosphoenolpyruvate (PTS) -dependent phosphotransferase system. There are no reports in the literature that mention that a pentose, such as xylose, can be transported by a PTS-type system, and there is no report where the ability to transport said pentose is attributed to the gatC gene (SEQ ID NO: 13). . To find out if gatC has any participation in the transport of xylose, the inactivation of gatC was carried out in the strains JU01 and JU15 and their effects were characterized. It was found that (figure 3): a) the growth rate for strain JU15 was reduced 69% by inactivating gatCS184L; b) for strain JU01 the growth rate was reduced by 14%; and c) the base consumption used to neutralize the organic acids in the fermentation at 48 hours was reduced 71% and 31% respectively (data not shown). With the results presented above, it is demonstrated that the gatC protein (SEQ ID NO: 12) has a role as a transporter of xylose and that in JU15, with the mutation generated by the adaptive evolution process, this participation is more important.

Example 5.- Elimination of the secondary transporter of xylose.

To carry out the inactivation of the xylE gene, the PCR product was obtained with the regions of homology to said gene using oligos xylEF (SEQ ID NO: 3) and xyIER (SEQ ID NO: 4) and the plasmid was used pKD3 as tempered (Datsenko and Wanner, 2000). This product was electroporated in cells induced with arabinose and carrying the helper plasmid (pKD46). Several colonies resistant to chloramphenicol were obtained and analyzed by PCR with oligos xyIEckF (SEQ ID NO: 5) and xyIEckR (SEQ ID NO: 6), which amplify a product of 1.6 Kpb, corresponding to regions of 50 bp river down and upstream, plus the gene xy / E (1.5 Kpb), when the interruption has been carried out, a product of 1.3 Kpb corresponding to the chloramphenicol resistance cassette and the adjacent regions already mentioned is obtained. By means of the previously detailed methodology, strains JU01 Axy / E and JU15 AxylE were obtained whose PCR products are observed in figure 4. Since JU01 and JIM 5 have inactivated the xylFGH genes (Table 1), the obtained strains have both systems of transport of xylose reported inactivated.

Example 6 - Growth and consumption of xylose of the strains with both xylose transport systems eliminated.

To investigate the participation of gatC as a transporter of xylose, strains JU01 AxylE and JU 5 AxylE were characterized with the two reported xylose transport systems eliminated, in their growth rate in xylose as the sole carbon source. As mentioned in the background, there is already experimental evidence of a basal transport of xylose in strains in which both transport systems have been eliminated. However, no study had described the involvement of gatC in the transport of xylose. The results show that strain JU01 AxylE had a 65% reduction in its growth rate with respect to strain JU01, whereas strain JU15 AxylE, which has the mutation in the gatC gene, (SEQ ID NO: 13) showed a reduction in the growth rate of only 26% compared to the strain to which the gene was not inactive (Figure 5). This shows that gatCS184L (SEQ ID NO: 14) has a more important participation than gatC (SEQ ID NO: 13), since the strain carrying said mutation grows at a considerable speed in xylose (0.13 IT1) even with both systems of inactivated xylose.

Example 7.- Cloning of the transcriptional unit gatYZABCD.

To check the functionality of the GatC proteins (SEQ ID NO: 15) and GatCS184L (SEQ ID NO: 16) as transporters of xylose, the cloning of their coding and regulatory sequences was carried out in an expression vector. To carry out the above, and to ensure the correct expression of the gatC gene (SEQ ID NO: 13) and gatCS184L (SEQ ID NO: 14) in the wild genomic context, the regions corresponding to the genes were amplified by PCR. gatYZABCD transcriptional units with their native promoter of strains JU01 and JIM 5, the latter has the gatCS184L gene (SEQ ID NO: 14) that carries the aforementioned mutation. The PCR product used was amplified with the oligos OpGatF (SEQ ID NO: 17) and OpGat® (SEQ ID NO: 18). The gel shown in figure 6, exemplifies the amplification of the transcriptional unit from chromosomal DNA of the strains JU01 and JIM 5. The obtained PCR product was ligated to the vector pCR2.1TOPO (Invitrogen®), using the Topo TA cloning Kit (Invitrogen®). Its analysis by restriction with Ncol and Mlul enzymes is shown in figure 7. The fragments obtained were 9.3 Kpb corresponding to the linearized vector and 5.4 and 3.9 kbp corresponding to the cloned product and the vector respectively. The obtained vectors, called pGafCS184L, are shown in figures 8 and 9.

Example 8.- Characterization of the functionality of GatCS184L protein to transport xylose.

To exemplify the capacity of the GatCS184L protein (SEQ ID NO: 16) in the transport of xylose, the JU15 AgaíC strain was used, which has a low growth rate in said pentose (see Example 4), and was transformed by electroporation ( Maniatis et al., 1982) with the vector pGa / CS184L (obtained in Example 7) that has cloned the transcriptional unit gatYZABCD with the mutated gene version (gatCS184L). It was characterized in mineral medium AM2 with xylose as the only carbon source and it was found to have a growth velocity 38% higher than strain JU15AgafC. The foregoing demonstrates that expression of the GatCS184L protein (SEQ ID NO: 16) complements the loss of the gatCS ^ 8AL · gene (SEQ ID NO: 14); however, it was expected to have a growth rate similar to strain JU15. The above could not be achieved due to the metabolic load that occurred when transforming the strain with the plasmid; this is experimentally demonstrated by transforming JU15AgaíC with the empty vector, causing the bacterium not to grow under the conditions evaluated (Experiment called Control in Figure 10), when it was expected to grow at a low speed, similar to that of strain JU15AgafC.

Example 9.- Cloning of gatC and gatCS184L into an expression vector for B. subtilis.

Cloning of gatC (SEQ ID NO: 13) and gatCS184L (SEQ ID NO: 14) was carried out in the expression vector for B. subtilis pHCMC05 (Nguyen et al., 2005), the oligos were designed to (SEQ ID NO: 19) and gat_1 R (SEQ ID NO: 20) to amplify the gatC genes (SEQ ID NO: 13) and gatCs184L (SEQ ID NO: 14) of strains JU01 and JU15 respectively. When designing the oligos, the recognition sites for the Xbal and Smal enzymes were added at their 5 'end. PCR products were amplified containing only the coding region of the gatC (SEQ ID NO: 13) and gatCS184L (SEQ ID NO: 14) genes of strains JU01 and JU15 respectively, with oligos gat_1 F (SEQ ID. NO: 19) and gat_1 R (SEQ ID NO: 20). Both the PCR products and the vector pHCMC05 were digested with the enzymes Xbal and Smal. Ligation was performed using the T4 DNA ligase enzyme and electroporated into electrocompetent E. coli cells. The correct cloning of the genes was verified by digestion with the enzymes Xbal and Smal and by sequencing with the oligos mentioned above. Figure 13 shows the gel with the digestion of the vector obtained with the enzymes Xbal and Smal, the empty vector is shown (8.3 Kbp) and the release of the cloned fragment (1.3 kbp). The vector pHCMgatC carries a copy of the gatC gene (SEQ ID NO: 13) for its expression in B. subtilis under the IPTG-inducible pSpac promoter (Figure 11), the vector pHCMgafCS184L carries a copy of the gafCS184L gene (SEQ ID NO. : 14) under the pSpac promoter inducible by IPTG (Figure 12).

Example 10.- Characterization of the use of pHCMgafCS184L to give B. subtilis the utilization capacity of xylose.

The strain of B. subtilis 168 trp + was transformed with the vector pHCMgaíCS184L using a standard transformation metholodology by natural competition (Harwood and Cutting 1990), in figure 14 the kinetics of growth and consumption of xylose in mineral medium, anaerobic conditions are shown and with xylose as the sole carbon source. It can be seen that when transformed with the vector expressing the GatCS184L protein (SEQ ID NO: 16) the strain acquires the ability to grow and ferment the xylose, the untransformed strain did not grow and was not able to use the xylose in the conditions evaluated as previously reported (Pariente T., 2007).

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Claims (7)

1. A purified and isolated fragment of DNA encoding the GatCS184L protein capable of transporting xylose into a microorganism using only 1 ATP molecule per transported xylose molecule, characterized in that it has a nucleotide sequence SEQ.ID.NO: 14.

2. A protein that, when expressed in a recombinant microorganism, confers the capacity to transport xylose into said recombinant microorganism using only 1 molecule of ATP per molecule of transported xylose, characterized in that it has an amino acid sequence SEQ.ID.NO: 15.

3. The use of an isolated and purified DNA fragment selected from the group consisting of SEQ.ID.NO.13 (gatC) and SEQ.ID.NO.14. { gatCS184L) in an expression vector to generate recombinant microorganisms capable of transporting xylose into said recombinant microorganism using only 1 molecule of ATP per molecule of transported xylose, where said capacity is greater when compared with the same microorganism before being transformed with said expression vector.

4. An expression vector that when transformed into a microorganism increases the transport capacity of xylose into said microorganism using only 1 ATP molecule per transported xylose molecule, characterized in that it comprises a DNA fragment selected from the group consisting of: the DNA fragment of claim 1 and a DNA fragment with nucleotide sequence SEQ.ID.NO: 13, inserted in operative form that allows the expression of said DNA fragment, where said capacity is greater when compared to the same microorganism before of being transformed with said expression vector.

5. A recombinant microorganism that has the ability to transport xylose to the cell interior using only 1 molecule of ATP per molecule of xylose transported, characterized in that it comprises the expression vector of claim 4, wherein said capacity is greater when compared to the same microorganism before being transformed with said expression vector.

6. The recombinant microorganism of claim 5, characterized in that the capacity to transport xylose to the cellular interior of the microorganism before its transformation is null.

7. A fermentation process, characterized in that it comprises the step of incubating the microorganism of claim 5 in the presence of xylose as the main carbon source.