MXPA01000490A

MXPA01000490A - Process and materials for production of glucosamine

Info

Publication number: MXPA01000490A
Application number: MXPA/A/2001/000490A
Authority: MX
Inventors: Alan Berry; Richard P Burlingame; James R Millis
Original assignee: Arkion Life Sciences Llc
Priority date: 1998-07-15
Filing date: 2001-01-15
Publication date: 2002-07-25

Abstract

The present invention relates to a method and materials for producing glucosamine by fermentation of a genetically modified microorganism. Included in the present invention are genetically modified microorganisms useful in the present method for producing glucosamine, as well as recombinant nucleic acid molecules and the proteins produced by such recombinant nucleic acid molecules.

Description

MATERIALS AND PROCESS FOR THE PRODUCTION OF GLUCOSAMINE FIELD OF THE INVENTION The present invention relates to a method for producing glucosamine by fermentation. The present invention also relates to genetically modified strains of microorganisms useful for producing glucosamine.

BACKGROUND OF THE INVENTION Aminosugars are usually found as monomer residues in polysaccharides and complex oligosaccharides.

Glucosamine is an amino derivative of simple sugar, glucose.

Glucosamine and other aminoasugars are important constituents of many natural polysaccharides. For example, polysaccharides containing aminoazucares can form structural materials of cells, analogous to structural proteins. Glucosamine is manufactured as a nutraceutical product wapplications in the treatment of osteoarthritic conditions in animals and humans. The market for glucosamine is experiencing tremendous growth. In addition, the significant erosion of the world market price for glucosamine is not expected. Glucosamine is currently obtained by acid hydrolysis of chitin, a complex carbohydrate derived from N- J ... i? X X R acetyl-D-glucosamine. Alternatively, glucosamine can also be produced by acid hydrolysis of variously acetylated chitosans. These processes also suffer from productions of deficient products (in the range of 50% conversion of substrate to glucosamine). In addition, the availability of raw material (ie, a source of chitin, such as crab shells) is becoming increasingly limited. Therefore, there is a need in the industry for an effective cost method for producing high yields of glucosamine for commercial use and sale.

SUMMARY OF THE INVENTION One embodiment of the present invention relates to a method for producing glucosamine by fermenting a microorganism. This method includes the steps of: (a) cultivating in a fermentation medium a microorganism having a genetic modification in a metabolic pathway of aminosugar; and (b) recovering a product produced from the cultivation step, which is selected from the group of glucosamine-6-phosphate and glucosamine. Such a metabolic pathway of aminosugar is selected from the group of one path to convert glucosamine-6-phosphate to another compound, a path to synthesize glucosamine-6-phosphate, a path to transport glucosamine or glucosamine-6-phosphate outside the microorganism, a path to transport glucosamine to the microorganism, and a competing path > wsubstrates included in the production of glucosamine-6-phosphate. The fermentation medium includes assimilable sources of carbon, nitrogen and phosphate. In a preferred embodiment, the microorganism is a bacterium or yeast, and more preferably, a bacterium of the genus Escherichia, and even more preferably, Escherichia coli. In one embodiment, the product can be recovered by recovering intracellular glucosamine-6-phosphate from the microorganism and / or recovering extracellular glucosamine from the fermentation medium. In In further embodiments, the recovery step may include purifying glucosamine from the fermentation medium, isolating glucosamine-6-phosphate from the microorganism, and / or dephosphorylating glucosamine-6-phosphate to produce glucosamine. In one embodiment, at least about 1 g / L of the product is recovered. In still another embodiment, the cultivation step includes the step of maintaining the carbon source at a concentration of from about 0.5% to about 5% in the fermentation medium. In another embodiment, the cultivation step is carried out at a temperature of from about 28 ° C to about 37 ° C. In yet another embodiment, the cultivation step is carried out in a fermenter. In an embodiment of the present invention, the microorganism has a modification in a gene encoding a protein that includes, but is not limited to, deacetylase N-25 acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase, llNag enzyme specific for? / - acetyl-glucosamine, glucosamine-6-phosphate synthase, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, PEG enzyme IIG | C: glucose 5 PTS, IEM, P / lllMan PEP: mannose PTS, and / or a phosphatase. In another embodiment, the genetic modification includes a genetic modification that increases the action of glucosamine-6-phosphate synthase in the microorganism. Such genetic modification includes the transformation of the microorganism with a Recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase to increase the action of glucosamine-6-phosphate synthase and / or to overexpress the glucosamine-6-phosphate synthase by the microorganism. In one embodiment, the recombinant nucleic acid molecule is linked Operatively to a transcription control sequence. In a further embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism. In yet another embodiment, the recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase has a genetic modification that increases the action of the synthase. Such genetic modifications can result in inhibition of the reduced glucosamine-6-phosphate product of glucosamine-6-phosphate synthase, for example. In one embodiment, a recombinant nucleic acid molecule of the present invention comprising a sequence nucleic acid encoding a glucosamine-6-phosphate synthase § | 3 ^ g ^ gtegs4i s ^^^^^^^ j ^^^^^ ii ^^^ encodes an amino acid sequence SEQ ID NO: 16. In another embodiment, such a recombinant nucleic acid molecule comprises a nucleic acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Preferred recombinant nucleic acid molecules of the present invention include pKLN23-28, nglmS-282Δ84 and nglmS -28? 8 o. Also included in the present invention are recombinant nucleic acid molecules encoding a glucosamine-6-phosphate synthase comprising a genetic modification that increases the action of glucosamine-6-phosphate synthase (i.e., a homolog of the synthase). of glucosamine-6-phosphate). Such genetic modification can reduce the inhibition of the glucosamine-6-phosphate product of the synthase, for example. In one embodiment, such genetic modification in a recombinant nucleic acid molecule of the present invention encoding a glucosamine-6-phosphate synthase results in at least one amino acid modification selected from the group of an addition, substitution, elimination and / or derivation. of an amino acid residue is a position of the amino acid sequence in the modified (i.e., homologous) protein that corresponds to one or more of the following amino acid positions in the amino acid sequence SEQ ID NO: 16: lle (4) , lle (272), Ser (450), Ala (39), Arg (250), Gly (472), Leu (469). In another embodiment, such an amino acid modification is selected from the group of a substitution of: (a) an amino acid residue having an aliphatic hydroxyl side group for Ile (4); (b) an amino acid residue having an aliphatic hydroxyl side group for Ile (272); (c) an amino acid residue having an aliphatic side group for Ser (450); (d) an amino acid residue having an aliphatic hydroxyl side group for Ala (39); (e) an amino acid residue having a sulfur-containing side group for Arg (250); (f) an amino acid residue having an aliphatic hydroxyl side group for Gly (472); (g) an amino acid residue having an aliphatic side group for Leu (469); and (h) combinations of (a) - (g). In still another embodiment of the present invention, an amino acid modification as described above is preferably a substitution selected from the group of: lle (4) to Thr, lle (272 to Thr), Ser (450) to Pro, Ala (39 ) to Thr, Arg (250) to Cys, Gly (472) to Ser, Leu (469) to Pro, and combinations thereof. In another embodiment, a genetically modified recombinant nucleic acid molecule of the present invention comprises a nucleic acid sequence encoding the glucosamine-6-phosphate synthase comprising an amino acid sequence selected from the group of SEQ ID NOJ 9, SEQ ID NO. : 22, SEQ ID NO: 25, SEQ ID NO: 28 OR SEQ ID NO: 31. In another embodiment, a genetically modified recombinant nucleic acid molecule of the present invention comprises a nucleic acid sequence selected from the group of SEQ ID NO: 1 7, SEQ ID NO: 1 8, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 30 The preferred genetically modified recombinant nucleic acid molecule of the present invention includes pKLN23-49, pKLN23-54, pKLN23-149, pKLN23-151, nglmS-492? 84, nglmS-49? 83o, nglmS-542? 84, nglmS -54183o, nglmS-1242184, nglmS-124183o, nglmS-1492? 8, nglmS-149? 83o, nglmS-1512184, nglmS-1 51? 83o- Another embodiment of the present invention relates to a glucosamine-6 synthase. phosphate having glucosamine-6-phosphate synthase action, such synthase being encoded by a nucleic acid sequence having a genetic modification resulting in increased glucosamine-6-phosphate synthase action. Such a nucleic acid sequence has been described above with respect to recombinant nucleic acid molecules of the present invention. Yet another embodiment of the present invention relates to a method for producing glucosamine by fermentation, such method comprising: (a) growing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a genetically modified microorganism having an action of Increased glucosamine-6-phosphate synthase, wherein the genetically modified microorganism is produced by a process comprising the steps of: 1) generating modifications in an isolated nucleic acid molecule comprising a nucleic acid sequence encoding glucosamine synthase -6-phosphate to create a plurality of modified nucleic acid sequences; (2) transforming the microorganism with the modified nucleic acid sequences to produce genetically modified microorganisms; (3) classify microorganisms ^^^ ¡^ ^^^^^^^^^^ t | ^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ genetically modified by glucosamine-6-phosphate synthase action; and, (4) select the genetically modified microorganisms that have increased the action of glucosamine-6-phosphate synthase; and (b) recover the product. The cultivation stage produces a product selected from the glucosamine-6-phosphate and glucosamine group of the microorganism. In another embodiment, a microorganism of the present invention has an additional genetic modification in genes encoding deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase,? / - acetyl-glucosamine specific llNag enzyme, phosphoglucosamine mutase , glucosamine-1-phosphate acetyltransferase -? / - acetylglucosam i na-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C PEP: glucose PTS, IEM, P / lllMan PEP: mannose PTS, where the modification reduces the action of such proteins. In another embodiment, a microorganism of the present invention has a further genetic modification in a gene encoding a phosphatase, wherein the modification increases the action of the phosphatase. In a preferred embodiment, a microorganism of the present invention has an additional genetic modification in the genes encoding the following proteins, deacetylase N-acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase, and? / - acetyl-specific llNag enzyme. glucosamine, such modifications include, but are not limited to, an elimination of at least a portion of such genes. Another embodiment of the present invention relates to a ^^^^^ j ^ ^^^ ¡jg * fe¡¡ ^^^^^^^^ j ^^^^^^^^^^^^^ J ^^? method for producing glucosamine by fermentation which includes the steps of (a) cultivating an Escherichia coli transformed with a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate to produce a product; and (b) recover the product. The product includes intracellular glucosamine-6-phosphate which is recovered from Escherichia coli and / or extracellular glucosamine which is recovered from the fermentation medium. In this modality, the nucleic acid molecule Recombinant increases the expression of glucosamine-6-phosphate synthase by Escherichia coli, and is operably linked to a transcription control sequence. In one embodiment, the recombinant nucleic acid molecule comprises a genetic modification that reduces the inhibition of the glucosamine-6-phosphate product of the glucosamine-6-phosphate synthase. In another embodiment, Escherichia coli has an additional genetic modification in at least one gene selected from the group of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmU, glmS, ptsG and / or phosphatase gene. In yet another embodiment, the additional modification comprises an elimination nagA, nagB, nagC, nagD, nagE and a mutation in manXYZ where the modification results in reduced enzymatic activity of deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase and lNa9 specific enzyme? / - acetyl-glucosamine. Still in another embodiment of the present invention, refers to a microorganism to produce glucosamine by a biosynthetic process. The microorganism is transformed with a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase, wherein the recombinant nucleic acid molecule is operably linked to a transcription control sequence. The recombinant nucleic acid molecule further comprises a genetic modification that increases the action of glucosamine-6-phosphate synthase. The expression of the recombinant nucleic acid molecule increases the production of glucosamine by the microorganism. In a preferred embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism. In yet another modality, the microorganism has at least one additional genetic modification in a gene encoding a protein selected from the group consisting of deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase, llNag specific enzyme? / - acetyl glucosamine , phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - acetyl Iucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C PEP: glucose PTS, IEM, P / lllMan PEP: mannose PTS, where the genetic modification reduces the action of the protein. In another embodiment, the microorganism has a modification in a gene encoding a phosphatase, wherein the genetic modification increases the action of the phosphatase. In yet another embodiment, the microorganism has a modification in the genes encoding deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase and N-specific lNa9 enzyme acetyl-glucosamine, where the genetic modification reduces the enzymatic activity of the protein. In a preferred embodiment, the genetic modification is a deletion of at least a portion of the genes. In a further embodiment, the microorganism is Escherichia coli having a modification in a gene selected from the group of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmU, glmS, ptsG and / or phosphatase gene. In one embodiment, such Escherichia coli has a deletion of the nag regution genes and in another embodiment, such Escherichia coli has an elimination of the nag regution genes and a genetic modification in the manXYZ genes in such a way that the proteins encoded by the genes ManXYZ genes have reduced the action. Yet another embodiment of the present invention is a microorganism as described above that produces at least about 1 g / L of glucosamine when cultured for about 10-60 hours at from about 28 ° C to about 37 ° C at a cell density of at least about 8 g / L by the weight of the dry cell; in a pH 7.0 fermentation medium comprising: 14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3Citrate-2H2O, 5 g / L (NH4) 2SO4, 20 g / L of glucose , 10 mM MgSO4, 1 mM CaCl2, and from approximately 0.2 mM to approximately 1 mM of IPTG. Another embodiment of the present invention is a microorganism for producing glucosamine by a process - ^ - *** &biosynthetic, which includes: (a) a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase operably linked to a transcription control sequence; and, (b) at least one genetic modification in a gene encoding a protein selected from the group of deacetylase? / - acetylglucosamine-6-phosphate, deaminase glucosamine-6-phosphate, enzyme specific llNag of? / - acetyl-glucosamine, mutase of phosphoglucosamine, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C of PEP: glucose PTS, IEM, P / lllMan of PEP: mannose PTS, where genetic modification reduces the action of protein. In another embodiment, the microorganism includes at least one genetic modification in a gene encoding a phosphatase, wherein the genetic modification increases the action of the phosphatase. The expression of the recombinant nucleic acid molecule increases the action of the glucosamine-6-phosphate synthase in the microorganism. In an addition mode, the recombinant nucleic acid molecule is integrated into the genome of the microorganism.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION Figure 1 is a schematic representation of the trajectories for the biosynthesis and catabolism of glucosamine and N-acetyl-glucosamine and their phosphorylated derivatives in Escherichia coli: Figure 2 is a schematic representation of the modifications to trajectories related to amino sugar metabolism for the overproduction of glucosamine in Escherichia coli. Figure 3 is a schematic representation of the production of strains of Escherichia coli containing combinations of the mutations of manXYZ, ptsG and Anag. Figure 4 is a line graph illustrating the glucosamine accumulation effects of additional feed glucose and ammonium sulfate to the cultures. Figure 5 is a line graph showing that the glucosamine-6-phosphate synthase is inhibited by glucosamine-6-phosphate and glucosamine. Figure 6 is a line graph illustrating the inhibition of the product of glucosamine-6-phosphate synthase activity in mutant glmS clones. Figure 7 is a schematic representation of the strategy for constructions of strains of Escherichia coli containing mutant glmS genes. Figure 8 is a line graph illustrating inhibition of the glucosamine-6-phosphate synthase product in strains of Escherichia coli with integrated mutant glmS genes. Figure 9 is a line graph showing the production of glucosamine in strains of Escherichia coli with mutant glmS genes. Figure 10 is a line graph showing the inhibition of glucosamine-6-phosphate synthase in strains that produce iiit.JA.-ii ^ .. itAjat., .- - .. ... ü-ú »á., ír.kÁ4 glucosamine. Figure 1A is a line graph showing the thermal stability at 45 ° C of glucosamine-6-phosphate synthase in strains that produce glucosamine. Figure 1 1 B is a line graph illustrating the thermal stability at 50 ° C of glucosamine-6-phosphate synthase in strains that produce glucosamine. Figure 12 is a line graph showing the effect of IPTG concentration on glucosamine production. Figure 13 is a line chart demonstrating the effects of IPTG concentration on glucosamine production. Figure 14A is a line graph illustrating glucosamine production and growth by the strain that produces glucosamine 21 23-54 at 30 ° C. Figure 14B is a line graph illustrating glucosamine production and growth by the strain that produces glucosamine 2123-54 at 37 ° C. Figure 15A is a line graph showing the production of glucosamine by strain 2123-49 at 30 ° C. Figure 15B is a line graph showing the production of glucosamine by strain 2123-124 at 30 ° C. Figure 16A is a line graph illustrating the production of glucosamine by the strain that produces glucosamine in a limited glucose fermenter at 37 ° C. 25 Figure 16B is a line graph illustrating the glucosamine production by the strain that produces glucosamine in a limited glucose fermenter at 30 ° C. Figure 16A is a line graph illustrating the production of glucosamine by the strain that produces glucosamine in a fermentor in excess of glucose at 30 ° C.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a biosynthetic method for producing glucosamine. Such a method includes the fermentation of a genetically modified microorganism to produce glucosamine. The present invention also relates to genetically modified microorganisms, such as strains of Escherichia coli, useful for producing glucosamine. As used herein, the terms glucosamine and / V-glucosamine can be used interchangeably. Similarly, the terms glucosamine-6-phosphate and? / - glucosamine-6-phosphate can be used interchangeably. Glucosamine can also be abbreviated as GlcN and glucosamine-6-phosphate can also be abbreviated as GlcN-6-P. The new method of the present invention for the production of glucosamine by fermentation is inexpensive and can produce a glucosamine production that exceeds the cost production of glucosamine produced by the current hydrolysis methods. In addition, when using a genetically modified microorganism as described herein, the method tUgjn ^ fc * .. of the present invention can be easily modified to adapt to particular problems or change the needs related to the production of glucosamine. Aminosugars,? / - acetylglucosamine (GIcNAc) and glucosamine (GlcN) are fundamentally important molecules in microorganism, because they are the precursors for the biosynthesis of larger macromolecules, and in particular, glycoconjugates (ie, macromolecules containing oligosaccharide chains). united covalently). For example, in Escherichia coli; N-acetylglucosamine and glucosamine are precursors for two macromolecules of the cell coat, peptidoglycan and lipopolysaccharide. Mutations that block the biosynthesis of peptidoglycan or lipopolysaccharide are lethal, resulting in loss of integrity of the cell envelope and ultimately in cell lysis. One embodiment of the present invention relates to a method for producing glucosamine by fermenting a microorganism. This method includes the steps of (a) culturing in a fermentation medium a microorganism that has a genetic modification in an amino sugars metabolic path that includes: a path to convert glucosamine-6-phosphate to another compound, a path to synthesize the glucosamine-6-phosphate, a path to transport glucosamine or glucosamine-6-phosphate out of the microorganism, a path to transport glucosamine to the microorganism, and a path that competes with substrates included in the production of glucosamine-6- phosphate to produce a product that may include intracellular glucosamine-6-phosphate and / or extracellular glucosamine of the microorganism; and (b) recovering the product, by recovering intracellular glucosamine-6-phosphate from the microorganism and / or recovering the extracellular glucosamine from the fermentation medium. The fermentation medium includes assimilable sources of carbon, nitrogen and phosphate. Another embodiment of the present invention relates to a method for producing glucosamine by fermentation. Such method includes the steps of: (a) cultivating in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate an Escherichia coli transformed with a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase operably binds to a transcription control sequence; and (b) recovering the product selected from the glucosamine-6-phosphate and glucosamine group. The recombinant nucleic acid molecule increases the expression of glucosamine-6-phosphate synthase by Escherichia coli. In a further embodiment, the recombinant nucleic acid molecule comprises a genetic modification that reduces the inhibition of the glucosamine-6-phosphate product of the glucosamine-6-phosphate synthase. In still another embodiment, Escherichia coli has an additional genetic modification in at least one gene selected from the group of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmil, glmS, ptsG and / or phosphatase gene . To produce significantly high glucosamine productions by the fermentation method of the present invention, a microorganism is genetically modified to increase glucosamine production. As used herein, a genetically modified microorganism, such as Escherichia coli, has a genome that is modified (i.e., mutates or changes) in its normal (i.e. wild type or naturally occurring) form. The genetic modification of a microorganism can be carried out using conventional strain development and / or molecular genetic techniques. Such techniques are generally described, for example, in Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. The reference Sambrook ef al., Is incorporated herein by reference in its entirety. Additionally, techniques for the genetic modification of a microorganism are described in detail in the Examples section. A genetically modified microorganism may include a natural genetic variant as well as a microorganism in which the nucleic acid molecules have been inserted, deleted or modified (i.e., mutated, eg, by insertion, removal, substitution, and / or reversal of nucleotides), such that such modifications provide the desired effect within the microorganism. According to the present invention, a genetically modified microorganism includes a microorganism that has been modified using recombinant technology. As used herein, genetic modifications that result in a reduction in gene expression, in gene function, or in the > . i I t & > * function of the gene product (ie, the protein encoded by the gene) can be referred to as inactivation (complete or partial), elimination, interruption, blocking or sub-regulation of a gene. For example, a genetic modification in a gene that results in a reduction in the function of the protein encoded by such a gene, may be the result of a complete elimination of the gene (ie, the gene does not exist, and therefore the protein it does not exist), a mutation in the gene that results in incomplete or no translation of the protein (for example, the protein is not expressed), or a mutation in the gene that reduces or ends the natural function of the protein (for example, a protein that has reduced or no activity or enzymatic action is expressed). Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, activation, increase, addition, or supra-regulation of a gene. In one embodiment of the present invention, a genetic modification of a microorganism increases or reduces the action of a protein included in an amino sugar metabolic pathway according to the present invention. Such a genetic modification includes any type of modification and specifically includes modifications made by recombinant technology and by classical mutagenesis. For example, in one embodiment, a microorganism of the present invention has a genetic modification that increases the action of glucosamine-6-phosphate synthase. It should be noted that the reference to the increase in the share (or activity) of glucosamine-6-phosphate synthase and other enzymes treated herein refers to any genetic modification in the microorganism in question, which results in increased functionality of the enzymes and includes higher activity of the enzymes (e.g. specific or enzymatic activity in vivo), reduced inhibition or degradation of enzymes and overexpression of enzymes. For example, a number of copies of the gene may be increased, expression levels may be increased by the use of a promoter that gives higher expression levels than those of the native promoter, or a gene may be altered by genetic engineering or classical mutagenesis to increase the action of an enzyme. Examples of nucleic acid molecules encoding glucosamine-6-phosphate synthase that has been genetically modified to increase the action of glucosamine-6-phosphate synthase are described in the Examples section. Similarly, the reference to the reduction of the action of the enzymes treated herein refers to any genetic modification in the microorganism in question that results in reduced functionality of the enzymes and includes reduced activity of the enzymes (eg, specific activity ), increased inhibition or degradation of the enzymes and a reduction or elimination of the expression of the enzymes. For example, the action of an enzyme of the present invention can be reduced by blocking or reducing the production of the enzyme, reducing the enzymatic activity, or inhibiting the activity of the enzyme. Blocking or reducing the production of an enzyme a ^^^^^^^ á ^ ?? a ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^ i ^^^^^^^^ iji ^^^^^^^^ fafc may include placing the gene encoding the enzyme under the control of a promoter that requires the presence of a composed of induction in the growth medium. By setting the conditions so that the inducer is reduced from the medium, the expression of the gene encoding the enzyme (and therefore, an enzyme synthesis) could be cut. Blocking or reducing the activity of an enzyme could also include using an excision technology approach similar to that described in the U.S. Patent. No. 4,743,546 incorporated herein by reference. To use this approach, the gene encoding the enzyme of interest is cloned among the specific genetic sequences that allow specific controlled excision of the genome gene. The cleavage could be indicated by, for example, a change in cultivation temperature of the culture, as in the U.S. Patent. No. 4,743,546 or by some other nutritional or physical signal. An "aminosugar" is an amino derivative of a saccharide (eg, a saccharide having an amino group instead of a hydroxyl group). According to the present invention, a metabolic pathway of aminosugar is any biochemical pathway included in, or affecting, the biosynthesis, anabolism or catabolism of an aminosugar. As used herein, metabolic aminosugar pathways include pathways included in the transport of aminoazucars and their precursors within and outside a cell, and may also include biochemical pathways, which compete with substrates included in biosynthesis or catabolism of an aminosugar. For example, the immediate precursor to one of the aminoazouzas formed earlier is fructose-6-phosphate (F-6-P), which, in a biochemical reaction with glutamine (Gln, the donor of the amino group), forms glucosamine-6 -phosphate. Fructose-6-phosphate is also an intermediate compound in the path of glycolysis. Therefore, the glycolytic path competes with the biosynthetic pathway of glucosamine-6-phosphate when competing for a substrate, fructose-6-phosphate. In addition, glucosamine-6-phosphate can be converted into other aminoasugars and form constituents in several macromolecules through a series of biochemical reactions. As such, the trajectory of fructose-6-phosphate / glucosamine-6-phosphate, the glycolytic trajectory of fructose-6-phosphate, to the extent that it affects the biosynthesis of glucosamine-6-phosphate, and the trajectory of glucosamine-6-phosphate / biosynthesis of macromolecule all are considered metabolic pathways of aminosugar in the present invention. In general, a microorganism having a metabolic pathway of genetically modified amino sugar has at least one genetic modification, as discussed above, which results in a change in one or more metabolic pathways of aminosugar as described above, as compared to a microorganism of wild type grown under the same conditions. Such modification in a metabolic pathway of aminosugar changes the ability of the microorganism to produce an aminosugar. According to the present invention, a genetically modified microorganism preferably has an increased ability to produce glucosamine in comparison to a wild-type microorganism grown under the same conditions. A metabolic pathway of amino sugar that affects glucosamine production can generally be categorized into at least one of the following kinds of trajectories: (a) trajectories to convert glucosamine-6-phosphate to other compounds, (b) trajectories to synthesize glucosamine-6 phosphate, (c) trajectories for transporting glucosamine to a cell, (d) trajectories for transporting glucosamine or glucosamine-6-phosphate out of a cell, and (e) trajectories competing for substrates included in the production of glucosamine-6-phosphate . A genetically modified microorganism useful in a method of the present invention typically has at least one modified gene included in at least one amino sugar metabolic pathway that results in (a) reduced ability to convert glucosamine-6-phosphate to other compounds (ie, inhibition of catabolic or anabolic glucosamine-6-phosphate trajectories), (b) an increased ability to produce (ie, synthesize) glucosamine-6-phosphate, (c) a reduced ability to transport glucosamine to the cell, (d) an increased ability to transport glucosamine-6-phosphate or glucosamine out of the cell, and / or (e) a reduced ability to utilize substrates included in the production of glucosamine-6-P to compete for biochemical reactions. It should be understood that the present invention describes a tL?.?. * Aá > *? t * áiJ ^ ^^^^^ ^^^^ ki¿i ^ ^ method comprising the use of a microorganism with an ability to produce commercially useful amounts of glucosamine in a fermentation process (ie, preferably a skill increased to produce glucosamine compared to a wild-type microorganism grown under the same conditions). This method is achieved by the genetic modification of one or more genes encoding a protein included in an amino sugars metabolic pathway that results in the production (expression) of a protein that has an altered (eg, increased or reduced) function in comparison to the corresponding wild-type protein. Such altered function increases the ability of the genetically engineered microorganism to produce glucosamine. It should be appreciated by those skilled in the art that the production of genetically modified microorganisms having a particular altered function as described elsewhere herein (eg, an increased ability to produce glucosamine-6-phosphate) such as by Specific selection techniques described in the Examples, can produce many organism that satisfy the given functional requirement, although by virtue of a variety of different genetic modifications. For example, different random deletions and / or substitutions in a given nucleic acid sequence can all be raised to the same phenotypic result (eg, reduced action of the protein encoded by the sequence). The present invention contemplates any genetic modification that results in the production of a microorganism having the characteristics set forth herein. For a variety of microorganisms, many of the metabolic pathways of aminosugar have been elucidated. In particular, the trajectories for the biosynthesis or catabolism of glucosamine and? / - acetylglucosamine and their phosphorylated derivatives have been elucidated in Escherichia coli. These trajectories include the multiple transport systems for the use of these aminoazucares as carbon sources. The genes that encode the enzymes and proteins directly related to the transport, catabolism or biosynthesis of aminoazucares in Escherichia coli have been cloned and sequenced. In addition, mutant strains of Escherichia coli blocked substantially in each stage of amino sugar metabolism have been isolated. The known trajectories for amino sugar metabolism for Escherichia coli are illustrated in Figure 1. As will be discussed in detail below, even though many of the trajectories and genes included in the amino sugars metabolic trajectories have been elucidated, until the present invention, no he knew which of the many possible genetic modifications might be necessary to generate a microorganism that can produce commercially significant quantities of glucosamine. In fact, the present inventors are the first to design and perform a glucosamine-producing microorganism that has glucosamine production capabilities that greatly exceed the glucosamine production capacity of any known mutant or wild-type microorganism. The present inventors are also the first to appreciate that such a genetically modified microorganism is useful in a method for producing glucosamine for commercial use. A microorganism for use in the fermentation method of the present invention is preferably a bacterium or a yeast. More preferably, such a microorganism is a bacterium of the genus Escherichia. Escherichia coli is the most preferred microorganism for use in the fermentation method of the present invention. Particularly preferred strains of Escherichia coli include K-12, B and E, and more preferably, K-12. Although Escherichia coli is more preferred, it should be understood that any microorganism that produces glucosamine and that can be genetically modified to increase the production of glucosamine can be used in the method of the present invention. A microorganism for use in the fermentation method of the present invention can also be referred to as a production organism. Metabolic trajectories of aminosugar of the microorganism, Escherichia coli, will be directed as specific embodiments of the present invention described below. It will be appreciated that other microorganisms, and in particular, other bacteria, have similar aminosugar metabolic pathways and genes and proteins that have similar function and structure within such ¿T fr í% i trajectories. As such, the principles discussed below with respect to Escherichia coli are applicable to other microorganisms. In one embodiment of the present invention, a genetically modified microorganism includes a microorganism that has an increased ability to synthesize glucosamine-6-phosphate. According to the present invention, "an increased ability to synthesize" a product refers to any increase, supra-regulation, in a metabolic pathway of aminosugar related to the synthesis of the product in such a way that the microorganism produces an increased amount of the product in comparison to the wild-type microorganism grown under the same conditions. In one embodiment of the present invention, the enhancement of a microorganism's ability to synthesize glucosamine-6-phosphate is accomplished by amplifying the expression of the glucose-6-phosphate synthase gene, which in Escherichia coli is the gene glmS, the product of which is glucosamine-6-phosphate synthase. The glucosamine-6-phosphate synthase catalyzes the reaction in which fructose-6-phosphate and glutamine form glucosamine-6-phosphate and glutamic acid. The amplification of glucosamine-6-phosphate synthase expression can be carried out in Escherichia coli, for example, by the introduction of a recombinant nucleic acid molecule encoding the glmS gene. The overexpression of glmS is crucial for the intracellular accumulation of glucosamine-6-phosphate and finally the production of glucosamine, since the level of glucosamine-6-phosphate synthase in the cell will control the redirection of the carbon flux away from glycolysis and towards the synthesis of glucosamine-6-phosphate. The glmS gene is located 84 min on the chromosome Escherichia coli, and the sequence analysis of this region of the chromosome reveals that glmS resides in an operon with the glmLI gene, which encodes the bifunctional enzyme, glucosamine-1-phosphate acetyltransferase-? / -acetylglucosamine-1-phosphate uridyltransferase. Glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase functions within the metabolic pathway of amino sugar in which glucosamine-6-phosphate is incorporated, through a series of biochemical reactions, into macromolecules. No promoter sequence is detected upstream of glmS; Transcription of the glmUS operon is initiated from two promoter sequences upstream of glmil. In this way, it is preferred that the glmS gene be cloned under the control of an artificial promoter. The promoter can be any suitable promoter that will provide a level of glmS expression required to maintain a sufficient level of glucosamine-6-phosphate synthase in the production organism. Preferred promoters are constitutive (rather than inducible) promoters, since the need for expensive inducer addition is therefore obviated. Such promoters include normally inducible promoter systems that have been made functionally constitutive or "cracked" by genetic modification, such as by using a mutant, debilitating repressor gene. Particularly preferred promoters for use with glmS are lac,? P and T7. The gene dose (number of copies) of ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^ 3¿ ^^^ & glmS can be varied according to the requirements for the formation of the maximum product. In one embodiment, the recombinant glmS gene is integrated into the E. coli chromosome. Therefore, it is an embodiment of the present invention to provide a microorganism, such as E. coli, which is transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase, which E. coli, for example, is encoded by the glmS gene. Recombinant nucleic acid molecules comprising such a nucleic acid sequence include recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase comprising an amino acid sequence SEQ ID NO: 16. Other molecules Preferred recombinant nucleic acid molecules of the present invention include nucleic acid molecules comprising a nucleic acid sequence selected from the group of SEQ ID NO: 1 3, SEQ ID NO: 14 and / or SEQ ID NO: 15. Particularly, the preferred recombinant nucleic acid molecules of the present invention include nucleic acid molecules comprising nucleic acid molecules nglmS-28218 and / or nglmS-28183o. A recombinant molecule of the present invention, referred to herein as plasmid pKLN23-28, includes SEQ ID Nos: 13, 14 and 15 and is particularly useful for expressing glucosamine-6-phosphate synthase in a microorganism. The nucleic acid molecules identified represent the nucleic acid molecules comprising acid sequences wild type nucleic (i.e., occurring naturally or endogenously) encoding the glucosamine-6-phosphate synthase proteins. Genetically modified nucleic acid molecules that include the nucleic acid sequences encoding the homologous (ie, modified and / or mutated) glucosamine-6-phosphate synthase proteins are also included by the present invention and are described in detail down. The reported Km's of glucosamine-6-phosphate synthase of Escherichia coli are 2mM and 0.4mM for fruclosa-6-phosphate and glutamine, respectively. These values are relatively high (ie, the affinity of the enzyme for its substrates is preferably weak). Therefore, it is another embodiment of the present invention to provide a microorganism having glucosamine-6-phosphate synthase with improved affinity for its substrates. 15 A glucosamine-6-phosphate synthase with improved affinity for its substrates can be produced by any suitable method of genetic modification or protein engineering. For example, computer-based protein engineering can be used to design a glucosamine-6-phosphate synthase protein with greater stability and better affinity for its substrate. See for example, Maulik ef al., 1997, Molecular Biotechonology: Therapeutic Applications and Strategies; Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. White (1986, Biochem J., 106: 847-858) first demonstrated that the glucosamine-6-phosphate synthase was inhibited by glucosamine-6 phosphate. The present inventors determined that this inhibition was a key factor limiting the accumulation of glucosamine in glucosamine production strains of the present invention, which have been designed for commercial use. Therefore, it is still another embodiment of the present invention to provide a microorganism having a glucosamine-6-phosphate synthase with feedback inhibition of the reduced glucosamine-6-phosphate product. A glucosamine-6-phosphate synthase with inhibition of the reduced product can be a mutated glucosamine-6-phosphate synthase gene (i.e., genetically modified), for example, and can be produced by any suitable method of genetic modification. For example, a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase can be modified by any method to insert, remove and / or replace the nucleotides, such as error-prone PCR. In this method, the gene is amplified under conditions that lead to a high frequency of errors of misincorporation by the DNA polymerase used for the amplification. As a result, a high frequency of mutations are obtained in the PCR products. This method is described in detail in Example 5. The mutants of the glucosamine-6-phosphate synthase gene can then be classified by the inhibition of the reduced product by testing the ability of the mutant genes to confer increased glucosamine production on a microorganism of test, compared to a microorganism that carries the nucleic acid molecule of the synthase t t.? t, 4 * n ¿. ^^^^^^ * ** > Of unmutated recombinant glucosamine-6-phosphate. It should be noted that the inhibition of the reduced product of glucosamine-6-phosphate synthase typically results in a glucosamine-6-phosphate synthase with increased action, even though the specific activity of the enzyme is to remain the same, or actually reduce, in relation to a glucosamine-6-phosphate enzyme that occurs naturally. Therefore, it is an embodiment of the present invention to produce a genetically modified glucosamine-6-phosphate synthase with increased action and increased in vivo enzymatic activity, which has specific, unmodified and even reduced activity compared to a glucosamine-6 synthase. -phosphate that occurs naturally. Also included by the present invention are genetically modified glucosamine-6-phosphate synthases with increased specific activity. Thus, it is an embodiment of the present invention to provide a microorganism, such as E. coli, which is transformed with a genetically modified recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase protein, mutant or homologous. Such glucosamine-6-phosphate synthase proteins can be referred to herein as glucosamine-6-phosphate synthase homologs. The protein homologs are described in detail below. Preferred recombinant nucleic acid molecules comprising such a nucleic acid sequence include recombinant nucleic acid molecules comprising a i •. * ^ and r - *. ,. faith i. *. t ^ JUt ^ A ^ M nucleic acid sequence, which encodes a glucosamine-6-phosphate synthase comprising an amino acid sequence selected from the group of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO; , SEQ ID NO: 28 and / or SEQ ID NO: 31. Other preferred recombinant nucleic acid molecules comprise a nucleic acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO. .24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 and / or SEQ ID NO: 30. Particularly, the genetically modified recombinant nucleic acid molecules useful in the present invention include nucleic acid molecules comprising nucleic acid molecules selected from the group of nglmS-492? 8, nglmS-49? 83o, nglmS-542184, nglmS-54? 830, nglmS-1242? 84, nglmS-1241830, nglmS-1492184, nglmS-1491830, nglmS-1512184 and nglmS-151 1830. The plasmids pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149 and pKLN23-1 51 are recombinant nucleic acid molecules of the present invention that are particularly useful for expressing glucosamine-6-phosphate sistase homologs in a microorganism. A suitable intracellular supply of glutamine (Gln) is critical for the glucosamine-6-phosphate synthase reaction. Inspection of the degradative and synthetic pathways for glucosamine-6-phosphate reveals the presence of a potential futile cycle, whereby the continuous intraconversion of fructose-6-phosphate and glucosamine-6-phosphate results in useless depletion of glutamine. In one embodiment of the present invention, the glutamine delivery "IL" can be increased either by genetic modification of the production organism to increase the production of glutamine in the cell, or by modifying the fermentation medium (i.e., adding glutamine to the fermentation medium), to ensure that the Glutamine supply will not limit the production of glucosamine-6-phosphate. In another embodiment of the present invention, the potential futile cycle of fructose-6-phosphate and glucosamine-6-phosphate is directed by inhibiting or blocking the reverse reaction, in which glucosamine-6-phosphate is converted to fructose-6-phosphate . In this modality, a microorganism is genetically modified to have an inactivation or elimination of the gene that catalyzes this conversion, deaminase glucosamine-6-phosphate, which in Escherichia coli is the nagB gene. NagB is one of several nag genes that are part of the Nag regution. The nag genes included in the degradation of glucosamine and N-acetyl-glucosamine exist as a reguion located 15 min from the chromosome Escherichia coli. In another modality, the complete nag regution is inactivated or eliminated. The advantages of eliminating the complete nag regution are discussed in more detail below. As discussed above, the overproduction of glucosamine-6-phosphate synthase results in the amusement of synthesis of fructose-6-phosphate in glucosamine-6-phosphate synthesis. However, many other enzymes can compete for the substrate, fructose-6-phosphate. Therefore, one embodiment of the present invention includes a microorganism in which these competitive side reactions »-a.- - *« -? »3Mífc ~, J., J SJijüjfcfa i ?. i are blocked. In a preferred embodiment, a microorganism is provided which has complete or partial inactivation of the gene encoding phosphofructokinase. The second stage in the glycolytic pathway is the conversion of fructose-6-phosphate to fructose-6- 5 diphosphate by phosphofructokinase, which in Escherichia coli exists as two isozymes encoded by the genes pfkA and pfkB. Complete or partial inactivation of any of the pfkA and pfkB genes decreases the entry of fructose-6-phosphate into the glycolytic pathway and increases the conversion of fructose-6-phosphate to glucosamine-6-phosphate. As used herein, inactivation of a gene can refer to any modification of a gene that results in a reduction in the activity (ie, expression or function) of such a gene, including attenuation of activity or complete elimination of the activity. In a further embodiment of the present invention, a genetically modified microorganism has a reduced ability to convert glucosamine-6-phosphate to other compounds. The inactivation of deaminase glucosamine-6-phosphate, as described above, represents such modification, however, glucosamine-6-phosphate serves as a substrate for other biochemical reactions. The first step performed in the path leading to the production of macromolecules such as lipopolysaccharide and peptidoglycan in Escherichia coli is the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate by the mutase of phosphoglucosamine, in which Escherichia coli is the product of the gene glmM. The incorporation of this enzyme activity in the biosynthetic pathway of lipopolysaccharides and peptidoglycan was recently confirmed with the cloning of the glmM gene. Consequently, the regulation of the glmM gene, and its cognate product, phosphoglucosamine 5 mutase, has not been studied in detail. It has been shown, however, that the phosphoglucosamine mutase, as well as enzymes of hexosephosphate mutase, is regulated by phosphorylation. This type of regulation at the enzyme level is typically exquisitely extensive at the levels of the end products of the path. This Thus, the flow of carbon through the phosphoglucosamine mutase can self-regulate and may not be a problem as glucosamine-6-phosphate accumulates. Since the sequence of the glmM gene is known, however, it is a preferred embodiment of the present invention to provide a microorganism, in which the one encoding phosphoglucosamine mutase is interrupted or eliminated. More preferably, the phosphoglucosamine mutase-encoding gel is sub-regulated, but not completely inactivated, by mutation, so as not to completely block the biosynthesis of the critical cell envelope components. 0 Another path that results in the conversion of glucosamine-6-phosphate into another compound is catalyzed by the enzyme, deacetylase? / - acetylglucosamine-6-phosphate, deacetylase N-acetylglucosamine-6-phosphate is able to catalyze the reverse reaction of converting glucosamine-6-phosphate (plus CoA acetyl) in? / - acetyl-5-glucosamine-6-phosphate. This could result in a futile cycle of glucosamine-6-phosphate and? / - acetyl-glucosamine-6-phosphate and result in a product composed of a mixture of glucosamine and? V-acetyl-glucosamine. Therefore, it is a further embodiment of the present invention to provide a genetically modified microorganism 5 in which the one encoding deacetylase? / -acetylglucosamine-6-phosphate, which is the nagA gene in Escherichia coli, is inactivated. It is a further embodiment of the present invention to inactivate the transport systems for glucosamine in a microorganism so that once the glucosamine is excreted by the cell is not taken again. This modification is useful to avoid an elevated intracellular level of glucosamine that could be toxic to the cells, and facilitates the recovery of the product, since the product remains intracellular. In a preferred embodiment of the present invention, the transportation systems of Glucosamine is inactivated to keep glucosamine out of the microorganism once it is excreted by the microorganism. During the growth of Escherichia coli in glucosamine as a single carbon source, glucosamine is transported to the cell pro the PEP system: mannose phosphotransferase (PTS), which does not is only able to transport glucosamine to the cell, but is also induced by glucosamine. It is therefore an embodiment of the present invention to provide a microorganism that lacks the ability to transport glucosamine to the cell. For example, a manXYZ mutant (ie, Escherichia coli) that lacks or has a mutation in the genes that code IEM, P / lMan of PEP: mannose PTS) can not transport glucosamine to the cell by this mechanism. PEP: PTS glucose from Escherichia coli, on the other hand, is capable of transporting both glucose and glucosamine to the cell, but glucosamine can not induce this system. In this way, in order to develop a manXYZ mutant in glucosamine, cells must first develop into glucose to induce the expression of the glucose system (alternate) and allow glucose (the preferred carbon source) to be transported into the cell. These induced cells are then able to transport glucosamine to the cell via the glucose transporter. A similar situation exists to transport glucosamine by PEP: fructose PTS, although in this case the transport of glucosamine by the enzyme llFru is deficient. Methods to inhibit these secondary glucosamine transport pathways are discussed below. It is still another embodiment of the present invention to provide a microorganism having a reduced function in PEP: glucose PTS (described above). Such modification may be necessary to avoid the "reabsorption of glucosamine from the culture medium". For example, a ptsG mutant (ie, Escherichia coli lacking or having a mutation in the genes encoding PEP enzimallGIC: glucose PTS). Since such microorganism will have reduced ability to grow using glucose as a carbon source, such organism can also be genetically modified to take glucose by an independent mechanism of PEP: glucose PTS. It has been shown, for example, that mutant microorganisms can be selected that are defective in PEP: glucose PTS and still have an ability to develop into glucose (Flores et al., 1996, Nature Biotechonology 14: 620-623). The DNA sequence of the nag regimen in Escherichia coli reveals that the nagA gene, which encodes the protein specific for? / - acetyl-glucosamine of the PEPJosfotransferase sugar (PTS) system, which is included in the transport of glucosamine to the cell, it resides at one end of the reguion and is transcribed divergently from the other nag genes (nagBACD) located at the other end of the reguion. Therefore, another genetic modification that could result in reduced ability of Escherichia coli to transport glucosamine to the cell is an inactivation or elimination of the nagE gene, or a gene encoding a similar enzyme in any microorganism used in a method of the present invention. . As discussed above, in one embodiment of the present invention, a genetically modified Escherichia coli microorganism useful in a method of the present invention has a complete nag regution elimination. Elimination of the complete chromosomal nag regution is preferred, because many genes that are harmful to the production of glucosamine-6-phosphate are inactivated together. The genes, nagA, nagB and nagE, have been discussed in detail above. The nagC gene encodes a regulatory protein that acts as a repressor of the nag regution as well as both an activator and i > A.t «t .Ít-.t-í j .. t r .. i, * ,. . ^^^^^ ^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The glm genes are discussed in detail above. The function of the nagD gene is not known, but it is thought to be related to the metabolism of the aminosugar since it resides within the nag regution. Thus, in Escherichia coli, a complete elimination of the nag regimen prevents the catabolism of the initial intracellular product (glucosamine-6-phosphate) in an Escherichia coli strain designed to overproduce glucosamine. A preferred mutant strain of Escherichia coli that has an elimination of the nag regimen is Escherichia coli that has an elimination / insertion of? NagEBACDr? C. With respect to the activation of the glnnUS operon (a function of nagC), although activation of the glmS gene, which encodes the glucosamine-6-phosphate synthase, an increase in the level of the glmU gene product, glucosamine-1 is desirable. -phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase could be harmful for the accumulation of glucosamine-6-phosphate as it would lead to siphoning the flow of carbon towards the components of the cell envelope. It is therefore an embodiment of the present invention to inactivate glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase in a microorganism useful in a method of the present invention. In a microorganism in which the glmS operon, or its equivalent, has been inactivated or eliminated, it is a further embodiment of the present invention to genetically modify the microorganism by recombinantly producing the gene encoding the glucosamine-6-phosphate synthase under control of an artificial promoter in the microorganism. The initial intracellular product in the genetically modified microorganism described herein is glucosamine-6-phosphate. In many microorganisms, including Escherichia coli, glucosamine-6-phosphate is typically dephosphorylated in glucosamine before transporting it out of the cell. However, it is still another embodiment of the present invention to provide a microorganism that is genetically modified to have a phosphatase activity suitable for the conversion of glucosamine-6-phosphate to glucosamine. Such a phosphatase may include, but is limited to, for example, alkaline phosphatase. In a preferred embodiment, such Escherichia coli has an increased (ie, increased) level of phosphatase activity (ie, phosphatase action). As noted above, in the method for producing glucosamine of the present invention, a microorganism having a metabolically modified pathway of genetically modified amino sugar is cultured in a fermentation medium for the production of glucosamine. An appropriate, or effective, fermentation medium refers to any medium in which a genetically modified microorganism of the present invention, when grown, is capable of producing glucosamine. Such a medium is typically an aqueous medium comprising assimilable sources of carbon, nitrogen and phosphate. Such a medium can also include salts, minerals, metals and other appropriate nutrients. An advantage of the genetic modifications to a microorganism described herein is that although such genetic modifications significantly alter the metabolism of the aminoazucares, they do not create any nutritional requirement for the production organism. In this way, the glucose containing the minimum salt medium as the single carbon source is preferably used as the fermentation medium. The use of a glucose-salts-minimum method for the glucosamine fermentation will also facilitate the recovery and purification of the glucosamine product. The microorganism of the present invention can be cultured in conventional fermentation bioreactors. Microorganisms can be grown through any fermentation process that includes, but is not limited to, continuous fermentation, cell recycling, discontinuous feed, discontinuous. Preferably, the microorganisms of the present invention are developed by batch or discontinuous batch fermentation processes. In one embodiment of the present invention, prior to inoculation, the fermentation medium is brought to the desired temperature, typically from about 20 ° C to about 40 ° C, preferably from about 25 ° C to about 40 ° C, at temperatures from about 28 ° C to about 37 ° C, and in some embodiments, about 30 ° C or about 37 ° C being more preferred. The present inventors have discovered that the production of glucosamine in microorganism of the present ^^^^ a ^ SJ ^ S ^^^ jjé &mfecta transfectados with a nucleic acid molecule under the control of the promoter 17-lac (see section Examples) continues after the growth has stopped when the microorganisms are grown to 30 ° C, while at 37 ° C, the growth and production of glucosamine occur in concrete. The growth at 37 ° C is slightly better than at 30 ° C, but the glucosamine production at 30 ° C is significantly better than at 37 ° C. It is noted that the optimum temperature for the growth and production of glucosamine by a microorganism of the present invention can vary according to a variety of factors. For example, the selection of a particular promoter for the expression of a recombinant nucleic acid molecule in the microorganism can affect the optimum culture temperature. One of ordinary skill in the art can readily determine the glucosamine production temperature and optimal growth for any microorganism of the present invention using standard techniques, such as those described in the Examples section for a microorganism of the present invention. The medium is inoculated with a culture that actively grows the genetically modified microorganism in an amount sufficient to produce, after a reasonable growth period, a high cell density. The cells are grown at a cell density of at least about 10 g / l, preferably between about 10 g / l and about 10 g / l, and more preferably at least about 40 g / l. East Prooeso typically requires approximately 10-60 hours. Sufficient oxygen must be added to the medium during the fermentation cure to maintain cell growth during initial cell growth and to maintain glucosamine production and metabolism. Oxygen is conveniently provided by agitation and aeration of the medium. Conventional methods, such as stirring or shaking, can be used to stir and aerate the medium. Preferably, the concentration of oxygen in the medium is greater than about 15% of the saturation value (ie, the solubility of oxygen in the medium at atmospheric pressure and about 30-40 ° C) and more preferably greater than about 20% of the Saturation value, although excursions at lower concentrations may occur if the fermentation is not adversely affected. The oxygen concentration of the medium can be monitored by conventional methods, such as with an oxygen electrode. Other sources of oxygen, such as undiluted oxygen gas and oxygen gas diluted with insertion gas instead of nitrogen, can be used. Since the production of glucosamine by fermentation is preferably based on the use of glucose as the sole carbon source, in a preferred embodiment, in Escherichia coli, PEP: glucose PTS will be induced. According to the above, even in the absence of EIIM, P / lllMan of PEP: mannose PTS (for example, in Escherichia coli having a mutation manXYZ., The product, t-it- ai a -á. *. i ^ 5. * -. ^^ ^ ^ ^ ^ ^ ^ ^ * Glucosamine will still be taken through the cells through the induced glucose transport system. In the presence of excess glucose, however, the glucosamine intake is severely suppressed. In this way, it is an embodiment of the present invention to prevent the intake of the glucosamine product by maintaining an excess of glucose in the fermentation bioreactor. As used herein, "an excess" of glucose refers to an amount of glucose above which the growth of the microorganism must be maintained under normal conditions, such as as the culture conditions described above. Preferably, the glucose concentration is maintained at a concentration of from about 0.5% to about 5% w / v of the fermentation medium. In another embodiment, the glucose concentration is maintained at a concentration of about 5 g / L to about 50 g / L of the fermentation medium, and even more preferably, from about 5 g / L to about 20 g / L of the fermentation medium. In one embodiment, the glucose concentration of the fermentation medium is monitored by any suitable method (by For example, when using glucose test tapes), and when the glucose concentration is at or near the elimination, the additional glucose can be added to the medium. In another embodiment, the glucose concentration is maintained by semi-continuous or continuous feeding of the fermentation medium. The parameters described herein for glucose can be applied to any > > amm »m & i > ""4" ? S ^^^? carbon source used in the fermentation medium of the present invention. It should be understood that the carbon source can be allowed to reach undetectable levels for any appropriate amount of time during fermentation if it increases the glucosamine production process. It is a further embodiment of the present invention to supplement and / or control other components and parameters of the fermentation medium, as necessary to maintain and / or increase the production of glucosamine by a production organism. For example, in one embodiment, the fermentation medium includes ammonium sulfate, and the concentration of ammonium sulfate in the culture medium is supplemented by the addition of excess ammonium sulfate. Preferably, the amount of ammonium sulfate is maintained at a level of from about 0.1% to about 1% (weight / volume) in the fermentation medium, and preferably, at about 0.5%. In yet another embodiment, the pH of the fermentation medium is monitored by fluctuations in pH. In the fermentation method of the present invention, the pH is preferably maintained at a pH of from about pH 6.0 to about pH 8.0, and more preferably, at about pH 7.0. In the method of the present invention, if the initial pH of the fermentation medium is pH 7.0, the pH of the fermentation medium is monitored by significant variations from pH 7.0, and is adjusted according to the above, for example, by addition of sodium hydroxide. ? ? ? ? ? t tiiAA k ___ A further embodiment of the present invention is to redirect the carbon flux from acetate production to the production of less toxic by-products. By such methods, the toxicity problems associated with an excess of glucose in the fermentation medium can be avoided. Methods for redirecting the carbon flux from acetate production are known in the art. In a batch fermentation process of the present invention, the fermentation is continued until the formation of glucosamine, as evidenced by the accumulation of extracellular glucosamine, essentially ceases. Total fermentation is typically from about 40 to about 60 hours, and more preferably, about 48 hours. In a continuous fermentation process, glucosamine can be removed from the bioreactor as it accumulates in the medium. The method of the present invention results in the production of a product which may include intracellular or extracellular glucosamine-6-phosphate and intracellular or extracellular glucosamine. The method of the present invention further includes recovering the product, which may be intracellular glucosamine-6-phosphate or extracellular glucosamine. The phrase "recover glucosamine" refers simply to collecting the product from the fermentation bioreactor and does not need to involve additional steps of separation or purification. For example, the recovery stage may refer to the removal of the entire crop (ie, the microorganism and the fermentation medium) of the bioreactor, removing the fermentation medium containing extracellular glucosamine from the bioreactor, and / or removal of the microorganism containing intracellular glucosamine-6-phosphate from the bioreactor. The steps can be followed by the additional purification steps. Glucosamine is preferably recovered in substantially pure form. As used herein, "substantially pure" refers to a purity that allows the effective use of glucosamine as a nutritional compound for commercial sale. In one embodiment, the glucosamine product is preferably separated from the production organism and other constituents of the fermentation medium. The methods for carrying out such separation are described below. Preferably, by the method of the present invention, at least about 1 g / L of product (ie, glucosamine and / or glucosamine-6-phosphate) are recovered from the microorganism and / or fermentation medium. More preferably, by the method of the present invention, at least about gg / L, and even more preferably, at least about 10 g / L, and even more preferably, at least about 20 g / L and even more preferably at least approximately 50 g / L of the product are recovered. In one embodiment, the glucosamine product is recovered in an amount from about 1 g / l to about 50 g / L. Typically, the majority of glucosamine produced in the ? tÍHá.A &. * ~ ét & H < rL To IkA-m. .- li ». . «Cttw t. . i. . . ," .,go? r ^ Ííi- - ^? ^ > »- J IjsSJt i A L tkMl? Mtt .: present process is extracellular. The microorganism can be removed from the fermentation medium by conventional methods, such as by filtration or centrifugation. In one embodiment, the step of recovering the product includes purifying glucosamine from the fermentation medium. Glucosamine can be recovered from the cell-free fermentation medium by conventional methods, such as chromatography, extraction, crystallization (e.g., evaporative crystallization), membrane separation, reverse osmosis and distillation. In a preferred embodiment, the glucosamine is recovered from the cell-free fermentation medium by crystallization. In another embodiment, the step of recovering the product includes the step of concentrating the extracellular glucosamine. In one embodiment, glucosamine-6-phosphate accumulates intracellularly, the product recovery step includes isolating glucosamine-6-phosphate from the microorganism. For example, the product can be recovered by destroying the microorganism cells by plant action by a method that does not degrade the glucosamine product, centrifuge the lysate to remove the insoluble cell debris, and then recover the glucosamine and / or glucosamine product. -6-phosphate by a conventional method as described above. The initial intracellular product in the genetically modified microorganism described herein is glucosamine-6-phosphate. It is generally accepted that intermediate compounds ttér t t? < t¡W »« m ...? The phosphorylates are defoforilated during the export of the microorganism, most likely due to the presence of alkaline phosphatase in the periplasmic space of the microorganism. In one embodiment of the present invention, glucosamine-6-phosphate is defoforilated before or during export of the cell by naturally occurring phosphatases in order to facilitate the production of the desired product, glucosamine. In this embodiment, the need for amplification of a phosphatase activity recombinantly provided in the cell or treatment of the fermentation medium with a phosphatase is obviated. In another embodiment, the level of phosphatase in the production organism is increased by a method that includes, but is not limited to, genetic modification of an endogenous phosphatase gene or by recombinant modification of the microorganism to express a phosphatase gene. In still another embodiment, the recovered fermentation medium is treated with a phosphatase after glucosamine-6-phosphate is released into the medium, such as when the cells are destroyed by the action of the Usinas as described above. As noted above, the process of the present invention produces significant amounts of extracellular glucosamine. In particular, the process produces extracellular glucosamine in such a way that more than about 50% of the total glucosamine is extracellular, more preferably more than about 75% of the total glucosamine is extracellular, and more preferably more than approximately 90% of the total glucosamine is extracellular. By the method of the present invention, the production of an extracellular glucosamine concentration can be achieved, which is greater than about 1 g / l, more preferably greater than about 5 g / l, even more preferably greater than about 10 g / l. , and even more preferably greater than about 20 g / L and even more preferably greater than about 50 g / L. One embodiment of the present invention relates to a method for producing glucosamine by fermentation which includes the steps of (a) cultivating Escherichia coli having a metabolically modified pathway of amino sugar in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate to produce a product, and (b) recover the product. The product includes intracellular glucosamine-6-phosphate which is recovered from Escherichia coli and / or extracellular glucosamine which is recovered from the fermentation medium. One embodiment of the present invention relates to a microorganism for producing glucosamine by a biosynthetic process. The microorganism is transformed with a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase operably linked to a transcription control sequence. The recombinant nucleic acid molecule has a genetic modification that reduces the inhibition of the glucosamine-6-phosphate product of the glucosamine-6-phosphate synthase. The " .1. .rtr ktÁ? ^^^^^^^^ i. ? Expression of the recombinant nucleic acid molecule increases the expression of the glucosamine-6-phosphate synthase by the microorganism. In a preferred embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism. In a further embodiment, the microorganism has at least one additional genetic modification in a gene that encodes a protein selected from the group of deacetylase? / - acetylglucosamine-6-phosphate, deaminase glucosamine-6-phosphate, enzyme lNa specific for? / - acetyl glucosamine, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-? F-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, PEG enzyme IIG | C: glucose PTS, IEM, P / lllMan of PEP: mannose PTS, and / or a phosphatase. Genetic modification reduces the action of the protein, except in the case of phosphatase, in which the action of the phosphatase is preferentially increased. In other In a preferred embodiment, the microorganism has a modification in the genes encoding deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase and N-acetyl-glucosamine-specific llNag enzyme, where genetic modification reduces the action of the protein. In one modality, the genetic modification is an elimination of at least a portion of the genes. In a preferred embodiment, the genetically modified microorganism is a bacterium or yeast, and more preferably, a bacterium of the genus Escherichia coli, and even more preferably, Escherichia coli. Escherichia coli genetically Preferably modified has a modification in a gene that A-1 ^ 1 »i u > * 'tt-Á' t > i •• i aattotet ter. n a. - > ? n. t. a ^ im i »includes, but is not limited to, nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmil, glmS, ptsG and / or phosphatase gene. In another embodiment, such genetically modified Escherichia coli has a deletion of the nag regution genes and in yet another embodiment, a deletion of the nag regution genes and a genetic modification in manXYZ genes in such a way that the proteins encoded by the manXYZ genes They have reduced the action. Still another embodiment of the present invention relates to a microorganism for producing glucosamine by a biosynthetic process having a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase in a gene encoding a protein selected from the deacetylase group? / -acetylglucosamine-6-phosphate, deaminase glucosamine-6-phosphate, enzyme-specific llNag of? / - acetyl-glucosamine, mutaf of phosphoglucosamine, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, enzyme PEG IIG | C of PEP: glucose PTS, IEM, P / lllMan of PEP: mannose PTS. The genetic modification reduces the action of said protein and the expression of the recombinant nucleic acid molecule increases the expression of the glucosamine-6-phosphate synthase by the microorganism. In another embodiment, the microorganism has at least one genetic modification in a phosphatase gene, such that the phosphatase encoded by such a gene has increased action. In a preferred embodiment, the recombinant nucleic acid molecule is integrated into the genome of the microorganism.

^^^^^ Aj ^^^^^ e * Another embodiment of the present invention relates to any of the microorganisms described above that produce at least about 1 g / L of glucosamine when cultured for approximately 24 hours at 37 ° C. at a cell density of at least about 8 g / L by dry cell weight, in a fermentation medium of pH 7.0 comprising: 14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3Citrate- 2H2O, 5 g / L (NH4) 2SO4, 20 g / L of glucose, 10 mM of MgSO, 1 mM of CaCl2, and from about 0.2 mM to about 1 mM of IPTG. A preferred embodiment of the present invention relates to any of the above-described microorganisms that produce at least about 1 g / L of glucosamine when grown for about 10 to about 60 hours at from about 28 ° C to about 37 ° C at about cell density of at least about 8 g / L per dry cell weight in a fermentation medium of pH 7.0 comprising: 14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3Citrate-2H2O, 5 g / L (NH4) 2SO4, 20 g / L of glucose, 10 mM of MgSO4, 1 mM of CaCl2, and from about 0.2 mM to about 1 mM of IPTG. In a preferred embodiment, the amount of IPTG is approximately 0.2 mM. Still another embodiment of the present invention relates to any of the genetically modified microorganisms described above that produce at least about 1 g / L, and preferably at least about 5 g / L, and more preferably, at least about 10 g / L, and even more preferably, at least about 20 g / L, and even more preferably, at least about 50 g / L of glucosamine and / or glucosamine-6-phosphate when grown under the culture conditions as described herein. Another embodiment of the present invention relates to any of the genetically modified microorganisms described above that produce at least about 2 times more glucosamine and / or glucosamine-6-phosphate, and preferably at least about 25 times, and more preferably at least about 50 times, and even more preferably at least about 10 times, and even more preferably, at least about 200 times more glucosamine and / or glucosamine-6-phosphate synthase than a wild-type (ie, unmodified, microorganism that occurs naturally) grown under the same conditions as the genetically modified microorganism. A specific number of microorganisms are identified in the Examples section. Additional embodiments of the present invention include these microorganisms and microorganisms that have the identification characteristics of the microorganisms specifically identified in the Examples. Such microorganisms are preferably yeast or bacteria, more preferably, they are bacteria, and more preferably they are E. coli. Such identification characteristics may include any of the genotypic and / or phenotypic characteristics of the microorganisms in the Examples, including their ability to produce glucosamine. Preferred microorganisms of the present invention include strains of Escherichia coli that have been transformed with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase. Preferably, such a nucleic acid molecule is integrated into the genome of the microorganism. A particularly preferred microorganism is the strain of Escherichia coli 2123-12. The strain 2123-12 has integrated in its genome a recombinant nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 15, which represents the coding region of a wild type (i.e., normal, unmodified, or naturally occurring) glucosamine-6-phosphate synthase enzyme having a sequence of amino acid SEQ ID NO: 16. Particularly preferred microorganisms of the present The invention has been transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase that has been genetically modified in such a way that the synthase has increased action (described above). More preferably, such genetic modification increases the The ability of the microorganism to produce glucosamine compared to a microorganism that has not been transformed with such a nucleic acid molecule. The genetically modified, particularly preferred microorganisms of the present invention are described in the Examples section, and include E. coli strains. 2123-49, 2123-54, 2123-124, 2123-149 and 2123-1 51.

-AaaafeiBMgtefct i l i i ü u i t. . S. j,. . . í. t * < * *. > . i. * > -fe ^ Jt i? The development of a microorganism with enhanced ability to produce glucosamine through genetic modification can be carried out using both classical strain development and molecular genetic techniques. In general, the strategy for creating a microorganism with increased glucosamine production is (1) to inactivate or eliminate at least one, or preferably more than one of the amino sugars metabolic pathways, in which the production of glucosamine-6-phosphate is affected negatively (for example, inhibits), and (2) amplifying at least one, and preferably more than one of the amino sugar sucrose metabolic pathways in which the production of glucosamine-6-phosphate is increased. As such, the genetically modified microorganisms of the present invention have a (a) reduced ability to convert glucosamine-6-phosphate to other compounds (ie, inhibition of catabolic and anabolic pathways of glucosamine-6-phosphate), (b) an ability increased to produce (i.e., synthesize) glucosamine-6-phosphate, (c) a reduced ability to transport glucosamine in the cell, (d) an increased ability to transport glucosamine-6-phosphate or glucosamine out of the cell, and / or (e) a reduced ability to use substrates included in the production of glucosamine-6-P to compete biochemical reactions. As previously discussed herein, in one embodiment, a genetically modified microorganism may be a microorganism in which the nucleic acid molecules have been removed, inserted or modified, such as by insertion, removal, substitution, and / or reversal of nucleotides, such that such modifications provide the desired effect within the microorganism. Such genetic modifications may, in some embodiments, be found within the coding region for a protein encoded by the nucleic acid molecule that results in amino acid modifications such as insertions, deletions, substitutions in the amino acid sequence of the protein provided by the nucleic acid molecule. desired effect within the microorganisms. A genetically modified microorganism can be modified by recombinant technology, such as by introducing an isolated nucleic acid molecule into a microorganism. For example, a genetically modified microorganism can be transfected with a recombinant acid molecule that encodes a protein of interest, such as a protein for which increased expression is desired. The transfected nucleic acid molecule can remain extrachromosomal or can be integrated into one or more sites within a chromosome of the transfected (i.e., recombinant) host cell such that its ability to express is maintained. Preferably, once a host cell of the present invention is transfected with a nucleic acid molecule, the nucleic acid molecule is integrated into the genome of the host cell. A significant advantage of integration is that the nucleic acid molecule is stably maintained in the cell. In a preferred embodiment, the molecule The integrated nucleic acid mAb is operably linked to a transcription control sequence (described below) that can be induced to control the expression of the nucleic acid molecule. A nucleic acid molecule can be integrated into the genome of the host cell either by directed or random integration. Such integration methods are known in the art, for example, as described in detail in Example 2, the E. coli strain ATCC 47002 (Table 1) contains mutations that confers an inability to maintain plasmids containing an origin Co1 E1 replication. When such plasmids are transferred to this strain, the selection of genetic markers contained in the plasmids results in the integration of the plasmid into the chromosome. This strain can be transformed, for example, with plasmids containing the gene of interest and a selectable marker flagged by the 5 'and 3' termini of the lacZ E. coli gene. The lacZ sequences direct the incoming DNA to the lacZ gene contained in the chromosome. The integration of the lacZ site replaces the intact lacZ gene, which encodes the enzyme β-galactosidase, with a partial lacZ gene interrupted by the gene of interest. Successful members can be selected for ß-galactosidase negativity. A genetically modified microorganism can also be produced by introducing nucleic acid molecules into a recipient cell genome by a method such as by using a bacteriophage of transduction. The use of recombinant technology and bacteriophage transduction technology to produce several microorganisms r .i .r,, í. * rr i? j? -. í. ., M ^ M .. A .., > .... - -., - genetically modified different from the present invention is known in the art and is described in detail in the Examples section. According to the present invention, a gene, for example the pstG gene, includes all the nucleic acid sequences related to a natural pstG gene such as the regulatory regions that control the production of the pstG protein (Enzyme IIG | C of PEP: glucose PTS) encoded by that gene (such as, but not limited to, transcription, translation, or post-translation regions) as well as the coding region itself. In another embodiment, a gene, for example the pstG gene, can be an allelic variant (ie, a naturally occurring allelic variant) that includes a sequence similar but not identical to the nucleic acid sequence encoding a given pstG gene. An allelic variant of a pstG gene having a given nucleic acid sequence is a gene that occurs in essentially the same place (or loci) in the genome as the gene having the given nucleic acid sequence, but which, due to to natural variations caused by, for example, mutation or recombination, it has a similar but not identical sequence. Allelic variants typically encode proteins that have activity similar to that of the protein encoded by the gene to which they are compared. Allelic variants may also comprise alterations in the 5 'or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given microorganism, such as E. coli, and / or between a group of two or more microorganisms. Although the phrase "nucleic acid molecule" refers primarily to the physical nucleic acid molecule and the phrase "nucleic acid sequence" refers primarily to the sequence of nucleotides in the nucleic acid molecule, the two phrases may be used in interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, which is capable of encoding a gene included in an amino sugars metabolic pathway. In addition, the phrase "molecule "Recombinant" refers primarily to a nucleic acid molecule operably linked to a transcription control sequence, but can be used interchangeably with the phrase "nucleic acid molecule" which is isolated and expressed in a host cell. By knowing the nucleic acid sequences of certain nucleic acid molecules of the present invention, and particularly the nucleic acid molecules Escherichia coli, allows a person skilled in the art, for example, (a) to make copies of those nucleic acid molecules and / or (b) obtain molecules of Nucleic acid including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules that include full length genes, full length coding regions, regulatory control sequences, truncated coding regions). Such nucleic acid molecules can obtained in a variety of ways including the techniques of Traditional cloning using oligonucleotide tests to classify appropriate libraries or DNA and PCR amplification of appropriate libraries or DNA using oligonucleotide primers. Preferred libraries for classifying or from which to amplify the nucleic acid molecule include libraries of yeast and bacterial genomic DNA, and in particular, libraries of E. coli genomic DNA. Techniques for cloning and amplifying genes are described, for example, in Sambrook et al., Ibid. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (i.e., that has been subjected to human manipulation). As such, "isolated" does not reflect the degree to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA or derivatives of either DNA or RNA. There is no limit, instead of a practical limit, to the maximum size of a nucleic acid molecule in which the nucleic acid molecule can include a portion of a gene, a complete gene, or multiple genes or portions thereof. An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as a whole (ie, complete) gene or a portion thereof capable of forming a stable hybrid with that gene. An isolated nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. The acid molecules j ^ g ^^^^ üAH, _______, isolated nucleic acids include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which the nucleotides have been inserted, deleted, 5 replaced and / or inverted in such a way that such modifications provide the desired effect within the microorganism. A homologue of the nucleic acid molecule can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., Ibid.). By For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, division of the Restriction enzyme of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, synthesis of mixtures of oligonucleotides and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. The homologs of the nucleic acid molecules can be selected from a mixture of modified nucleic acids by classifying the function of the protein encoded by the nucleic acid and / or by hybridization with a wild-type gene. Examples of such techniques are described in detail in the Examples section.

In a preferred embodiment of the present invention, a nucleic acid nucleic acid homologue a nucleic acid molecule of the present invention preferably comprises a genetic modification resulting in a modification of the action of the protein encoded by the nucleic acid homologue. For example, in one embodiment of the present invention, there is provided a genetically modified recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase protein homologue, wherein the genetic modification increases the action of the glucosamine-6 synthase homologue phosphate, preferably in comparison to a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate synthase that occurs naturally in the absence of such genetic modification. Such a genetic modification can increase the action of glucosamine-6-phosphate synthase, for example, by encoding a glucosamine-6-phosphate synthase, which has an inhibition of the reduced glucosamine-6-phosphate product and / or increased specific activity. . Such recombinant nucleic acid molecules having genetic modifications are referred to herein as "nucleic acid homologs" of wild-type nucleic acid molecules encoding glucosamine-6-phosphate synthase. According to the present invention, proteins that have modifications as a result of genetic modifications in the nucleic acid molecules encoding the proteins are referred to herein as protein homologs, or protein homologs ? *, j ^? M ?. .- «,.," • .., - ^ _- JlBß- ^^^^^^^ given. According to the above, a glucosamine-6-phosphate synthase protein, for example, having glucosamine-6-phosphate synthase activity and is useful in the present invention, can be a glucosamine-6 synthase protein. full-length phosphate, an enzymatically active portion of a full-length glucosamine-6-phosphate synthase protein, or any homologue of such proteins, such as glucosamine-6-phosphate synthase protein having at least one or a few of nucleic acid modifications, in which the amino acid residues have been removed (eg, a truncated version of the protein, such as a peptide), inserted, substituted and / or derivatized (eg, by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation, and / or addition of glycosylphosphatidyl inositol). A protein homologue of any of the proteins within the amino sugars metabolic pathways as described in the present invention is a protein having an amino acid sequence that is sufficiently similar to a natural protein amino acid sequence (i.e., that occurs naturally, unmodified, or wild-type) that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (ie, with) a nucleic acid molecule encoding the native protein (i.e., the complement of the nucleic acid strain encoding the amino acid sequence of , i. to . i. ^ • &** ** * *. ., ^, ^ m ^, ...,, «^. «.« A ^, .. - ,. > .. _ ... ^ t -nj ^^^ mtiflltt- natural protein). A complement of the nucleic acid sequence of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (ie, it can form a double helix with the molecule integer) filament for which the sequence is cited. It should be noted that a double-stranded nucleic acid molecule of the present invention for which a nucleic acid sequence has been determined for a filament that is represented by a SEQ ID NO, also comprises a filament complementary that has a sequence that is a complement of that SEQ ID NO. As such, the nucleic acid molecules of the present invention, which can be either double-stranded or single-filament, include those nucleic acid molecules that form stable hybrids under conditions of demanding hybridization. with either a SEQ ID NO given, denoted above and / or with the complement of that SEQ ID NO, which may or may not be denoted herein. Methods for deducting a complementary sequence are known to those skilled in the art. The minimum size of a protein homologue of the The present invention is a sufficient size to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding native protein. Additionally, the minimum size of a protein homolog of the present invention is a sufficient size to have glucosamine-6-phosphate action *? iMki? H? l .. «? ? *, **%, *, * ».". , ^, -. . ... ... ^ ^, ...., r, ^ _ IJfc1¡_tlÍL (for example, an active catalytic or enzymatic portion) and preferably, increased glucosamine-6-phosphate phosphate synthase action. As such, the size of the nucleic acid molecule encoding such a protein homologue depends on the nucleic acid composition and percent homology between the nucleic acid molecule and the complementary sequence as well as the conditions of hybridization per se ( for example, temperature, salt concentration, and formamide concentration). There is no limit, instead of a practical limit, on the maximum size of such molecule of nucleic acid in which the nucleic acid molecule can include a portion of a gene, a total gene, or multiple genes, or portions thereof. Similarly, the minimum size of a protein homologue of the present invention is from about 4 to about 6 amino acids in length, with preferred sizes depending on whether functional, multivalent portions (i.e., fusion protein having more than one domain each of which has a function) or full length of such proteins are desired. As used herein, the demanding hybridization conditions refer to hybridization conditions standard under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are described, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1 989. Sambrook et al. , ibid is incorporated for reference in its totality (see specifically, pages 9.31 -9.62, 1 1 .7 and 1 1 .45- 1 1 .61). In addition, Aas formulas are described for calculating proper hybridization and washing conditions to achieve hybridization that allows varying degrees of nucleotide mismatch, for example, in Meinkoth et al. , 1884, Anal. Biochem, 1 38,. 267-284; Meinkoth et aK | ibid, is incorporated herein by reference in its entirety. More particularly, the stringent hybridization conditions, as referred to herein, refer to conditions that allow the isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule using to test the hybridization reaction, more particularly at least about 75%, and more particularly at least about 80%. Such conditions will vary, depending on whether DNA: RNA or DNA: DNA hybrids are formed. The melting temperatures calculated for DNA: DNA hybrids are 10 ° C less for DNA: RNA hybrids. In particular embodiments, the stringent hybridization conditions for DNA: DNA hybrids include hybridization at an ionic strength of 0.1 X SSC (0.157 M Na +) at a temperature between about 20 ° C and about 35 ° C, more preferably, between about 28 ° C and about 40 ° C, even more preferably, between about 35 ° C and about 45 ° C. In particular embodiments, the demanding hybridization conditions for DNA: RNA hybrids include hybridization at an ionic intensity of 0J X SSC (0.157 M Na +) at a temperature between about 30 ° C and about 45 ° C, more preferably, between about 38 ° C and about 50 ° C, even more preferably, between about 45 ° C and about 55 ° C. These values are based on calculations of a melting temperature for molecules longer than about 100 nucleotides, 0% formamide and a G + C content of about 50%. Alternatively, Tm can be calculated empirically as established in Sambrook et al., Supra pages 1 1 .55 to 1 1 .57. The protein homologs of the proteins included in an amino sugars metabolic pathway according to the present invention can be the result of natural allelic variation or natural mutation. Protein homologs of the present invention can also be produced using techniques known in the art, including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, recombinant DNA techniques or classics to effect directed or random mutagenesis, as discussed above. In one embodiment of the present invention, a genetic modification in a recombinant nucleic acid molecule of the present invention encoding a glucosamine-6-phosphate synthase results in at least one amino acid modification (i.e., modification in the amino acid sequence of the encoded protein) selected from the group of an addition, substitution, elimination and / or derivation of an amino acid residue of the glucosamine-6-phosphate synthase. Such modification in the amino acid sequence of the encoded protein can be determined in comparison to a naturally occurring or wild-type glucosamine-6-phosphate synthase, such as a glucosamine-6-phosphate synthase having an amino acid sequence SEQ. ID NO: 16. One or more of the amino acid modifications results in increased action of glucosamine-6-phosphate synthase in Comparison to the naturally occurring glucosamine-6-phosphate synthase having amino acid sequence SEQ ID NO: 16. In one embodiment, such an amino acid modification is found at an amino acid sequence position in the modified protein (i.e. homologous) corresponding to one or more of the following amino acid positions in amino acid sequence SEQ ID NO: 16: lie (4), lie (272), Ser (450), Ala (39), Arg (250), Gly (472), Leu (469). In another embodiment, such an amino acid modification is selected from the group of a substitution of: (a) a residue of amino acid having an aliphatic hydroxyl side group for Ile (4); (b) an amino acid residue having an aliphatic hydroxyl side group for Ile (272); (c) an amino acid residue having an aiiphatic side group for Ser (450); (d) an amino acid residue having an aliphatic hydroxyl side group for Ala (39); (e) a waste amino acid that has a side group that contains sulfur for He has a great deal of interest in his work. 1. I n n •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• (f) an amino acid residue having an aliphatic hydroxyl side group for Gly (472); (g) an amino acid residue having an aliphatic side group for Leu (469); and (h) combinations of (a) - (g). According to the present invention, amino acid residues having an aliphatic hydroxyl group include serine and threonine, and amino acid residues having aliphatic side groups include glycine, alanine, valine, leucine, isoleucine and proline. In yet another embodiment of the present invention, an amino acid modification as described above is preferably a substitution selected from the group of: lle (4) to Thr, lle (272) to Thr, Ser (450) to Pro, Ala (39 ) to Thr, Arg (250 aCys), Gly (472) to Ser, Leu (469) to Pro, and combinations thereof. Specific examples of recombinant nucleic acid molecules having genetic modifications that result in amino acid modifications are described in detail in the Examples section. Genetically-modified recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase having increased action include recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a glucosamine-6 synthase. -phosphate comprising an amino acid sequence selected from the group of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28 and / or SEQ ID NO: 31. Other molecules of genetically recombinant nucleic acid t .., *, j * ,. í. * .. "r. > * jir, rM. ^ ... w -, uii ... IÜ? M, ^ ?? -f nfn ^ _ ^? _ JuL t ± jíJ_Aia¡ÍÍÉ modified, preferred comprise a nucleic acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 1 8, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO. 26,. SEQ ID NO: 27, SEQ ID NO: 29 and / or SEQ ID NO: 30. Genetically modified recombinant nucleic acid molecules useful in the present invention include nucleic acid molecules comprising nucleic acid molecules selected from the group of PKLN23-49, pKLN23-54, pKLN23-149, pKLN23-151, nglmS-492184, nglmS- 491830, nglmS-542184, nglmS-541830, nglmS-1242184, nglmS-1241830, nglmS-1492? 84, nglmS-149? 830, nglmS-1 512? 84, nglmS-1 51 1830. The present invention includes a recombinant vector , which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule in a bacterial cell. Such a vector may contain bacterial nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules to be inserted into the vector. The vector can be either RNA or DNA and is typically plasmid. The recombinant vectors can be used in the cloning, sequencing, and / or otherwise manipulation of nucleic acid molecules. A recombinant type vector, referred to herein as a recombinant nucleic acid molecule and described in more detail below, is herein referred to a recombinant nucleic acid molecule and described in more detail below, can be used in the expression of recombinant nucleic acid molecules. nucleic acid.

Preferred recombinant vectors are capable of being duplicated in a transformed yeast or bacteria cell, and in particular, in an Escherichia coli cell. The transformation of a nucleic acid molecule into a cell can be carried out by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation and microinjection. The recombinant molecules of the present invention, which may be either DNA or RNA, may also contain additional regulatory sequences, such as translational regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. One or more of the recombinant molecules of the present invention can be used to produce an encoded product (eg, a glucosamine-6-phosphate synthase). In one embodiment, an encoded product is produced by expressing a nucleic acid molecule of the present invention under conditions effective to produce the protein. Such conditions (i.e., culture conditions) have been described above and are further discussed in the Examples section. A preferred method for producing an encoded protein is by transferring a host cell with one or more recombinant molecules of the present invention to form a recombinant cell. As discussed above, the recombinant molecules _. «- Í * -As? ** *? ~ * - - ft *** - preferred of the present invention include, nglmS-282? 84, nglmS-28? 830) nglmS-492? 84, nglmS-49? 830, nglmS-54218, nglmS-541830 , nglmS-1242184, nglmS-1 241830, nglmS-1492? 84, nglmS-149? 830, nglmS-1 512? 84, nglmS-1 51 1830, pKLN23-28, pKLN23-49, pKLN23-54, pKLN23-124 , 5 pKLN23-149 and pKLN23-1 51. A recombinant cell is preferably produced by transforming a bacterial cell (i.e., a host cell) with one or more of the recombinant molecules, each comprising one or more nucleic acid molecules operably linked to an expression vector containing one or more transcription control sequences. The phrase "linked operative" refers to the insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is capable of expression when transformed into a host cell. As used in the In this embodiment, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting the expression of a specific nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. In the present invention, expression vectors are typically plasmids. The expression vectors of the present invention include any of the vectors that function (i.e., expression of the direct gene) in a yeast host cell or a host cell of bacteria, preferably an Escherichia coli host cell. Preferred recombinant cells of the present The invention is set forth in the Examples section.

The nucleic acid molecules of the present invention can be operably linked to expression vectors containing regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and which control the expression of the nucleic acid molecules of the present invention. In particular, the recombinant molecules of the present invention include transcription control sequences. The 0 transcript control sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those that control the initiation of transcription, such as promoter sequences, augmentators, operators and repressors. Suitable transcription control sequences include any transcriptional control sequence that can function in bacteria or yeast cells and preferably, Escherichia coli. A variety of such transcription control sequences are known to those skilled in the art. It can be appreciated by one skilled in the art that the use of recombinant DNA technologies can improve the expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficacy with the transcripts is translate, and the effectiveness of post-translational modifications. Recombinant techniques useful for increasing the expression of the nucleic acid molecules of the present invention include, but are not limited to, nucleic acid molecules that are operably linked to plasmids with high copy number, integration of the nucleic acid molecules into the host cell chromosome, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (eg, promoters, operators, enhancers), substitutions or modifications of translational control signals, modification of nucleic acid of the present invention to correspond to the use of the host cell codon, elimination of the sequences that destabilize the transcripts, and the use of control signals that temporarily separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention can be improved by fragmenting, modifying, or deriving the nucleic acid molecules encoding such a protein. Such modifications are described in detail in the Examples section. The following experimental results are provided for the purpose of illustration and are not intended to limit the scope of the invention EXAMPLES Example 1 The following example describes the production of mutant Escherichia coli strains that are blocked in the amino acid sugar metabolic pathways that include the degradation of glucosamine. The start strain for the construction of all the glucosamine overproducing strains described herein was E. coli W31 10 (publicly available from American Type Culture Collection as ATCC No. 25947), a derivative of F "?", Prototrophic E Coli K-12 (Bachmann, 1987, "Escherichia coli and Salmonella typhimurium", Cellular and Molecular Bioloqv, pp. 1 190-1219, incorporated herein by reference in its entirety). A list of all strains used and produced in the following examples is provided in Table 1. Table 1 Bacterial strains llUtt? -? i lAÁíJ Í, «,,,." i-jj -at? ít.-á 10 15 20 fifteen The blocked host strains for glucosamine collection and degradation were constructed by introducing mutations in the genes nagE, manXYZ and ptsG, which block the transport of glucosamine, and the genes nagA, -B, -C, and -D. which are include in glucosamine-6-phosphate metabolism. Each of these genes has been described in detail previously herein. Mutations in these genes are introduced into strains using the transduction bacteriophage P1 v? R (as described in Miller, 1972, "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, which is incorporated herein by reference in its entirety).

In this technique, the genes or mutations of a strain (the donor strain) are transferred to a recipient strain using the bacteriophage Pv? R. When the Pv? R bacteriophage develops in the donor strain, a small portion of the phage particles that are produced contain chromosomal DNA from the donor in place of the normal complement of phage DNA. After infection of the recipient strain with the bacteriophage developed in the donor strain, those bacteriophage particles containing chromosomal DNA from the donor strain can transfer that DNA to the recipient strain. If there is a strong selection for the DNA of the donor strain, the recipient strains containing the appropriate mutation or gene of the donor strain can be selected. To develop Pv? R in a donor strain, an existing bacteriophage group was used to infect a culture of that strain. The recipient strain develops at 37 ° C in LBMC medium (1 0 g / L Bacto tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1 mM MgCl 2, 5 mM CaCl2) until the absorbance at 600 nm was approximately 1.0, which corresponds to approximately 6x108 cells per mL of culture. One mL of the culture was then infected with an elimination of the phage group in a ratio of about one phage per 10 cells. The mixture was incubated without stirring for 20 minutes at 37 ° C, then transferred to 10 mL of pre-warmed LBMC broth in a deviated Erlenmeyer flask. The resulting culture was vigorously stirred for 2-3 hours at 37 ° C. During this period, it was generally observed that the crop would be ^^^ more cloudy, indicating bacterial growth. Towards the end of the incubation period, the culture would be clear, indicating cell lysis due to bacteriophage growth. After the lysis has occurred, the culture was cooled in ice, a few drops of chloroform were added, and the flask was shaken briefly. The contents of the flask that were then centrifuged to remove cell debris and chloroform, and the resulting supernatant generally contained between 108 and 109 of bacteriophage per mL. Mutations were transferred to recipient strains By transduction with Pvir developed in the appropriate donor strain as described above, for transduction with pVr bacteriophage, a culture of the recipient strain was grown overnight at 37 ° C in LBMC broth. OJ ml_ of culture were mixed with OJ mL of bacteriophage lysate or an elimination in series of lysate in a sterile test tube and incubated at 37 ° C for 20 minutes. 0.2 mL of 1 M sodium citrate was added to the tube, and the mixture was plated on the selective medium. For each transduction, contrcontaining uninfected cells and bacteriophage lysates without cells were carried out as described above. For the production of blocked strains in glucosamine degradation, the selections were for tetracycline resistance as described below. The mutants resistant to tetracycline are selected by plating in the LB medium (10 g / L of Bacto Tryptone, 5 g / L of yeast extract, 10 g / L of NaCl) containing 12.5 μg / mL tetracycline and 10 mM sodium citrate.

Mutations in the nag genes were introduced simultaneously as an elimination mutation (? Nag:: TcR). In strain IBPC590 (Plumbridge, Table 1), which contains this mutation, the nag genes have been replaced by a determinant of tetracycline resistance (TcR). As a result, the mutation that removes the functions of nag was transferred to appropriate recipient hosts by selection of tetracycline resistance. In this case, since the determinant of TcR is contained within the mutation of interest, the Anag and TcR mutations are linked 100%. That is, all recipient strains receiving the TcR determinant of IBPC590 also received the Anag mutation. This was confirmed by examining the growth phenotype of the tetracycline resistant strains that results from infection with Pv? R developed in IPBC590. All the strains were unable to grow in media containing glucosamine or N-acetylglucosamine as carbon sources, indicating the presence of the Anag mutation. Mutations in the manXYZ and pstG genes were also introduced by P 1 v? R transduction using the known phage in strains IBPC566 and IBPC522 (Plumbridge, Table 1), respectively. These strains also contain determinants of tetracycline resistance linked to the mutations of interest (designated zdj-225 :: Tn 10 and zcf-229 :: Tn 10, respectively). In these strains, the determinants of TcR are not found within the gene but are linked to the gene. In accordance with the above, not all container strains receiving the TcR determinant contained the l? g M * É * É ^ É ^ MfaÉhl mutations of interest. The degree of binding is indicative of the distance in the chromosome between the TcR determinant and the mutation of interest. As a result, it was necessary to classify the tetracycline resistant strains for manXYZ and ptsG. ManXYZ strains grew slowly in mañosa and stopped growing in glucosamine as unique carbon sources for growth. The pstG strains grew slowly in glucose as a single carbon source. Because all selections for the mutations described above were resistant to tetracycline, it was necessary to convert sensitive tetracycline strains between the stages if multiple mutations are to be introduced. To accomplish this, the tetracycline resistant strains were plated in the TCS medium (15 g / L agar, 5 g / L Bacto tryptone, 5 g / L yeast extract, 50 mg / L chlortetracycline hydrochloride, 10 g / L of NaCl, 10 g / L of NaH2PO4 H2O, 12 mg / mL of fusaric acid, and OJ mM of ZnCl2) which selects the tetracycline sensitive mutants (described in Maloy and Jun, 1981, J. Bacteriol., 145 1 1 10-1 1 12, which is incorporated herein by reference in its entirety). The colonies that originate in this medium were purified upon re-moving in the same medium, then checking the individual colonies for the sensitivity of tetracycline to plaque in the LB medium with and without 12.5 μg / mL of tetracycline. The scheme described above for the production of strains containing combinations of mutations of manXYZ, ptsG and Anag is presented schematically in Figure 3. ,.. *? .. ^ ^., .. ^,. ..m * j * .-. i j .. . .... ... .. "^.,. ^^ 8 'ftffffiafíf Example 2 The following examples describe the cloning and overexpression of the glmS gene and the integration of the T7-glmS gene cassette into the chromosome of E. co // J 5 Cloning and overexpression of the almS gene Using the information from the published sequence of the glmS gene (Waiker ef al., 1984, Biochem J. 224: 799-815, which is incorporated herein by reference in its entirety), the primers were synthesized to amplify the genomic DNA gene isolated from Strain W31 10 (Table 1) using the polymerase chain reaction (PCR). The primaries used for the PCR amplification were designated Up1 and Lo8 and had the following sequences: Up1: 5'-CGGTCTCCCATGTGTGGAATTGTTGGCGC-3 (SEQ ID NO: 1) Lo8: 5'-CTCTAGAGCGTTGATATTCAGTCAATTACAAACA-3 '(SEQ ID 15 NO: 2 ). The primary Up1 contained sequences corresponding to the first twenty nucleotides of the glmS gene crepressed at nucleotides 10-29 of SEQ ID NO: 1) preceded by a Bsal restriction endonuclease recognition site (GGTCTC, represented in nucleotides 2). -7 of SEQ ID NO: 1). The primary Lo8 contained the sequences corresponding to the positions between bases 145 and 1 71 downstream of the glmS gene (represented at nucleotides 8-34 of SEQ ID NO: 2) preceded by a restriction endonuclease site Xbal (TCTAGA , represented in the 25 nucleotides 2-7 of SEQ ID NO: 2). The PCR amplification is m. m. ,? ^ ¡* MiíMi * JMlk ». . . i M fc- .., - > ,,. ** I H, UÍ. . * "*. *." ""! »" * ~ T? T *? M *.. R.. Tm ±. ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ containing the glmS gene with base pairs 171 of DNA downstream of the gene flagged by the Bsal and Xbal sites.This DNA fragment was cloned into the pCR-Script ™ SK (+) vector (Stratagene Cloning Systems, La Joya, California) using the materials and instructions supplied by the manufacturer The resulting plasmid was designated pKLN23-20 One consequence of this cloning was that it was placed in a unique Sacl restriction endonuclease site downstream of the gene.This allowed the excision of a DNA fragment containing the glmS gene of pKLN23-20 using Bsal and Sacl restriction endonucleases This fragment was then cloned between the Ncol and Sacl sites of the pET-24d (+) expression vector (Novagen, Inc., Madison, Wisconsin) to generate the plasmid pKLN23-23 The expression vector pET-24d (+) is based on the promoter system T7 (Studier and Moffatt, 1986, J. Mol. Biol., 189: 1 13-130). Cloning in this manner resulted in the placement of the glmS gene behind the 17-lac promoter contained in pET-24d (+). The T7- / ac promoter is specifically recognized by the T7 RNA polymerase and is only expressed in strains containing a cloned 1 T7 gene, which encodes the T7 RNA polymerase. The cloned T7 polymerase gene is contained in a phage? of defective bacteriophage designated? D3. The strains in which the? D3 element is integrated into the chromosome contain the T7 RNA polymerase gene driven by the lacUVd promoter. In those strains, the expression of the polymerase gene of iii H 1 1I11 f lfajiiinif? -? í ruiM'ilrt. .nii niiiiii m. 1 mVthá M ámá b m? t ^ Am Mt Á? t á? i T7 RNA can be induced using isopropylthio-β-D-galactopyranoside lactose analogue (IPTG). Accordingly, the addition of IPTG to such cultures results in the induction of the T7 DNA polymerase gene and the expression of any gene controlled by the T7- / ac or T7 promoter. To verify that pKLN23-23 contained the glmS gene driven by the 17-lac promoter, the plasmid was transferred to strain BL21 (DE3) (Novagen, Inc.) (Table 1). Strain BL21 (DE3) / pKLN23-23 was developed in duplicate in LB medium containing 50 mg / L of kanamycin (resistance to kanamycin is encoded by the plasmid). One of the duplicates is induced with 1 mM of IPTG, the other does not. when the total proteins were examined from these two cultures by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a prominent protein of approximately 70,000 of molecular weight, which corresponds to the predicted sfor the gene product glmS, was observed in the cells of the induced culture but not in the non-induced culture cells. A preliminary enzyme analysis of an induced culture indicated the activity of glucosamine-6-phosphate synthase several hundred times higher in the induced culture that has typically been observed in a wild-type strain. Integration of the 77-glmS gene cassette in the E. coli chromosome The glmS gene driven by the T7- / ac promoter (the cassette gel T7-glmS) was transferred to the E. coli strains of chromosome through a multiple process. First, the cassette was cloned into the pBRINT-Cm plasmid (Balbás ef al., Gene 96: 65-69), which is incorporated herein by reference in its entirety). The gene cassette was then integrated into the chromosome of strain ATCC47002 (Table 1) 5 by the techniques described by Balbás ef al., Supra to generate strains T-71 and T-81 (Table 1). Finally, the gene cassette was transferred to the strains of interest by transduction with PVIR, as described below. The 17-glmS cassette was excised from pKLN23-23 by carrying carried out a partial digestion of the restriction endonuclease plasmid BgA \ and a complete digestion with restriction endonuclease HinO \\\. Plasmid pKLN23-23 contains a Bgr / ll site about 20 base pairs upstream of the T7 promoter. The glmS gene also contains two Bg ?. A partial digestion with this enzyme was necessarily to cut the plasmid upstream of the T7 promoter while avoiding the two internal sites. The plasmid pKLN23-23 also contains a single site - / Vi DIN downstream of the glmS gene. The excised T7-glmS cassette was then cloned between the unique SamHI and HinDIW sites of pBRINT-Cm. This resulted in the production of designated plasmids pKLN23-27 and pKLN23-28. The plasmids pKLN23-27 and pKLN23-28 contain the cassette of J7-glmS in addition to a determinant chloramphenicol resistance flanked by the 5 'and 3' termini of the E. coli lacZ gene. Strain ATCC 47002 (Table 1) contains the mutations that confer an inability to maintain such plasmids as pBRINT-Cm that contain a Co1 E1 origin of replication. When such plasmids are transferred to this strain, the selection of genetic markers contained in the plasmid results in the integration of the plasmid into the chromosome (Balbás et al., 1996 supra). As mentioned above, plasmids pKLN23-27 and -28 containing the T7-glmS cassette and a chloramphenicol resistance determinant flanked by the 5 'and 3' termini of the E. coli lacZ gene. The lacZ sequences direct the incoming DNA to the lacZ gene contained in the chromosome. The integration in the lacZ site replaces the intact lacZ gene, which encodes the β-galactoside enzyme, with a partial lacZ gene interrupted by the T7-g / mS-Cm cassette. As a result, the integration of lacZ results in the strain becoming negative β-galactosidase. The plasmid also contains a determinant of ampicillin resistance far from the cassette of 5'-lacZ-T7-glmS-Cm-lacZ-3 '. The integration in lacZ and the loss of plasmid also results in sensitivity to ampicillin. Plasmids pKLN23-27 and -28 were transferred to strain ATCC 47002 and the cells plated in the LB medium containing 10 μg / ml chloramphenicol, 1 mM IPTG, and 40 μg / ml substrate. -bromo-chloro-3-idolyl-β-galactosidase. Hydrolysis of X-gal by β-galactosidase results in a blue derivative. The inclusion of X-gal and IPTG, which induces the native lacZ gene, result in positive colonies of blue lacZ and negative colonies of white lacZ. The white chloramphenicol resistant colonies (lacZk negative) were then selected and purified. The strains were verified ~ * .- > ** xibt. .- ~ »< »~? Then, for the sensitivity to ampicillin when plating in the LB medium with or without 100 μg / mL of ampicillin. The integration of DNA was further confirmed using a PCR scheme as described by Balbás et al., 1 996 above. Members T-71 and T-81 (Table 1) resulted from the integration of the desired plasmid segments pKLN23-27 and pKLN23-28, respectively, into chromosome ATCC 47002. The T7-g / mS-Cm cassette it was then transferred to strains W31 10 (DE3), 71 01 -9 (DE3), 7101 -17 (DE3) and 21 23-4 (DE3) by transduction P1 vjr, as described in Example 1, using the lysates prepared in strains T-71 and T-81. These strains contain various combinations of the Anag manXYZ and ptsG mutations in addition to the? DE3 element necessary for the expression of the T7- / ac promoter. The? DE3 element was introduced to these strains using the? DE3 lysogenization equipment by Novagen, Inc. (Madison, Wisconsin). The transducers were selected on AgrA LB plates containing 30 μg / mL of chloramphenicol and 10 mM of sodium citrate. The loss of β-galactosidase activity was verified on plates containing X-gal and IPTG and the integration of DNA was further confirmed using a PCR scheme as described by Balbás et al., 1 996 supra. The activity of the glucosamine-6-phosphate synthase was measured in the production strains containing integrated 17-glmS cassettes after growth in LB medium with and without IPTG (Table 2). The glucosamine-6-phosphate synthase was analyzed in '' i -? SBiifc crude cell extr using either colorimetric or spectrophotomeric analyzes (Badet et al., 1987, Biochemistry 26: 1940-1948) as described below. The extr used for those analyzes were prepared by suspending cell pellets rinsed in 5 mL of OJ M of KH2PO / K2HPO4, pH 7.5 per gram of wet cell paste, which passes the suspension through a French press at 16,000 psi, and centrifuges the cell suspension interrupted at 35,000-40,000 xg for 15 to 20 minutes. The supernatant was used as the enzyme source for the analysis. For a colorimetric analysis, 1 mL of reons were prepared containing 45 mM of KH2PO / K2HPO, 20 mM of fructose-6-phosphate, 15 mM of L-glutamine, 2.5 mM of IΔDTA, pH 7.5, and cell extr The reons were incubated at 37 ° C for 20 minutes and stopped when boiled for 4 minutes. The resulting precipitate was removed by centrifugation and the supernatant was analyzed for glucosamine-6-phosphate by a modification of the method of Elson and Morgan (1933, Biochem J. 27: 1824-1828) essentially as described by Zalkin (1985, Meth. Enzymol 1 13: 278-281), both publications of which are incorporated herein by reference in their entirety. To 100 μL of the above supernatant are added 12.5 μL of saturated NaHCO3, and 12.5 μL aqueous acetic anhydride 5%, barely prepared, cold. After incubation for 3 minutes at room temperature, the mixture was boiled for 3 minutes to remove excess acetic anhydride. After cooling to room temperature, 150 μL of 0.8 M ^^ potassium borate, pH 9.2 (0.8 M H3BO3 adjusted to pH 9.2 with KOH) was added and the mixture boiled for 3 minutes. After cooling to room temperature, 1.25 mL of Ehrlich reagent (1% p-dimethylaminebenzaldehyde in glacial acetic acid containing 0.125 N HCl) was added to each tube. After incubation at 37 ° C for 30 minutes, the absorbance at 585 nm was measured and the amount of glucosamine-6-phosphate formed was determined using a standard curve. In the absence of the substrate, fructose-6-phosphate, or when the boiled extr are used in the analysis, no significant absorbance was observed at 585 nm. In the spectrometric analysis, 1 mL of reons containing 50 mM KH2PO / K2HPO4, 10 mM fructosad-phosphate, 6 mM L-glutamine, 10 mM KCl, 0.6 mM acetylpyridine adenine dinucleotide (APAD), and 50-60 L-glutamic dehydrogenase units (Sigma Type II from bovine liver) were passed at room temperature. The vity was monitored by monitoring the absorbance at 365 nm after the addition of the extrand corrected for the small increase in absorbency in the absence of the extr The vity was calculated using a molar extinction coefficient for APAD of 9100.

Hey? Table 2 Glucosamine-6-Phosphate Synthase vity in Production Strains Containing Integrated T7-glmS Cassettes Table 2 shows that, on average, the glucosamine-6-phosphate synthase vity in production strains containing integrated T7-glmS cassettes was approximately three to four times higher with induction of ITPG than without it. The vities were substantially higher than those obtained with a wild-type glmS strain directed by its native promoter, which are typically in the range of 0.05-0.1 μmole per minute per mL of extr One of the strains, 2123-6, apparently suffers from an aberrant integration event since the vity is lower than that observed in other strains and was not influenced by the presence of IPTG in the medium. Example 3 The following example shows the effect of the strain genotype on glucosamine accumulation. Strains with members of T7-glmS, produced as described in Example 2, as well as the origin strains ^^^^ p ^ ^ j ^ ^^^^^ a ^? * A Éj ^, corresponding without integrated DNA, grew in shake flasks containing M9A medium (14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3 Citrate2H2O, 5 g / L of (NH4) 2SO4, pH 7.0) supplemented with 20 g / L of glucose, 10 mM of MgSO, 1 mM of CaCl2, and 1 mM of IPTG. The samples were taken periodically during the course of two days and the concentration of glucosamine in the culture supernatant was measured using the modified Elson-Morgan analysis as described in Example 2. The samples were analyzed with or without acetic anhydride treatment, and the amount of glucosamine present was determined from the net absorbency using a standard curve. The concentrations of glucosamine after 24 hours of cultivation, at which time the maximum concentration, is indicated in Table 3. The results shown in Table 3 indicate that for significant glucosamine production to occur, the T7-glmS cassette must be present. The data also indicate that the presence of the Anag mutation in the host results in a significant increase in glucosamine accumulation compared to its absence. Little effect of the manXYZ mutation was observed in this experiment. In additional shake flask experiments, however, strain 2123-12 accumulated slightly higher glucosamine concentrations on a consistent basis.

Table 3 Glucosamine in Culture Supernatants of Production Strains Strain Genotype Concentration of Glucosamine, mg / L (24 hours) 2123-5 DE3, member T-71 21 2123-7 DE3, member T-71 23 2123-9 DE3 Anag, member T-71 267 2123-10 DE3 Anag, member T-81 80 2123-11 DE3? nag manXYZ, member T-71 65 2123-12 DE3 Anag manXYZ, member T-8 63 W3110 (DE3) DE3 Anag manXYZ, member T-71 4 7101-9 (DE3) DE3 Anag, without member 0 7101-17 (DE) DE3 Anag manXYZ, without member 0 EXAMPLE 4 The following examples demonstrate the effect of feeding nutrients to the cultures that it has in glucosamine accumulation.

In previous experiments, it was observed that glucosamine accumulation ceased when glucose was decreased from the cultures. In the experiment summarized in Table 4 and Figure 4, it was found that the increased glucosamine accumulation could be carried out by feeding additional glucose and ammonium sulfate and decreasing. For this experiment, strain 2123-12 was grown in M9A medium supplemented with 1.0mM MgSO4, 1mM CaCl2, and 1mM IPTG. Additional glucose concentrations and feed rates were varied as indicated in Table 4. In one of the flasks, the initial ammonium sulfate concentration was 10 g / L instead of 5 g / L normally used in the medium M9A. The glucose concentration was monitored in shake flasks during cultivation using Diastrix® glucose test tapes (Bayer Corporation, Diagnostics Division, Elkhart, Indiana). When the glucose concentration is decreasing or near the decrease (<5 g / L remaining), glucose and / or ammonium sulfate were supplemented as indicated in Table 4. pH was also monitored during cultivation. When the pH varied significantly from the initial pH of 7.0, it was adjusted to 7.0 with sodium hydroxide. Table 4 Agitation Flask Experiment to Examine the Effect of Glucose Feeding As Figure 4 indicates, the increase in glucose supply had a positive effect on glucosamine accumulation. When fed periodically with glucose and ammonium sulfate (additions of 20 g / L and 5 g / L, respectively), a maximum accumulation of 0.4 g / L of glucosamine was observed, approximately four times higher than when observed in the absence of food. Example 5 The following examples describe the isolation of mutant glmS genes encoding the glucosamine-6-phosphate synthase enzymes with reduced sensitivity to inhibition of the glucosamine-6-phosphate product. White (1968, Biochem, J. 106: 847-858) first demonstrated that glucosamine-6-phosphate synthase was inhibited by glucosamine-6-phosphate. Using the spectrophotometric analysis for the glucosamine-6-phosphate synthase as described in Example 2, the effects of glucosamine-6-phosphate and glucosamine or glucosamine-6-phosphate synthase were measured. For the determination of product inhibition, the analyzes were carried out in the presence of various concentrations of added glucosamine-6-phosphate. As indicated in Figure 5, the enzyme is significantly inhibited by glucosamine-6-phosphate and is slightly inhibited by glucosamine. These results are similar to those obtained by White, 1968, supra. This inhibition may be a key factor in limiting the accumulation of glucosamine in the glucosamine production strains. To further increase the synthesis of glucosamine in production strains, efforts were made to isolate the mutants of the glmS gene encoding the glucosamine-6-phosphate synthase variants with inhibition of the reduced product. To accomplish this, the cloned gene was amplified using the error prone PCR technique. In this method, the gel is amplified under conditions that lead to a high frequency of misincorporation errors by the DNA polymerase used for the amplification. As a result, a high frequency of mutations are obtained in the PCR products.

Plasmid pKLN23-28 contains a unique Spel restriction endonuclease site of 25 base pairs upstream of the T7 promoter and 1 1 1 base pairs upstream of the start of the glmS gene. The plasmid also contains a unique Hin \\\ site of 1 17 base pairs downstream of the glmS gene. The PCR primaries of the following sequences were synthesized to correspond to the regions just upstream of the Spel site and downstream of the Hin site \\\, respectively: 5'-ATGGATGAGCAGACGATGGT-3 '(SEQ ID NO: 3) 5'CCTCGAGGTCGACGGTATC -3 '(SEQ ID NO: 4). Amplification with these primaries (SEQ ID NO: 3 and SEQ ID NO: 4) allowed the mutagenesis of a region of 2247 base pairs that included the total glmS gene. PCR conditions are as described by Moore and Arnold, 1996, Nature Biotechnology 14: 458-467, which is incorporated herein by reference in its entirety. Briefly, a solution of 1 00 μL was prepared containing 1 mM of each of the four deoxynucleotide triphosphate, 16.6 mM ammonium sulfate, 67 mM Tris-HCl, pH 8.8, 6.1 mM MgCl2, 6.7 μM EDTA, 10 mM of β-mercaptoethanol, 10 μL of DMSO, 30 mg of each of the primaries (SEQ ID NO: 3 and SEQ ID NO: 4), either 7 or 35 ng of plasmid pKLN23-28 linearized with Kpn I, and 2.5 Units of Taq DNA polymerase (Perkin Elmer-Cetus, Emervylle, Califormin). The reaction mixture was covered with 70 μL of mineral oil and placed in a thermal cycler, where the following stages were repeated for 25 cycles: JILALfeL. "Use 1 minute at 94 ° C 1 minute at 42 ° C 2 minutes at 72 ° C Under these conditions, an error frequency of about one mutation per 1000 base pairs has been reported (Moore and Arnold, 1996, supra) . The product of the reaction was recovered, purified and digested with Spel and HinDIW, and cloned into the Spel-H / nDIII backbone fragment of pKLN23-28, which effectively replaces the wild type glmS gene in the Spel-H fragment. DIII of pKLN23-28. The cloned DNA was used to transform the NovaBlue strain (Novagen, Inc., Madison, Wisconsin), and the transformed cells were plated on an LB agar containing ampicillin. A total of 9,000 plasmid-containing colonies were pooled from the ampicillin plates and the plasmid DNA was prepared from the pooled cells to generate a library of plasmids derived from pKLN23-28 containing mutations in the cloned glmS gene. The mutant plasmids generated by error-prone PCR were classified by their ability to confer increased glucosamine production on a host structure Anag manXYZ DE3. This selection was found in the form of a bioanalysis in which the ability of strains containing plasmids to feed strains requiring glucosamine from E. coli were assessed. The strains of E. coli (Sarvas, 1971, J. Bacteriol. 105: 467- ^ jg ^ 471; Wu and Wu, 1971, J. Bacterio], 105: 455-466) and Bacillus subtilitis (Freese et al., 1970, J. Bacteriol. 1 01: 1046-1 062) which are defective for glucosamine-6 synthase. -phosphate require glucosamine or N-acetylglucosamine for its growth. A strain that requires glucosamine from E. coli was isolated after mutagenesis with N-methyl -? / '- nitro -? / - nitrosoguanidine (NG). Strain LE392 (Table 1) was grown in LB medium at a cell density of 36 x 108 cells per mL. 50 μL of 2.5 mg / ml of NG dissolved in methanol was added to 2 mL of this culture and the mixture was incubated at 37 ° C for 20 minutes. The treatment resulted in approximately 10% survival of the strain. The mutagenized cells were harvested by kelp, and the cells were rinsed twice by suspension in 0.9% NaCl and recentrifugation. The rinsed cells were diluted and plaques were placed in nutrient agar medium (NA, 5 g / L of Bacto peptone, 3 g / L of beef extract, 15 g / L of agar) containing 0.2 g / L of N-acetylglucosamine at a density of between 50 and 2000 units that form colonies per plate. Approximately 1 3, 000 hills were placed on plates. These colonies were replicated by plate to NA agar with or without 0.2 g / L of N-acetylglucosamine. Twenty-two colonies grew in NA with 0.2 g / L of N-acetylglucosamine but not in NA without 0.2 g / L of N-acetylglucosamine. These colonies were purified by moving to NA with 0.2 g / L of N-acetylglucosamine, and their growth phenotype was checked again. If the 22 original colonies were selected, five had the expected phenotype of a glmS mutant of Le392. They stopped growing in NA • • * * - * - - ** • * "* *** ~ ** - • * - -, r ?? .h? -?. - BM JS ** - 100 - but they grew in NA supplemented with 0.2 g / l of glucosamine or 0.2 g / L of N-acetylglucosamine TasfjbJén stopped growing on minimal glucose agar, but grew on minimal glucose agar supplemented with 0.2 g / L of N-acetylglucosamine One of these 5 mutants was designated 2123-16 (Table 1) For the transverse feed analysis, agar plates containing either glycero or fructose as the main carbon source for growth were coated with cells from a culture of strain 2123-16, the strain which requires glucosamine isolated as described above. The strains that produce glucosamine were traversed in the agar and the ability to produce glucosamine was assessed based on the size of the growth "halo" of the indicator strain that surrounds the gap. Those holes surrounded by longer halos are considered to produce larger quantities of glucosamine. The media used for the cross-feed analyzes consisted of M9 minimal medium (6 g / L of Na2HPO4, 3 g / L of KH2PO4, 0.5 g / L of NaCl, 1 g / L of NH4CI, 1 mM of MgSO4, 0J mM of CaCl2) supplemented with 40 mg / L of L-methionine (required for the growth of strains LE392 or 2123-16) and 2 g / L of either glycerol or fructose. These plates were covered with strain 2123-16 as follows. A culture of strain 2123-16 was grown overnight at 37 ° C in LB medium containing 1 g / L of N-acetylglucosamine. The culture was collected by centrifugation, and cells were rinsed twice by suspension in 0.9% NaCl ? V * 101 and recentrifugation. The rinsed cells were suspended in the original volume of 0.9% NaCl. For each plate to be covered, OJ mL of rinsed cell suspension is mixed with 3 mL of upper melted F agar or (50 ° C) (8 g / nL of NaCl, 8 g / L of agar) and poured 5 onto the license plate. The mutant plasmid library pKLN23-28 was transferred to strain 7101-17 (DE3) and the transformed cells were plated on LB agar containing 100 μg / mL ampicillin. Each colony originating in these plates contained an individual membrane of the mutant plasmid library. The colonies were classified by choosing them from the LB + ampicillin plates and sequentially traversed in: (1) LB + ampicillin agar: (2) minimal glycerin agar covered with strain 2123-16; and 15 (3) minimum fructose agar covered with strain 2123-16. All plates were incubated for approximately 24 hours at 37 ° C. After this incubation period, the growth halos of indicator strain 2123-16 could be observed surrounding the gaps in the covered plates. Those colonies that give rise to the larger halos are harvested from the corresponding LB + ampicillin plate and move for purification. In an initial classification, 4368 candidate mutants are classified, and 96 initial candidates were identified. In the reclassification those, 30 seemed to be superior to the rest, that is, they resulted in Gauls largest of the indicator strain.

Enzyme analyzes carried out with six strains containing isolated plasmid as described above indicated that three of the strains were less sensitive to glucosamine-6-phosphate inhibition than the enzyme of control strain 7101-17 (DE3) / pKLN23-28. The strains were grown overnight in LB broth containing 100 μg / mL of ampicillin and 1 mM of IPTG. Extracts prepared from the cells harvested from those cultures were analyzed for the glucosamine-6-phosphate synthase using the spectrophotometric analysis (described in Example 2) in the presence or absence of added glucosamine-6-phosphate. The mutant clones designated 1 1 C, 65A and 8A were significantly less sensitive to glucosamine-6-phosphate than the control strain (Figure 6). Other mutants were not distinguished from the control by this analysis. Example 6 The following example describes the construction and characterization of glucosamine production strains with mutations in glmS that result in inhibition of the reduced product. Plasmid DNA was isolated from clones 1 1 C, 52B and 8A described above, were transferred to ATCC 47002, which has previously been used to integrate the construction of T7-glmS into the chromosome of E. coli. The integration was carried out easily using the methods described in Example 2, and the integrated DNA was transferred to strain 7101-17 (DE3) by transduction P1 as described in Example 1. These procedures produced strains that have the same genotype as strain 2123-12 except for ??? áá ^ áÉÉ i the presence of mutations in the glmS gene generated by PCR. These new mutant production strains were designated 2123-49, 2323-51 and 2123-54, respectively. A summary of the strain construction strategy is presented in Figure 7. Strains 2123-12, 2123-49, 2123-51 and 2123-54 were grown overnight in LB broth containing 1 mM of IPTG. Extracts prepared from the cells harvested from those cultures were analyzed for glucosamine-6-phosphate synthase using the spectrophotometric analysis described in Example 2 in the presence and absence of added glucosamine-6-phosphate. The results of these analyzes are shown in Figure 8. The production of glucosamine in these mutants was significantly increased compared to that in 2123-12. When the glucosamine production was analyzed in the shake flask the cultures were developed using the ammonium sulfate feed process and the glucose previously described in Example 4, when the cultures were grown at a cell density (measured as OD eoo) of approximately 14 (approximately 8.4 g / L in dry cell weight), strains 2123-49, 2123-51 and 2123-54 produced 1.5, 2.4 and 5.8 g / L of glucosamine, respectively (Figure 9) compared to 0.3 g / L. L of 2123-12. Example 7 The following example describes the production of yeast from another strain with a mutation in glmS resulting in inhibition of the reduced product. 'ifT ^^ tMBfr, ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 6,344 additional colonies containing mutant plasmids generated by error-prone PCR as described in Example 5 were classified using the cross-feed analysis, also described in Example 5. Fifty-four hills resulted in larger halos than the rest of the colonies. The DNA was isolated from all 54 colonies and isogenic strains at 2123-12 except for the mutations in glmS that were constructed as described in Example 6. The glucosamine production in most of these mutants was significantly increased compared to the strain 2123-12. Among the recently isolated mutants, the strain that produced the most glucosamine was a designated strain 2123-24. This strain produced 3.6 g / L of glucosamine when the production was analyzed in shake flasks using the ammonium sulfate feed process and the glucose described in Example 4 compared to 4.3 g / L for the strain 2123-54 in a experiment from side to side. EXAMPLE 8 The following examples describe the sncing of the cloned wild type glmS gene present in the pásmid pKLN23-28. In addition, the snces present in the plasmids pKLN23-49, pKLN23-54, and pKLN23-124, which contains the mutant glmS genes used for the construction strains 21 23-49, 2123-54 and 2123-124, respectively, were snced and They describe. The DNA sncing reactions were carried out ^^^ out using the sncing method of Tintan Prism Cycle of Applied Biosystems (ABl) with DNA AmpliTaq polymerase. The extended products were separated by gel electrophoresis in an ABI 373A or 377 DNA sncer. The snces were analyzed using software 3.0 for the ABI Sncing Analysis of AB1 and Sncher 3.1 of Gene Codes. The primaries used for sncing are as follows: PK-1: 5'-TGGATGAGCAGACGATGG-3 '(SEQ ID NO: 5) PK-2: 5'-TCCGTCACAGGTATTTATTC-3' (SEQ ID NO: 6) PK-3: 5 '-AGCTGCGTGGTGCGTAC-3' (SEQ ID NO: 7) PK-4: 5'-GGACCGTGTTTCAGTTCG-3 '(SEQ ID NO: 8) PK-5A: 5'-GCCGTGGCGATCAGTAC-3' (SEQ ID NO: 9) PK -6A: 5'-GCCAATCACCAGCGGAC-3 '(SEQ ID NO: 10) PK-7: 5'-ATGGTTTCCCGCTACTGG-3' (SEQ ID NO: 1 1) PK-8: 5'-GAACCAAGGTAACCCAGC-3 '(SEQ ID NO: 12) Plasmid pKLN23-28 nucleotide snces, which contain the wild-type glmS gene, were determined to be a 7408 bp nucleic acid snce depicted herein as SEQ ID NO: 13. The 2184 base pairs between positions 1 1 30 and 3313 of SEQ ID NO. 13 were determined using the primaries described above. The nucleic acid molecule representing positions 1 1 30-331 3 of SEQ ID NO: 1 3 is herein referred to nglmS-282? 84 and is further identified as SEQ ID NO: 14. It was shown that nglmS- 28218 includes the Spel and H / nDIII sites used to a -a ftj. It is possible to construct the mutant plasmids as described in Example 5. The remaining DNA sequence of SEQ ID NO: 13 is based on the known sequences of the vectors used for the construction of pKLN23-28. The same region of 21 84 base pairs was sequenced in plasmids pKLN23-49, pKLN23-54 and pKLN23-124. It is noted that for discussion of the mutant glmS genes of these pyramids (Table 5), the nucleotide position specific for mutations in the nucleotide sequence of the plasmids containing mutant glmS will be described using SEQ ID NO: 1 3 as a reference. SEQ ID Nos. 13 and 14 contain an open reading frame that encodes the glmS gene product (ie, GlcN6P synthase enzyme) which is a nucleic acid molecule referred to herein as nglmS-28Δ830, the sequence of nucleic acid of which SEQ ID NO: 15 is represented. SEQ ID NO: 15 measures nucleotides 1253 to 3082 of SEQ ID NO: 13, with a codon measurement of nucleotides 1253-1255 and a codon measurement of termination of nucleotides 3080-3082. SEQ ID NO: 1 5 encodes the 609 amino acid protein referred to herein as GlcN6P-S-28, the deduced amino acid sequence which is represented herein as SEQ ID NO: 16. It is noted that for the discussion of the mutant glmS gene products produced by the mutant strains described herein, specific mutations in the amino acid sequence of the mutant glmS gene products will be described using SEQ ID NO: 16. The primaries described above correspond to the i »following nucleotide positions of SEQ ID NO: 13: PK-1 (SEQ ID NO: 5): positions 1087-1 104 of SEQ ID NO: 1 3; PK-2 (SEQ ID NO: 6): positions 3378-3359 of a nucleic acid sequence complementary to SEQ ID NO: 1 3; PK-3 (SEQ ID NO: 7): positions 1707-1723 of SEQ ID NO: 13; PK-4 (SEQ ID NO: 8): positions 2772-2755 of a nucleic acid sequence complementary to SEQ ID NO: 13; PK-5A (SEQ ID NO: 9): positions 2667-2686 of SEQ ID NO: 13; PK-6A (SEQ ID NO: 10): positions 1798-1782 of a nucleic acid sequence complementary to SEQ ID NO: 13. PK-7 (SEQ ID NO: 1 1): positions 2177-2194 of SEQ ID NO: 1 3. PK-8 (SEQ ID NO: 12): positions 2364-2347 of a nucleic acid sequence complementary to SEQ ID NO: 13. The nucleic acid sequence of the nglmS-28? 830 nucleic acid molecule (SEQ. ID NO: 15, or positions 1253-3082 of SEQ ID NO: 1 3) of pKLN23-28, differs from the published sequence (Waiker, JE ef al., 1984"DNA sequencing around the Escherichia coli une operon", Biochem J. 224: 799-815) at positions 2509 and 2510 (with reference to SEQ ID NO: 13). The nucleotides for pKLN23-28 in these positions as determined in this example were G and C, respectively, while those reported in the published sequence were C and G. Otherwise, the published and determined sequences of the glmS gene were identical. The sequences determined upstream and downstream of the gel glms are those expected based on the known sequences of the vectors i i used for the construction of pKLN23-28 and the methods used to construct the plasmid. The nucleotide sequences for the glmS genes for plasmids pKLN23-49, pKLN23-54 and pKLN23-124 were determined as described above for pKLN23-28. Mutations were found in each of the plasmids. The mutations and amino acid changes predicted in the corresponding mutant glmS gene products, compared to the wild-type sequence determined by pKLN23-28 (SEQ ID NO: 13) are summarized in Table 5. Table 5 GlmS gene mutations of Strains that produce Glucosamine Plasmid Position * Base Change Amino Acid Change (Position **) pKLN23-49 1263 T a C lie a Thr (4) 2067 T a C He a Thr (272) 2600 T a C lie a Thr (450) PKLN23-54 1367 G a A Wing to Thr (39) 2000 C to T Arg a Cys (250) 2239 T a C Silence (329) 2666 G aA Gly to Ser (472) 3264 A to G External Gen PKLN23-124 1525 T a C Silence (91) 2658 T a C Leu a Pro (469) 3280 G a A External gene * Refers to nucleic acid position as indicated in sequence pKLN23-28 (SEQ ID NO: 13) ** The glmS gene (nglmS-281 ß3o; SEQ ID NO: 15) encodes a protein of 609 amino acids in length (SEQ ID NO: 16); the methionine residue in position 1 is removed by a hydrolase Plasmid pKLN23-49 contains a 2184 bp nucleic acid molecule referred to herein as nglmS-49218, which comprises a mutant glmS gene. The nucleic acid sequence of nglmS-492β8 is represented herein as SEQ ID NO: 17. A measurement of the nucleic acid molecule of nucleotide 124 to 1953 of SEQ ID NO: 17, referred to herein as nglmS -49? 83o, represents an open reading frame encoding a mutant glucosamine-6-phosphate synthase of the present invention, with a measurement of the start codon of nucleotides 124-126 and a measurement of the final codon of nucleotides 1951-1 953 of SEQ ID NO: 17. The nucleic acid sequence of nglmS-49183o is depicted herein as SEQ ID NO: 18. SEQ ID NO: 18 encodes a mutant glucosamine-6-phosphate synthase protein of 609 amino acids referred to herein as GlcN6P-S-49, the deduced amino acid sequence which is represented herein as SEQ ID NO: 19. SEQ ID NO: 17 has a nucleic acid sequence that is identical in the positions 1 130 to 331 3 of SEQ ID NO: 13 (from cir, SEQ ID NO: 14), except for the mutations indicated for the plasmid pKLN23-49 in Table 5. SEQ ID NO: 18 15 has a nucleic acid sequence that is identical at positions 1253 to 3082 of SEQ ID NO: 1 3 (ie, SEQ ID NO: 15) except that the mutations indicated for the plasmid pKLN23-49 in Table 5. Plasmid pKLN23-54 contains a 2184 bp nucleic acid molecule referred to herein as nglmS -542184, which comprises a mutant glmS gene. The nucleic acid sequence of nglmS-542? 84 is represented herein as SEQ ID NO: 20. A measurement of the nucleic acid molecule of nucleotide 124 to 1953 of SEQ ID NO: 20, referred to herein as nglmS-541830, represents an open reading frame encoding a synthase i ^^^ & ^^^^^ ßj ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ »^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ of the start codon of nucleotides 124-126 and a measurement of the final codon of nucleotides 1951 -1953 of SEQ ID NO: 20. The nucleic acid sequence of nglmS-541830 is represented herein as SEQ ID NO: 21. SEQ ID NO: 21 encodes a mutant glucosamine-6-phosphate synthase protein of 609 amino acids referred to herein as GlcN6P-S-54, the deduced amino acid sequence which is depicted herein as SEQ ID NO: 22. SEQ ID NO: 20 has a nucleic acid sequence that is identical at positions 130 to 3313 of SEQ ID NO: 13 (ie, SEQ ID NO: 14), except for the mutations indicated for the plasmid pKLN23- 54 in Table 5. SEQ ID NO: 21 has a nucleic acid sequence that is identical at positions 1253 to 3082 of SEQ ID NO: 13 (ie, SEQ ID NO: 15) except that the mutations indicated for the Plasmid pKLN23-54 in Table 5. Plasmid pKLN23-124 contains a 2184 bp nucleic acid molecule referred to herein as nglmS-1242184, which comprises a mutant glmS gene. The nucleic acid sequence of nglmS-1242? 8 is represented herein as SEQ ID NO: 23. A measurement of the nucleic acid molecule of nucleotide 124 to 1953 of SEQ ID NO: 23, referred to herein as nglmS-1241830) represents an open reading frame encoding a mutant glucosamine-6-phosphate synthase of the present invention, with a measurement of the start codon of nucleotides 124-126 and a OR? M > . * .A ^? , Measurement of the final codon of nucleotides 1951 -1953 of SEQ ID NO: 23. The nucleic acid sequence of nglmS-1241830 is represented herein as SEQ ID NO: 24. SEQ ID NO: 24 encodes a mutant glucosamine-6-phosphate synthase protein of 5 609 amino acids referred to herein as GlcN6P-S-124, the deduced amino acid sequence which is depicted herein as SEQ ID NO: 25 . SEQ ID NO: 23 has a nucleic acid sequence that is identical at positions 130 to 3313 of SEQ ID NO: 13 (ie, SEQ ID NO: 14), except for mutations indicated for the plasmid pKLN23-124 in Table 5. SEQ ID NO: 24 has a nucleic acid sequence that is identical at positions 1253 to 3082 of SEQ ID NO: 13 (ie, SEQ ID NO: 15) except that the mutations indicated for the plasmid pKLN23-124 in Table 5. 15 To verify that the same mutations occur in the strains in which the mutant glmS genes were integrated into the chromosome, the PCR products were generated from isolated genomic DNA of strains 2123-49, 2123-54 and 2123-124. For PCR amplification, the primary ones lit in Example 3 for the mutagenesis of the gene (SEQ ID NO: 3 and SEQ ID NO: 4) were used. The PCR reactions were carried out in 50 μL reactions consisting of 20 mM Tris: HCI (pH 8.8), 10 mM KCl, 10 mM (NH4) 2 SO4; 20 mM MgSO4, 0.1% Triton X-100, 0J mg / mL bovine serum albumin without nuclease, 0.05 mM each triphosphate of deoxynucleotide, 2 μM of each primary, 1.25 polymerase of Cloned pfu DNA U (Stratagene) and 160 ng of genomic DNA. The complete reactions were placed in a Gradient 96 RoboCycler Temperature Cycle (Stratagene). After 3 minutes at 94 ° C, the following three stages were repeated for 30 cycles: (1) 30 seconds at 94 ° C; (2) 30 seconds at 47 ° C, and (3) 2 minutes at 72 °. This was followed with a 7 minute incubation at 72 ° C. The resulting DNA contained the expected amplification product in addition to the foreign products. The product containing the glmS gene was purified using a QIAquick PCR purification kit, followed by electrophoresis of the purified product on an agarose gel, isolation of the correct band using QIAquick gel extraction equipment, and re-amplification using this isolated DNA as a tempered. The reactions with the isolated DNA were amplified in a manner similar to the original amplification described above, except that 40 mg of DNA was used as a template, and only 20 cycles of amplification were carried out. The product of this second amplification reaction was recovered as described above. The presence of mutations in the genomic DNA was verified using the primaries specific for the DNA regions containing the mutations identified in the plasmids. For 2123-49, these include the primary PK-1 (SEQ ID NO: 5), PK-3 (SEQ ID NO: 7), PK-4 (SEQ ID NO: 8) and PK-5A (SEQ ID NO. : 9). For 2123-24, the primary PK-1 (SEQ ID NO: 5), PK-4 (SEQ ID NO: 8) and PK-5 (SEQ ID NO: 9) were used. For 2123-54, the entire PCR product was sequenced using the eight primaries described above (SEQ ID NOS: 5-12). Sequencing of the PCR products confirmed the presence of the mutations identified from the plasmids and are listed in Table 5. Example 9 This example describes the construction of strains containing a mutant glmS gene encoding a product containing only the alteration of glycine to serine at position 472 (SEQ ID) NO: 22) of strain 2123-54. As indicated in Table 5, the amino acid change only in the GlcN6P synthase enzyme for strain 2123-24 (GlcN6P-1 24) is an alteration of leucine to proline at position 469 (SEQ ID NO: 25) , which unambiguously defines this mutation as being responsible for the overproduction of glucosamine by strain 2123-124. This would suggest the possibility that the alteration of glycine to sera at position 472 (gly? Ser472; SEQ ID NO: 22) of GlcN6P-S-54 in strain 2123-54 was probably responsible for the overproduction phenotype of glucosamine for this strain In an effort to demonstrate this, the alteration was isolated away from the other two amino acid alterations in the amino acid sequence GlcN6P-S-54 (SEQ ID NO: 22) of strain 2123-54 (ie, Ala? Thr39 and Arg? Cys250) by digesting the plasmid pKLN23-54 with EcoRI and Hindi. These enzymes each have unique cleavage sites on the plasmid and are cut at positions 2241 and 3305, respectively (positions indicated with respect to the positions -to . .? 'k & equivalent in SEQ ID NO: 13 for pKLN23-28), resulting in fragments of 1064 and 6334 base pairs. The smallest fragment contains the mutations in which the alteration of glyser472 is the only amino acid change in this portion of GlcN6P-S-54. This smaller fragment was ligated to the corresponding longer fragment of pKLN23-28 containing the wild-type glmS gene. Two plasmids resulting from this ligation were designated pKLN23-149 and pKLN23-151. The DNA sequencing of These plasmids using the primary PK-1 (SEQ ID NO: 5), PK-3 (SEQ ID NO: 7) and PK-4 (SEQ ID NO: 8), verified that these plasmids contained the mutation at position 2666 present in plasmid pKLN23-54 but not mutations at positions 1367 and 2000 (Table 5 with reference to SEQ ID NO: 13). The nucleic acid sequence of the 2184 base pairs between positions 1 130 and 3313 of plasmid pKLN23-149 (these positions being determined relative to the equivalent positions in SEQ ID NO: 13) are referred to herein as a nucleic acid molecule nglmS-1492? 8, the nucleic acid sequence that is represents by SEQ ID NO: 26. SEQ ID NO: 26 contains a measurement of nucleotide nucleic acid sequence 124 to 1953, referred to herein as nglms-149? 830, which represents an open reading frame encoding a glucosamine-6-phosphate synthase of the present invention, with a codon measurement of start of nucleotides 124-126 and a codon measurement of termination of nucleotides 1951 -1953 of SEQ ID NO: 26. The nglms-149? 830 nucleic sequence is depicted herein as SEQ ID NO: 27. SEQ ID NO: 27 encodes a 609 amino acid glucosamine-6-phosphate synthase protein referred to as GlcN6P-5 S-149, the deduced amino acid sequence is depicted herein as SEQ ID NO: 28. The nucleic acid sequence of the 21 84 base pairs between positions 1 130 and 3313 of plasmid pKLN23-151 (these positions being determined relative to the equivalent positions in SEQ ID NO: 13) are referred to herein as nucleic acid molecule nglmS-1512Δ84, the nucleic acid sequence which is represented by SEQ ID NO: 29. SEQ ID NO: 29 contains a measurement of nucleic acid nucleotide sequence 124 to 1953, referred to herein as nglms-151? 830, which represents a The open reading frame encoding a glucosamine-6-phosphate synthase of the present invention, with a start codon measurement of nucleotides 124-126 and a nucleotide termination coding measurement 1951 -1953 of SEQ ID NO: 29. The nucleic sequence of nglms-1511830 is represented in this as SEQ ID NO: 30. SEQ ID NO: 30 encodes a 609 amino acid glucosamine-6-phosphate synthase protein referred to as GlcN6P-S-151, the deduced amino acid sequence is depicted herein as SEQ ID NO: 31. Strains isogenic to strain 2123-12 except for the mutations that confer the alteration gly? Ser472 were constructed ^? ^^^^^^^^^^^^^^ A ^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^^^^^ 4 using the scheme indicated in Figure 7. Strains 2123-2149 and 2123-151 were generated from plasmids pKLN23-149 and pKLN23- 151, respectively. The presence of a mutation at position 2666 (SEQ ID NO: 13) and the absence of mutations at positions 1367 5 and 2000 were verified by sequencing the PCR products of genomic DNA of these strains using the methods described in Example 8. Example 10 This example compares the properties of the enzymes of synthase of GIcNdP strains 2123-12, 2123-49, 2123-54, 2123-124, 2123-149 and 2123-151. Strains 2123-12, 2125-49, 2123-54, 2123-124, 2123-149 and 2123-151, described in the previous examples, are grown overnight in LB broth at 37 ° C then transferred to broth LB fresh. The cultures were grown at an absorbance at 600 nm from 0.8 to 0.9, then induced to produce GlcN6P synthase by the addition of 1 mM of IPTG. The cultures were grown for an additional three hours at 37 ° C and harvested. The extracts were prepared from the cells harvested from those cultures as described Example 2 and were analyzed for glucosamine-6-phosphate synthase using the spectrometric analysis as described in Example 2 except that a concentration of 20 mM fructose-6-phosphate is used. The enzyme was analyzed in the presence and absence of added glucosamine-6-phosphate. In the absence of glucosamine-6-phosphate, specific activities measurements for these enzymes were similar except for that of strain 2123-124. The data in Table 6 suggest that the last strain encodes at least one active variant of the enzyme. Table 6 Specific Activities of GIcNßP Synthase from Strains that Produce Glucosamine Strain Specific Activity, μmol min "1 mg" 1 2123-12 0.385 2123-49 0.375 2123-54 0.416 2123-124 0.0076 2123-149 0.494 2123-151 0.515 10 Figure 10 shows that the GlcN6P synthase enzymes of strains 2125-49, 2123-54 and 2123-124 are significantly less inhibited by GlcN6P than the enzyme of strain 2123-13. Enzymes of strains 2123-149 and 2123-51 are inhibited slightly less for GlcN6P than the 2123-12 enzyme. The thermal stability of the enzymes was also examined using these extracts. The extracts were incubated at 45 ° C (Figure 1 1 A) or 50 ° C (Figure 1 1 B) for several periods then analyzed using spectrophotometric analysis. Figures 1 1A and 1 1 B show that the enzymes of 2123-49 and 2123-124 are much less stable than the wild-type enzyme of strain 2123-12. The enzyme of this strain 2123-124 is comparable in stability with the wild-type enzyme and the enzymes of 2123-149 and 2123-151 are slightly less stable under the incubation conditions described herein.

Example 1 1 The following example illustrates the effects of the concentration of isopropylthio-β-D-galactoside (IPTG) and temperature of glucosamine production. Cultures of strains 2123-54 and 2123-124 were grown for 20 hours at 37 ° C in M9A medium (14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3 Citrate2H2O, 5 g / L). L (NH4) 2SO4, pH 7.0) supplemented with 20 g / L glucose, 10 mM MgSO, 1 mM CaCl2, and various amounts of IPTG. At the end of the growth period, a sample is taken and the concentration of glucosamine in the culture supernatant is analyzed using the Elson-Morgan analysis described in Example 2. The results shown in Figure 12 indicate that the optimal IPTG concentration for The production is approximately 0.2 mM. Subsequently, strain 2123-54 was grown in the same medium as described above in shake flasks with either 0.2 or 1 mM IPTG at 30 ° C or 37 ° C. These flask cultures also feed glucose and ammonium sulfate as described in Example 4. At various intervals, the samples are taken and the glucosamine concentrations in culture supernatants are analyzed using the Elson-Morgan analysis described in Example 2. Figure 13 shows that under the conditions of this experiment, there was little difference in glucosamine production associated with differences in IPTG concentration. However, growth at 30 ° C resulted in glucosamine production more «Tet-Ot» elevated than in growth at 37 ° C. The results shown in Figures 14A and 14B further indicated that at 30 ° C (Figure 14A), the glucosamine production continues after the growth has ceased, while at 37 ° C (Figure 14B), the growth of the production of Glucosamine occurred at the time. When strains 2123-49 and 2123-124 are grown with 0.2 mM IPTG at 30 ° C, glucosamine production also occurred after the growth has ceased, as shown in Figures 1A (2123-49) and 15B (2123-124). As observed at 37 ° C, the highest concentrations of glucosamine were obtained with strain 21 23-54, followed by 2123-24 and 2123-49. strains 2123-149 and 2123-151 were also tested, which produce negligibly higher concentrations of glucosamine than 2123-12 (Table 7). Table 7 Glucosamine Production at 30 ° C Cepa Glucosamine Production Maximum, g / L 2123-12 0.3 2123-49 46 2123-54 7.2 2123-124 5.3 2123-149 0.6 2123-151 0.6 Example 12 The following example illustrates that glucosamine can be produced in higher concentrations in fermenting cultures of strain 2123-54 compared to shake flasks. This example also illustrates that in fermenters, strain 2123-54 produces more glucosamine at 30 ° C than at 37 ° C.

The fermentation cultures of strain 2123-54 were cultured in the medium shown in Table 8. The fermentations were carried out using NaOH for pH control at pH 6 7 and were fed to a 33% glucose mixture. % of ammonium sulfate. The aeration and agitation were adjusted to maintain a dissolved oxygen concentration of more than 20% air saturation. Table 8 Fermentation Medium Component Quantity, g / L K2HP04 14 KH2P04 16 Na3C? Trata2H20 1 (NH4) 2S04 5 MgSO4 0 12 CaCI2 0011 Mazu 204 Foam 05 mL / L IPTG 0 048 20 Ogometal Glucose * The composition of the oligometal is 0.7 mg / L CoCl2, 1.7 mg / mL H3BO3), 0.6 mg / L CuCl2 2H2O, 10.5 mg / L FeCl36H2O, 12 mg / L MnCl2 4H2O, 1.5 mg / L Na2Mo4.2H2O, 1.5 mg / L ZnCI2. In the following experiment, three fermentations were carried out in one liter containers containing an initial volume of 600 mL. The variables tested are as follows: Fermentor # 1: The mixture of 33% glucose and 8% ammonium sulfate was fed at such a rate that no glucose was accumulated in the fermenter. The growth was at 37 ° C. Fermentor # 2: As with fermentation # 1 except that the growth was at 30 ° C. Fermentor # 3: As with fermentation # 2 except . that the feeding speed was increased to maintain a constant glucose concentration in the fermenter from 5 to 10 g / L. The results of these fermentations are shown in Figures 16A, 16B and 16C. Comparison of the results of fermentors 1 (Figure 16A), and 2 (Figure 16B) shows that the major glucosamine compounds are markedly higher at 30 ° C than at 37 ° C, as observed in the shake flasks. The maximum observed glucosamine concentration was in the fermentor with excess glucose 3 developed at 30 ° C (Figure 16C), at 10.9 g / L. At 30 ° C, the growth and concentration of glucosamine seem to coincide, and it seems to be a slight advantage to develop low excess glucose. In subsequent fermentation experiments, operation under conditions similar to those of fermenter # 3, glucosamine concentrations in excess of 12 g / L have been obtained (data not shown). In summary, the present inventors have described herein the use of metabolic engineering to create the first over-production strains of glucosamine from E. coii. The concept, tested herein, will generally apply to any microorganism that has a trajectory for the production of aminoazucares, or any recombinant microorganism in which a path for the production of aminoazucares has been introduced. In addition, present this strategy to create a strain that produces glucosamine (ie, eliminate the degradation of glucosamine and take and increase the expression of the glmS gel), l £. ^ < t ú j i MftAu, ^ «MM-rt - - t * * j -. ^ '^^ .. ~~ . *. . ^ .t i i * j¿L¿ ** ¿. present inventors have also established that the inhibition of the glucosamine-6-phosphate synthase reduction product by glucosamine-6-phosphate improves the production of glucosamine. Although various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. However, it should be expressly understood that such modifications and adaptation are within the spirit and scope of the present invention, as set forth in the following claims. r *? A¡Íík £ m &? ~. .-. ,. tm *. * l.L «ü .. ^. .,. , »^ ^ A ^,. .. " ^ y. . .. ",,, _.,.," ^ .. j. i =? ^ ,. < 110 > Berry, Alan Burlingame, Richard P. Milus, James R. < 120 > PROCESS AND MATERIALS FOR THE PRODUCTION OF GLUCOSAMINE < 130 > 3161-18-C1-PCT < 140 > < 141 > < 150 > PCT / US98 / 00800 < 151 > 1998-01-14 < 150 > 60 / 035,494 < 151 > 1997-01-14 < 160 > 31 < 170 > Patentln Ver. 2. 0 < 210 > 1 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence.primary < 400 > 1 cggtctccca tgtgtggaat tgttggcgc 29 < 210 > 2 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence.primary < 400 > 2 ctctagagcg ttgatattca gtcaattaca aaca 34 < 210 > 3 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primary ^^^ ¿^^^ < 400 > 3 atggatgagc agacgatggt 20 < 210 > 4 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 4 cctcgaggtc gacggtatc 19 < 210 > 5 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 5 tggatgagca gacgatgg 18 < 210 > 6 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 6 tccgtcacag gtatttattc 20 < 210 > 7 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 7 agctgcgtgg tgcgtac 17 < 210 > 8 < 211 > 18 < 212 > DNA < 213 > Artificial sequence . ? A Í tMiiJi .r H.A & .., < 220 > < 223 > Description of the Artificial Sequence: primary < 400 > 8 ggaccgtgtt tcagttcg 18 < 210 > 9 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 9 gccgtggcga tcagtac 17 < 210 > 10 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Si < 400 > 10 gccaatcacc agcggac 17 < 210 > 11 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primary < 400 > 11 atggtttccc gctactgg 8 < 210 > 12 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the ArtificiahPrimary Sequence < 400 > 12 gaaccaaggt aacccagc 18 < 210 > 13 < 211 > 7408 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > RBS < 222 > (1240) .. (1245) < 220 > < 221 > promoter < 222 > (1165). . (118W < 220 > < 221 > conflict < 222 > (2509) .. (2510) < 400 > 13 gaattgatcc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 60 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 120 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg 180 caccagaagc ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg 240 tcgtcccctc aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtaacct 300 atcccattac ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc 360 tgttgatgaa tcacatttaa agctggctac aggaaggcca gacgcgaatt atttttgatg 420 gcgttaactc ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac ggccaggaca 480 gtcgtttgcc gtctgaattt gacctgagcg catttttacg cgccggagaa aaccgcctcg 540 cggtgatggt gctgcgttgg agtgacggca gttatctgga agatcaggat atgtggcgga 600 tgagcggcat tttccgtgac gtctcgttgc tgcataaacc gactacacaa atcagcgatt 660 tccatgttgc cactcgcttt aatgatgatt tcagccgcgc tgtactggag gctgaagttc 720 agatgtgcgg cgagttgcgt gactacctac gggtaacagt ttctttatgg cagggtgaaa 780 cgcaggtcgc cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag cgtggtggtt 840 atgccgatcg cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg agcgccgaaa 900 tcccgaatct ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg ctgattgaag 960 cagaagcctg cgatgtcggt ttccgcgagg tgcggattga aaatggtctg ctgctgctga 1020 acggcaagcc gttgctgatt cgaggcgtta accgtcacga gcatcatcct ctgcatggtc 1080 aggtcatgga tgagcagacg atggtgcagg atctccaccg cggtggcggc cgctctagaa 1140 ctagtggatc tcgatcccgc gaaattaata CGACTCACTA taggggaatt gtgagcggat 1200 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata ccatgtgtgg 1260 aattgttggc gcgatcgcgc aacgtgatgt agcagaaatc cttcttgaag gtttacgtcg 1320 tctggaatac cgcggatatg actctgccgg tctggccgtt gttgatgcag aaggtcatat 1380 gacccgcctg cgtcgcctcg gatgctggca gtaaagtcca caggcagcgg aagaacatcc 1440 tctgcatggc ggcactggta ttgctcacac tcgctgggcg acccacggtg aaccttcaga 1500 agtgaatgcg catccgcatg tttctgaaca cattgtggtg gtgcataacg gcatcatcga 1560 aaaccatgaa ccgctgcgtg aagagctaaa agcgcgtggc tataccttcg tttctgaaac 1620 cgacaccgaa gtgattgccc atctggtgaa ctgggagctg aaacaaggcg ggactctgcg 1680 tgaggccgtt ctgcgtgcta tcccgcagct gcgtggtgcg tacggtacag tgatcatgga 1740 ctcccgtcac ccggataccc tgctggcggc acgttctggt agtccgctgg tgattggcct 1800 gaaaacttta ggggatgggc tcgcttctga ccagctggcg ctgttgccgg tgacccgtcg 1860 ctttatcttc cttgaagagg gcgatattgc ggaaatcact cgccgttcgg taaacatctt 1920 cgataaaact ggcgcggaag taaaacgtca atcgaa gga tccaatctgc aatatgacgc 1980 gggcgataaa ggcatttacc gtcactacat gcagaaagag atctacgaac agccgaacgc 2040 gatcaaaaac acccttaccg gacgcatcag ccacggtcag gttgatttaa gcgagctggg 2100 accgaa cgcc gacgaactgc tgtcgaaggt tgagcatatt cagatcctcg cctgtggtac 2160 ttcttataac tccggtatgg tttcccgcta ctggtttgaa tcgctagcag gtattccgtg 2220 cgacgtcgaa atcgcctctg aattccgcta tcgcaaatct gccgtgcgtc gtaacagcct 2280 gatgatcacc ttgtcacagt ctggcgaaac cgcggatacc ctggctggcc tgcgtctgtq 2340 gaaagagctg ggttaccttg gttcactggc aatctgtaac gttccgggtt cttctctggt 2400 gcgcgaatcc gatctggcgc taatgaccaa cgcgggtaca gaaatcggcg tggcatccac 2460 taaagcattc accactcagt taactgtgct gttgatgctg gtggcgaagc tgtctcgcct 2520 gaaaggtctg gatgcctcca ttgaacatga catcgtgcat ggtctgcagg cgctgccgag 2580 ccgtattgag cagatgctgt ctcaggacaa acgcattgaa gcgctggcag aagatttctc 2640 tgacaaacat cacgcgctgt tcctgggccg tggcgatcag tacccaatcg cgctggaagg 2700 cgcattgaag ttgaaagaga tctcttacat tcacgctgaa gcctacgctg ctggcgaact 2760 gaaacacggt ccgctggcgc taattgatgc cgatatgccg gttattgttg ttgcaccgaa 2820 caacgaattg ctggaaaaac tgaaatccaa cattgaagaa gttcgcgcgc gtggcggtca 2880 gttgtatgtc ttcgccgatc aggatgcggg ttttgtaagt agcgataaca tgcacatcat 2940 cgagatgccg catgtggaag aggtgattgc accgatcttc tacaccgttc cgctgcagct 3000 gctggcttac catgtcgcgc tgatcaaagg caccgacgtt gaccagccgc gtaacctggc 3060 aaaatcggtt acggttgagt aataaatgga tgccctgcgt aagcggggca tttttcttcc 3120 tgttatgttt ttaatcaaac atcctgccaa caaaccgtca ctccatgtga tcttcggcta 3180 ctttttctct gtcacagaat gaaaattttt ctgtcatctc ttcgttatta atgtttgtaa 3240 ttgactgaat atcaacgctc tagaggggct agagcggccg ccaccgcggt ggagctccgt 3300 cgacaagctt atcgataccg tcgacctcga gggggggccc ggtaccgagg acgcgttcga 3360 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga 3420 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg 3480 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttatcgag 3540 gctaaggaag attttcagga gaaaaaaatc ctaaaatgga ccaccgttga actggatata 3600 tggcatcgta tatatcccaa aagaacattt tgaggcattt cagtcagttg ctcaatgtac 3660 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa 3720 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgaa 3780 attccgtatg gcaatg AAAG acggtgagct ggtgatatgg gatagtgttc acccttgtta 3840 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga 3900 tttctacaca tttccggcag tatattcgca agatgtggcg tgttacggtg aaaacctggc 3960 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag 4020 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac 4080 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ttcaggttca ccgctggcga 4140 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg 4200 cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc 4260 tggtgctacg cctgaataag tgataataag cggatgaatg gcagaaattc ggacgcgtca 4320 attcgagctc ctgcactgga tggtggcgct ggatggtaag ccgctggcaa gcggtgaagt 4380 gcctctggat gtcgctccac aaggtaaaca gttgattgaa ctgcctgaac taccgcagcc 4440 ggagagcgcc gggcaactct ggctcacagt acgcgtagtg caaccgaacg cgaccgcatg 4500 gggcacatca gtcagaagcc gcgcctggca gcagtggcgt ctggcggaaa acctcagtgt 4560 gacgctcccc gccgcgtccc acgccatccc gcatctgacc accagcgaaa tggatttttg 4620 catcgagctg ggtaataagc g ttggcaatt taaccgccag tcaggctttc tttcacagat 4680 gtggattggc gataaaaaac aactgctgac gccgctgcgc gatcagttca cccgtgcacc 4740 gctggataac gacattggcg taagtgaagc gacccgcatt gaccctaacg cctgggtcga 4800 acgctggaag gcggcgggcc attaccaggc cgaagcagcg ttgttgcagt gcacggcaga 4860 tacacttgct gatgcggtgc tgattacgac cgctcacgcg tggcagcatc aggggaaaac 4920 cttatttatc agccggaaaa cctaccggat tgatggtagt ggtcaaatgg cgattaccgt 4980 tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg attggcctga actgccagct 5040 ggcgcaggta gcagagcggg taaactggct cggattaggg actatcccga ccgcaagaaa 5100 ccgccttact gccgcctgtt ttgaccgctg ggatctgcca ttgtcagaca tgtatacccc 5160 gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg cgcgaattga attatggccc 5220 acaccagtgg cgcggcgact tccagttcaa catcagccgc tacagtcaac agcaactgat 5280 ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc acatggctga atatcgacgg 5340 tttccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 5400 tcctcgctca ctcttccgct ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 5460 atcagctcac tcaaaggcgg taatacggtt atccacagaa acgcaggaaa tcaggggata 5520 gaacat GTGA gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 5580 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 5640 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 5700 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 5760 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 5820 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 5880 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 5940 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 6000 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 6060 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 6120 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 6180 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 6240 ttatcaaaaa ggtcatgaga ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 6300 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 6360 ^^^^^^^^ f ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^? fí tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 6420 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 6480 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 6540 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 6600 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 6660 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 6720 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 6780 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 6840 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 6900 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 6960 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 7020 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 7080 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggt gagcaaa 7140 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 7200 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 7260 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7320 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 7380 7408 gcgtatcacg aggccctttc gtcttcaa < 210 > 14 < 211 > 2184 < 212 > DNA < 213 > Escherichia coli < 400 > 14 ccgctctaga actagtggat ctcgatcccg cgaaattaat acgactcact ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaattgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtctggaata ccgcggatat gactctgccg gtctggccgt tgttgatgca 240 gaaggtcata fcgacccgcct gcgtcgcctc ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttctgaac acattgtggt ggtgcataac 420 ^ r £ t ^ z2 ^ Z &M tá &^ r ^ _j_ ^ í_i ,, rl r? rj j .. ^ ¿. . ..irStmr? r. r r- .. ^ ^ faith,. ^ ... .. .. í. . ... ¿¿. ¿¿. ^. ^. ¿, M r ^ jr ^ r ^. ggcatcatcg aaaaccatga accgctgcgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atcccgcagc tgcgtggtgc gtacggtaca 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt atcgcttctg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atccaatctg 840 caatatgacg cgggcgataa cgtcactaca aggcatttac gatctacgaa tgcagaaaga 900 cgatcaaaaa cagccgaacg cacccttacc ggacgcatca ggttgattta gccacggtca 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 cttcttataa gcctgtggta ctccggtatg gtttcccgct atcgctagca actggtttga 1080 ggtattccgt gcgacgtcga aatcgcctct gaattccgct atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 ctgcgtctgt cgaaagagct gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 t cttctctgg tgcgcgaatc cgatctggcg ctaatgacca acgcgggtac agaaatcggc 1320 gtggcatcca ctaaagcatt caccactcag ttaactgtgc tgttgatgct ggtggcgaag 1380 ctgtctcgcc tgaaaggtct gga gcctcc attgaacatg acatcgtgca tggtctgcag 1440 gccgtattga gcgctgccga tctcaggaca gcagatgctg aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgctg ttcctgggcc gtggcgatca gtacccaatc 1560 gcgcattgaa gcgctggaag gttgaaagag atctcttaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 gttgcaccga gctggaaaaa acaacgaatt acattgaaga ctgaaatcca agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagcgataac 1800 atgcacatca tcgagatgcc gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actccatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 aatgtttgta attgactgaa tatcaacgct ctagaggggc tagagcggcc gccaccgcgg 2160 - • -..- Jiktílr,!: - ^: SíáiS? Ír .. £. ÍMÍ? S? t í. , ... ....-i., *. , ". . r,,, «. »> . . . »M. .. J r. "The t. jr i í. * ¿> ~ r. , Jlna¿ < . tggagctccg tcgacaagct tatc 2184 < 210 > 15 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1830) < 400 > 15 atg tgt gga att gtt ggc gcg ate gcg caa cgt gat gta gea gaa ate 48 Met Cys Gly lie Val Gly Ala lie Ala Gln Arg Asp Val Ala Glu lie 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tet gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gat gea gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa gaa cat ect ctg 192 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att gct falls act cgc tgg gcg acc falls ggt gaa 240 His Gly Gly Thr Gly He Wing His Thr Arg Trp Wing Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tet gaa falls att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 aaa gcg cgt ggc tat acc tt gtt tet gaa acc gac acc gaa gtg att 38 4 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg cgt gct ate ccg cag ctg ggt gcg tac ggt here gtg 480 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt cae ccg gat acc ctg ctg gcg gea cgt tet ggt 528 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 gj¡j ^ fcj ^ i ^^ J ^ i ^^^^ fefc ^ > ^^ a | ^^ ftri ^ agt ccg ctg gtg att ggc ctg ggg atg ggc gaa aac ttt ate gct tet 576 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 gac cag ctg gcg ctg ttg ccg gtg acc cgt cgc ttt ate tte ctt gaa 624 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc teg gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tec aat ctg ca 720 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt falls tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac acc ctt acc gga cgc ate 816 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 age falls ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala As p Glu 275 280 285 ctg ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt act tet 912 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tet gaa tte cgc tat cgc aaa tet 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tet ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat action ctg gct ggc ctg cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tet tet ctg gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gl and Thr Glu He Gly Val 385 390 395 400 . ,. i i-.?"? 'Ütíá & gca tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Ser Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tet cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser He Glu His 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 ctg tet cag gac aaa cgc att gaa gcg ctg gea gaa gat tte tet gac 1392 Leu Ser Gln Asp Lys Arg He Glu Wing Leu Wing Glu Asp Phe Ser Asp 450 455 460 aaa cat falls gcg ctg tte ctg ggc cgt ggc gat cag tac cea ate gcg 1440 Lys His His Wing Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tet tac att cae gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct gct ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1536 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu He Asp 500 505 510 gcc gat atg ccg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tec aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg gtt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Being Ser Asp Asn Met 545 550 555 560 falls ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Glu Glu Val He Wing Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 4 M i * íé $ á s ¿. ^ Fc. ^^^^^^^^^ * < 210 > 16 < 211 > 609 < 212 > PRT < 213 > Escherichia coli < 400 > 16 Met Cys Gly He Val Gly Ala He Ala Gln Arg Asp Val Ala Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Wing Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gl and Asp He Ala Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Ala «l Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 I ír -? I., Mr & ia.j! Í .., .. * ».. ^ i ^^^^ ¡íri! & ^ ia¡ Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His He Gln He Leu Wing Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Wing Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 Asp He Val His Gly Leu Gln Wing Leu Pro Ser Arg He Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg He Glu Wing Leu Wing Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro He Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Wing Tyr His Val Wing Leu He Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 Glu < 210 > 17 < 211 > 2184 < 212 > DNA < 213 > Escherichia coli < 400 > 17 ccgctctaga actagtggat ctcgatcccg cgaaattaat acgactcact ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaactgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtctggaata ccgcggatat gactctgccg gtctggccgt tgttgatgca 240 gaaggtcata tgacccgcct gcgtcgcctc ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttctgaac acattgtggt ggtgcataac 420 ggcatcatcg aaaaccatga accgctgcgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atcccgcagc tgcgtggtgc gtacggtaca 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt atcgcttctg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atccaatctg 840 cgggcgataa caatatgacg aggcatttac cgtcactaca gatctacgaa tgcagaaaga 900 cgatcaaaaa cagccgaacg cacccttacc ggacgcacca gccacggtca ggttgattta 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 gcctgtggta ettettataa ctccggtatg gtttcccgct ategetagea actggtttga 1080 ggtattecgt gcgacgtcga aatcgcctct gaattceget atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 ctgcgtctgt cgaaagagct gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 ^^^^^^^^^^^^^^^^^^^ M ^^^^^^ | ^^^^ ¿¿^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ tcttctctgg tgcgcgaatc cgatctggcg ctaatgacca acgcgggtac agaaateggc 1320 gtggcatcca ctaaagcatt caccactcag ttaactgtgc tgttgatgct ggtggegaag 1380 ctgtctcgcc tgaaaggtet ggatgcctcc attgaacatg acatcgtgca tggtetgeag 1440 gccgtattga gcgctgccga gcagatgctg cctcaggaca aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgctg ttcctgggcc gtggcgatca gtacccaatc 1560 gcgcattgaa gcgctggaag gttgaaagag atetettaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 gttgcaccga acaaegaatt ctgaaatcca gctggaaaaa acattgaaga agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagegataac 1800 tegagatgee atgeacatca gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca to ctccatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 aatgtttgta attgactgaa tatcaaeget ctagaggggc tagagcggcc gccaccgcgg 2160 tggagctccg tegacaaget tatc 2184 < 210 > 18 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1830) < 400 > 18 atg tgt gga act gtt ggc gcg ate gcg ca gt gat gta gea gaa ate 48 Met Cys Gly Thr Val Gly Ala He Wing Gln Arg Asp Val Ala Glu He 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tet gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gtt gat gea gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa cat ect ctg 192 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu , 4 i, i -i i riA'.f., I - 50 55 60 cat ggc ggc act ggt att gct falls act cgc tgg gcg acc cae ggt gaa 240 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tet gaa falls att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 aaa gcg cgt ggc tat acc tt gtt tet gaa acc gac acc gaa gtg att 384 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg cgt gct ate ccg cag ctg cgt ggt gcg tac ggt here gtg 480 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt falls ccg gat acc ctg ctg gcg gea cgt tet ggt 528 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg atg ggc gaa aac ttt ate gct tet 576 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He A Ser 180 185 190 gac cag ctg gcg ctg ctg ccg gtg acc cgt cgc ttt ate tte ctt gaa 624 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc cgt teg gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate ate gaa tec aat ctg ca 720 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp He Glu Being Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt falls tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac acc ctt acc gga cgc acc 816 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg Thr 260 265 270 age falls ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 »Í * k? T? F». r * ctg ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt act tet 912 Leu ueu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tet gaa tte cgc tat cgc aaa tet 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tet ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat acc ctg gct ggc ctg cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tet tet ctg gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 gea tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tet cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Wing Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 ctg ect cag gac aaa cgc att gaa gcg ctg gea gaa gatte tet gac 1392 Leu Pro Gln Asp Lys Arg He Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 aaa cat falls gcg ctg tte ctg ggc cgt ggc gat cag tac cea ate gcg 1440 Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tet tac att cae gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct gct ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1536 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pr or Leu Ala Leu He Asp 500 505 510 gcc gat atg ccg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tec aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg gtt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Be Ser Asp Asn Met 545 550 555 560 fall ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 < 210 > 19 < 211 > 609 < 212 > PRT < 213 > Escherichia coli < 400 > 19 Met Cys Gly Thr Val Gly Wing He Wing Gln Arg Asp Val Wing Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Ala His Leu Val Asn Trp G? U Itu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 t? 140 Ala Val Leu Arg Ala He Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg Thr 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Wing He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Wing Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser He Glu His Wí ^^ j ^ * »^. 420 425 430 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 Leu Pro Gln Asp Lys Arg He Glu Ala Leu Wing Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 Leu Glu Gly Wing Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 Wing Asp Met Pro Val He Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Being Ser Asp Asn Met 545 550 555 560 His He He Glu Met Pro His Val Glu Glu Val He Wing Pro He Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 Glu < 210 > 20 < 211 > 2184 < 212 >; DNA < 213 > Escherichia coli < 400 > 20 ccgctctaga actagtggat ctcgatcccg cgaaattaat acgactcact ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaattgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtetggaata ccgcggatat gactctgccg gtctggccgt tgttgataca 240 gaaggtcata tgacccgcct gcgtcgcctc ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttctgaac acattgtggt ggtgcataac 420 j »-Ata ^ a ^^ j ^. ^^^. ^ Mj ^^ M ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^ ^^ »^. ^^^^^. ggcatcatcg aaaaccatga accgctggpgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atc? cgcagc gtacggtaca tgcgtggtgc 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt atcgcttctg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atccaatctg 840 cgggcgataa caatatgacg tgtcactaca aggcatttac gatctacgaa tgcagaaaga 900 cgatcaaaaa cagccgaacg cacccttacc ggacgcatca gccacggtca ggttgattta 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 gcctgtggta ettettataa ctccggtatg gtttcccgct ategetagea actggtttga 1080 ggtattecgt gcgacgtcga aatcgcctcc gaattceget atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 ctgcgtctgt cgaaagagct gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 tcttctctgg tgc gcgaatc cgatctggcg ctaatgacca acgcgggtac agaaateggc 1320 gtggcatcca ctaaagcatt caccactcag ttaactgtgc tgttgatgct ggtggegaag 1380 ctgtctcgcc tgaaaggtet ggatgcctcc attgaacatg acatcgtgca tggtetgeag 1440 gccgtattga gcgctgccga tctcaggaca gcagatgctg aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgctg ttcctgagcc gtggcgatca gtacccaatc 1560 gcgcattgaa gcgctggaag gttgaaagag atetettaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 gttgcaccga acaaegaatt getggaaaaa acattgaaga ctgaaateca agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagegataac 1800 tegagatgee atgeacatca gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actecatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 '• A' * - 'a- * "- * -' -'- ~ ** * - - - - - -» - * - * * * * * * ?? Ü ^^ á aatgtttgta attgactgaa tatcaaeget ctaggggggc tagagcggcc gccaccgcgg 2160 tggagctccg tegacaaget tatc 2184 < 210 > 21 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1). (1830) < 400 > 21 atg tgt gga att gtt ggc gcg ate gcg ca gt gta gta gat gta gea gaa ate 48 Met Cys Gly He Val Gly Ala He Wing Gln Arg Asp Val Ala Glu He 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tet gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gtt gat here gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Thr Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa cat ect ctg 192 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att gct falls act cgc tgg gcg acc falls ggt gaa 240 His Gly Gly Thr Gly He Wing His Thr Arg Trp Wing Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tet gaa falls att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 1 00 105 110 aaa gcg cgt ggc tat acc tt gtt tet gaa acc gac acc gaa gtg att 384 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg: gt gct ate ccg cag ctg cgt ggt gcg tac ggt here gtg 480 Wing Val Leu Arg Ala He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt falls ccg gat acc ctg ctg gcg gea cgt tet ggt 528 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly ^^ Ü ^ gjjg ... { i-. . ,,. -. z ***** ^ 165 170 175 agt ccg ctg gtg att ggc ctg; gga atg ggc gaa aac ttt ate gct tet 576 Ser Pro Leu Val He Gly Leu G? and Met Gly Glu Asn Phe He Wing Ser 180 185 190 gac cag ctg gcg ctg ttg ccg gtg acc cgt cgc ttt ate tte ctt gaa 624 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc teg gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tec aat ctg caa 720 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac tgt falls tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Cys His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac acc ctt acc gga cgc ate 816 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 age falls ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 ctg ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt act tet 912 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tec gaa tte cgc tat cgc aaa tet 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tet ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat acc ctg gct ggc ctg cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tet tet ctg gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 gea tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tet cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 ctg tet cag gac aaa cgc att gaa gcg ctg gea gaa gat tte tet gac 1392 Leu Ser Gln Asp Lys Arg Zle Glu Ala Leu Wing Glu Asp Phe Ser Asp 450 455 460 aaa cat falls gcg ctg tte ctg age cgt ggc gat cag tac cea ate gcg 1440 Lys His His Ala Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tet tac att cae gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct get ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1536 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 gcc gat atg ccg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tec aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg ggt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 falls ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 __________ t > ., «J a ^? J jj ^ < 210 > 22 < 211 > 609 < 212 > PRT < 213 > Escherichia coli < 400 > 22 Met Cys Gly He Val Gly Ala He Ala Gln Arg Asp Val Ala Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Thr Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Wing Gly Asp Lys Gly He Tyr Cys His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His He Gln He Leu Wing Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Wing Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 Asp He Val His Gly Leu Gln Wing Leu Pro Being Arg He Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg He Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro He Phe 565 570 575 ; .. ,, ^ ¿it », faith > Íia ... i > i- «tt? t,» *. ,. ,. . i, m ^ .. ^ ... a? fe ..,. ", .. ^ a-3 -.-. *.? s. < , **. *. - ^ A ".,". ",. TO., .,,_ . , M. "..; , i. J ^ MiÍMÍÉi Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 Glu < 210 > 23 < 211 > 2184 < 212 > DNA < 213 > Escherichia coli < 400 > 23 ccgctctaga actagtggat ctcgatcccg cgaaattaat acgactcact ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaattgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtetggaata ccgcggatat gactctgccg gtctggccgt tgttgatgca 240 gaaggtcata tgacccgcct gcgtcgcctc ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttccgaac acattgtggt ggtgcataac 420 ggcatcatcg aaaaccatga accgctgcgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atcccgcagc tgcgtggtgc gtacggtaca 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt ategettetg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atecaatetg 840 caatatgacg cgggcg ataa cgtcactaca aggcatttac tgcagaaaga gatetaegaa 900 cgatcaaaaa cagccgaacg ggaegeatca cacccttacc ggttgattta gccacggtca 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 gcctgtggta ettettataa ctccggtatg gtttcccgct ategetagea actggtttga 1080 ggtattecgt gegaegtega aatcgcctct gaattceget atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 ctgcgtctgt cgaaagagct gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 tcttctctgg tgcgcgaatc cgatctggcg ctaatgacca acgcgggtac agaaateggc 1320 gtggcatcca ctaaagcatt caccactcag ttaae ^ tgc tgttgatgct ggtggegaag 1380 ctgtctcgcc tgaaaggtet ggatgeetco attgaacatg acatcgtgca tggtetgeag 1440 gccgtattga gcgctgccga tctcaggaca gcagatgctg aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgccg ttcctgggcc gtggcgatca gtacccaatc 1560 gcgcattgaa gcgctggaag gttgaaagag atetettaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 acaaegaatt gttgcaccga getggaaaaa ctgaaateca acattgaaga agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagegataac 1800 atgeacatca tegagatgee gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actecatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 aatgtt tgta attgactgaa tatcaaeget ctagaggggc tagagcggcc accaccgcgg 2160 tggagctccg tegacaaget tatc 2184 < 210 > 24 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1830) < 400 > 24 atg tgt gga att gtt ggc gcg ate gcg ca gt gat gta gea gaa ate 48 Met Cys Gly He Val Gly Ala He Wing Gln Arg Asp Val Ala Glu He 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tet gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gtt gat gea gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa gaa cat ect ctg 192 -i - ^ - MUHfa -? - iáafattHÍriHkíMMtt ^ iiÉ «Étt ^^ Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att gct cae act cgc tgg gcg acc ggt gaa 240 His Gly Gly Thr Gly He Wing His Thr Arg Trp Wing Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tec gaa fall att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 aaa gcg cgt ggc tat acc tte gtt tet gaa acc gac acc gaa gtg att 384 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg cgt gct ate ccg cag ctg cgt gcg gcg tac ggt here gtg 480 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt falls ccg gat acc ctg ctg gcg gea cgt tet ggt 528 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg atg ggc gaa aac ttt ate gct tet 576 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 gac cag ctg gcg ctg ttg ccg gtg acc cgt cgc ttt ate ate ctt gaa 624 Asp Gln Leu Ala L eu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc teg gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr.Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tec aat ctg caa 720 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt cae tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac acc ctt acc gga cgc ate 816 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 age falls ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu ll ^^^ & ^ i 275 280 285 ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt act tet 912 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tet gaa tte cgc tat cgc aaa tet 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tet ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat acc gtc cct gct gcc gt cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tet tet ctg gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu M et Thr Asn Wing Gly Thr Glu He Gly Val 385 390 395 400 gea tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tet cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 ctg tet cag gac aaa cgc att gaa gcg ctg gea gaa gat tte tet gac 1392 Leu Ser Gln Asp Lys Arg He Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 aaa cat drops gcg ccg tte ctg ggc cgt ggc gat cag tac cea gcg gcg 1440 Lys His His Wing Pro Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tet tac att falls gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct gct ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1 536 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu He Asp 500 505 510 gcc gat atg ceg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tec aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg gtt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Be Ser Asp Asn Met 545 550 555 560 falls ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Val Glu Vallu He Ala Pro Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 < 210 > 25 < 211 > 609 < 212 > PRT < 213 > Escherichia coli < 400 > 25 Met Cys Gly He Val Gly Ala He Ala Gln Arg Asp Val Ala Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His He Gln He Leu Wing Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg He Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Pro Phe Leu Gly Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 Leu Glu Gly Wing Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 Wing Tyr Ala Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Ser Being Asp Asn Met 545 550 555 560 His He He Glu Met Pro His Val Glu Glu Val He Wing Pro He Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 Glu < 210 > 26 < 211 > 2184 < 212 > DNA < 213 > Escherichia coli < 400 > 26 acgactcact ccgctctaga actagtggat ctcgatcccg cgaaattaat ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaattgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtetggaata ccgcggatat gactctgccg gtctggccgt tgttgatgca 240 gcgtcgcctc gaaggtcata tgacccgcct ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttctgaac acattgtggt ggtgcataac 420 ggcatcatcg aaaaccatga accgctgcgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atcccgcagc tgcgtggtgc gtacggtaca 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt atcgcttctg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atccaatctg 840 cgggcgataa caatatgacg aggcatttac cgtcactaca gatctacgaa tgcagaaaga 900 cgatcaaaaa cagccgaacg cacccttacc ggacgcatca gccacggtca ggttgattta 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 gcctgtggta ettettataa ctccggtatg gtttcccgct ategetagea actggtttga 1080 ggtattecgt gegaegtega aatcgcctct gaattceget atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 cgaaa ctgcgtctgt GAGCT gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 tcttctctgg tgcgcgaatc egatetggcg ctaatgacca acgcgggtac agaaateggc 1320 gtggcatcca ctaaagcatt caccactcag ttaactgtgc tgttgatgct ggtggegaag 1380 ctgtctcgcc tgaaaggtet ggatgcctcc attgaacatg acatcgtgca tggtetgeag 1440 gccgtattga gcgctgccga tctcaggaca gcagatgctg aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgctg ttcctgagcc gtggcgatca gtacccaatc 1560 gcgcattgaa gcgctggaag gttgaaagag atetettaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 acaaegaatt gttgcaccga ctgaaateca getggaaaaa acattgaaga agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagegataac 1800 tegagatgee atgeacatca gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actecatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 __mWmW MM MMíh ?? m, ^ ii? > ? M? ^ Aatgtttgta attgactgaa tatcaaeget ctaggggggc tagagcggcc gccaccgcgg 2160 tggagctccg tegacaaget tatc 2184 < 210 > 27 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1830) < 400 > 27 atg tgt gga att gtt ggc gcg ate gcg ca gg gat gta gea gaa ate 48 Met Cys Gly He Val Gly Ala He Wing Gln Arg Asp Val Ala Glu He 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tet gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gtt gat gea gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa cat ect ctg 192 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att gct falls act cgc tgg gcg acc falls ggt gaa 240 His Gly Gly Thr Gly He Wing His Thr Arg Trp Wing Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tet gaa falls att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 10 0 105 110 aaa gcg cgt ggc tat acc tt gtt tet gaa acc gac acc gaa gtg att 384 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg cgt gct ate ccg cag ctg cgt ggt gcg tac ggt here gtg 480 Wing Val Leu Arg Ala He Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt falls ccg gat acc ctg ctg gcg gea cgt tet ggt 528 ^ - "" ji ^^^ ¡£££ g ^ £ _ k.?. »... ... J.l.i? s * ^ Aaa." ...... *. > ? a JL. »- .. ^ MÉIUI • -" * "* • --'- * Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg tgg atg ggc gaa aac ttt ate gct tet 576 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 gac cag ctg gcg ctg ctg gtg gtg gtg ct ct ct gtg ctt gtg gtg 624 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc te gta te gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tec aat. ctg ca 720 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt falls tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac ac ctact gga cgc ate 816 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 age cae ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 ctg ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt. act tet 912 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tet gaa tte cgc tat cgc aaa tet 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330"335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tct ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat acc ctg gct ggc ctg cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tct tct ectf gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 gea tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tct cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Wing Lys Leu Being Arg Leu Lys Gly Leu Asp Wing Being He Glu Hls 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Wing Leu Pro Being Arg He Glu Gln Met 435 440 445 ctg tct cag gac aaa cgc att gaa gcg ctg gea gaa gat tte tct gac 1392 Leu Ser Gln Asp Lys Arg He Glu Ala Leu Wing Glu Asp Phe Ser Asp 450 455 460 aaa cat falls gcg etg tte ctg age cgt ggc gat cag tac cea ate gcg 1440 Lys His His Ala Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tct tac att cae gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct gct ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1536 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 gcc gat atg ccg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tech aac att gaa gaa gtt cgc gcg cgt ggc ggt cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg gtt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Being Ser Asp Asn Met 545 550 555 560 falls ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Glu Glu Val He Wing Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 i ^ _ ^ _ í_ _________m - * - "taA -J, t <210> 28 <211> 609 <212> PRT <213> Escherichia coli <400> 28 Met Cys Gly He Val Gly Wing He Wing Gln Arg Asp Val Wing Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His He Gln He Leu Wing Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Wing Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 Asp He Val His Gly Leu Gln Wing Leu Pro Ser Arg He Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg He Glu Wing Leu Wing Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 Wing Tyr Wing Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu He Asp 500 505 510 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro He He Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Wing Tyr His Val Wing Leu He Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Wing Lys Ser Val Thr Val 595 600 605 * Glu < 210 > 29 < 211 > 2184 < 212 > DNA < 213 > Escherichia coli < 400 > 29 ccgctctaga actagtggat ctcgatcccg cgaaattaat acgactcact ataggggaat 60 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat 120 accatgtgtg gaattgttgg cgcgatcgcg caacgtgatg tagcagaaat ccttcttgaa 180 ggtttacgtc gtetggaata ccgcggatat gactctgccg gtctggccgt tgttgatgca 240 gaaggtcata tgacccgcct gcgtcgcctc ggtaaagtcc agatgctggc acaggcagcg 300 gaagaacatc ctctgcatgg cggcactggt attgctcaca ctcgctgggc gacccacggt 360 gaaccttcag aagtgaatgc gcatccgcat gtttctgaac acattgtggt ggtgcataac 420 ggcatcatcg aaaaccatga accgctgcgt gaagagctaa aagcgcgtgg ctataccttc 480 ccgacaccga gtttctgaaa agtgattgcc catctggtga actgggagct gaaacaaggc 540 gggactctgc gtgaggccgt tctgcgtgct atcccgcagc tgcgtggtgc gtacggtaca 600 gtgatcatgg actcccgtca cccggatacc ctgctggcgg cacgttctgg tagtccgctg 660 gtgattggcc tggggatggg cgaaaacttt ategettetg accagctggc gctgttgccg 720 gtgacccgtc gctttatctt ccttgaagag ggcgatattg cggaaatcac tcgccgttcg 780 gtaaacatct tcgataaaac tggcgcggaa gtaaaacgtc aggatatcga atecaatetg 840 caatatgacg cgggcg ataa cgtcactaca aggcatttac tgcagaaaga gatetaegaa 900 cgatcaaaaa cagccgaacg ggaegeatca cacccttacc ggttgattta gccacggtca 960 agcgagctgg gaccgaacgc cgacgaactg ctgtcgaagg ttgagcatat tcagatcctc 1020 gcctgtggta ettettataa ctccggtatg gtttcccgct ategetagea actggtttga 1080 ggtattecgt gegaegtega aatcgcctct gaattceget atcgcaaatc tgccgtgcgt 1140 cgtaacagcc tgatgatcac cttgtcacag tctggcgaaa ccgcggatac cctggctggc 1200 Ma bátM ^ ?? m. ,., .. ,,. t? .. - * "ctgcgtctgt cgaaagagct gggttacctt ggttcactgg caatctgtaa cgttccgggt 1260 tcttctctgg tgcgcgaatc egatetggcg ctaatgacca acgcgggtac agaaateggc 1320 gtggcatcca ctaaagcatt caccactcag ttaactgtgc tgttgatgct ggtggegaag 1380 ctgtctcgcc tgaaaggtet ggatgcctcc attgaacatg acatcgtgca tggtetgeag 1440 gccgtattga gcgctgccga gcagatgctg tctcaggaca aacgcattga agcgctggca 1500 ctgacaaaca gaagatttct tcacgcgctg ttcctgagcc gtggcgatca gtacccaatc 1560 gcgctggaag gcgcattgaa gttgaaagag atetettaca ttcacgctga agcctacgct 1620 gctggcgaac tgaaacacgg tccgctggcg ctaattgatg ccgatatgcc ggttattgtt 1680 acaaegaatt gttgcaccga ctgaaateca getggaaaaa acattgaaga agttcgcgcg 1740 cgtggcggtc agttgtatgt cttcgccgat caggatgcgg gttttgtaag tagegataac 1800 tegagatgee atgeacatca gcatgtggaa gaggtgattg caccgatctt ctacaccgtt 1860 ccgctgcagc tgctggctta ccatgtcgcg ctgatcaaag gcaccgacgt tgaccagccg 1920 cgtaacctgg caaaatcggt tacggttgag taataaatgg atgccctgcg taagcggggc 1980 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actecatgtg acaaaccgtc 2040 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt 2100 aatgtttgta attgactgaa tatcaaeget ctaggggggc tagagcggcc gccaccgcgg 2160 tggagctccg tegacaaget tatc 2184 < 210 > 30 < 211 > 1830 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1830) < 400 > 30 atg tgt gga att gtt ggc gcg ate gcg ca gt gat gta gea gaa ate 48 Met Cys Gly He Val Gly Ala He Wing Gln Arg Asp Val Ala Glu He 1 5 10 15 ctt ctt gaa ggt tta cgt cgt ctg gaa tac cgc gga tat gac tct gcc 96 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 ggt ctg gcc gtt gtt gat gea gaa ggt cat atg acc cgc ctg cgt cgc 144 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 ctc ggt aaa gtc cag atg ctg gea cag gea gcg gaa cat ect ctg 192 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Wing Glu Glu His Pro Leu 50 55 60 cat ggc ggc act ggt att gct falls act cgc tgg gcg acc ggt gaa 240 His Gly Gly Thr Gly He Wing His Thr Arg Trp Wing Thr His Gly Glu 65 70 75 80 ect tea gaa gtg aat gcg cat ccg cat gtt tct gaa fall att gtg gtg 288 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 gtg cat aac ggc ate ate gaa aac cat gaa ccg ctg cgt gaa gag cta 336 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 aaa gcg cgt ggc tat acc ttt gtt tct gaa acc gac acc gaa gtg att 384 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 gcc cat ctg gtg aac tgg gag ctg aaa ca ggc ggg act ctg cgt gag 432 Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 gcc gtt ctg cgt gct ate ccg cag ctg ggt gcg tac ggt here gtg 480 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 ate atg gac tec cgt falls ccg gat acc ctg ctg gcg gea cgt tct ggt 528 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 agt ccg ctg gtg att ggc ctg ggg atg ggc gaa aac ttt ate gct tct 576 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 gac cag ctg gcg ctg ttg ccg gtg acc cgt cgc ttt ate ate ctt gaa 624 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 gag ggc gat att gcg gaa ate act cgc cgt teg gta aac ate tte gat 672 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn He Phe Asp 210 215 220 aaa act ggc gcg gaa gta aaa cgt cag gat ate gaa tec aat ctg caa 720 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Being Asn Leu Gln 225 230 235 240 tat gac gcg ggc gat aaa ggc att tac cgt falls tac atg cag aaa gag 768 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 ate tac gaa cag ccg aac gcg ate aaa aac ac ctact gga cgc ate 816 He Tyr Glu Gln Pro Asn Ala He Lys Asn Thr Leu Thr Gly Arg H e 260 265 270 age falls ggt cag gtt gat tta age gag ctg gga ccg aac gcc gac gaa 864 «*,» .. «. , .. tt.A. ^ tu, ..... ^. ^. ».. üteriHfeMriÉlÉtii? ittiliiiiitf-f- * i ^ fc¿ * i ^» amiritttfMi Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 ctg teg aag gtt gag cat att cag ate ctc gcc tgt ggt act tct 912 Leu Leu Ser Lys Val Glu His He Gln He Leu Ala Cys Gly Thr Ser 290 295 300 tat aac tec ggt atg gtt tec cgc tac tgg ttt gaa teg cta gea ggt 960 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 att ccg tgc gac gtc gaa ate gcc tct gaa tte cgc tat cgc aaa tct 1008 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 gcc gtg cgt cgt aac age ctg atg ate acc ttg tea cag tct ggc gaa 1056 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 acc gcg gat acc ctg gct ggc ctg cgt ctg teg aaa gag ctg ggt tac 1104 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 ctt ggt tea ctg gea ate tgt aac gtt ccg ggt tct tct ctg gtg cgc 1152 Leu Gly Ser Leu Ala He Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 gaa tec gat ctg gcg cta atg acc aac gcg ggt here gaa ate ggc gtg 1200 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 gea tec act aaa gea tte acc act cag tta act gtg ctg ttg atg ctg 1248 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 gtg gcg aag ctg tct cgc ctg aaa ggt ctg gat gcc tec att gaa cat 1296 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Wing Ser He Glu His 420 425 430 gac ate gtg cat ggt ctg cag gcg ctg ccg age cgt att gag cag atg 1344 Asp He Val His Gly Leu Gln Ala Leu Pro Ser Arg He Glu Gln Met 435 440 445 ctg tct cag gac aaa cgc att gaa gcg ctg gea gaa gatte tct gac 1392 Leu Ser Gln Asp Lys Arg He Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 aaa cat falls gcg ctg tte ctg age cgt ggc gat cag tac cea ate gcg 1440 Lys His His Ala Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro He Wing 465 470 475 480 ctg gaa ggc gea ttg aag ttg aaa gag ate tct tac att cae gct gaa 1488 Leu Glu Gly Ala Leu Lys Leu Lys Glu He Ser Tyr He His Wing Glu 485 490 495 gcc tac gct gct ggc gaa ctg aaa falls ggt ccg ctg gcg cta att gat 1536 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu He Asp *. ~ j ¿,? ? * JK ~ ¿¿M »500 505 510 gcc gat atg ccg gtt att gtt gtt gea ccg aac aac gaa ttg ctg gaa 1584 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 aaa ctg aaa tec aac att gaa gaa gtt cgc gcg cgt ggc ggt. cag ttg 1632 Lys Leu Lys Ser Asn He Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 tat gtc tte gcc gat cag gat gcg gtt ttt gta agt age gat aac atg 1680 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Be Ser Asp Asn Met 545 550 555 560 fall ate ate gag atg ccg cat gtg gaa gag gtg att gea ccg ate tte 1728 His He He Glu Met Pro His Val Glu Glu Val He Ala Pro Pro He Phe 565 570 575 tac acc gtt ccg ctg cag ctg ctg gct tac cat gtc gcg ctg ate aaa 1776 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu He Lys 580 585 590 ggc acc gac gtt gac cag ccg cgt aac ctg gea aaa teg gtt acg gtt 1824 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 gag taa 1830 Glu 610 < 210 > 31 < 211 > 609 < 212 > PRT < 213 > Escherichia coli < 400 > 31 Met Cys Gly He Val Gly Wing He Wing Gln Arg Asp Val Wing Glu He 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Wing 20 25 30 Gly Leu Wing Val Val Asp Wing Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly He Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His He Val Val 85 90 95 Val His Asn Gly He He Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Wing Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val He 115 120 125 Wing His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Wing Val Leu Arg Wing He Pro Gln Leu Arg Gly Wing Tyr Gly Thr Val 145 150 155 160 He Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Wing Arg Ser Gly 165 170 175 Ser Pro Leu Val He Gly Leu Gly Met Gly Glu Asn Phe He Wing Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe He Phe Leu Glu 195 200 205 Glu Gly Asp He Wing Glu He Thr Arg Arg Ser Val Asn Zle Phe Asp 210 215 220 Lys Thr Gly Wing Glu Val Lys Arg Gln Asp He Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Wing Gly Asp Lys Gly He Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 He Tyr Glu Gln Pro Asn Wing He Lys Asn Thr Leu Thr Gly Arg He 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Wing Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His Zle Gln Zle Leu Wing Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Wing Gly 305 310 315 320 He Pro Cys Asp Val Glu He Wing Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Wing Val Arg Arg Asn Ser Leu Met He Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Wing Asp Thr Leu Wing Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Be Leu Ala-Zle Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Be Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu He Gly Val 385 390 395 400 Wing Being Thr Lys Wing Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser He Glu His 420 425 430 Asp Zle Val His Gly Leu Gln Ala Leu Pro Ser Arg Zle Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg Zle Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His Wing Leu Phe Leu Ser Arg Gly Asp Gln Tyr Pro Zle Wing 465 470 475 480 Leu Glu Gly Wing Leu Lys Leu Lys Glu Zle Ser Tyr Zle His Wing Glu 485 490 495 Wing Tyr Ala Wing Gly Glu Leu Lys His Gly Pro Leu Wing Leu Zle Asp 500 505 510 Wing Asp Met Pro Val He Val Val Wing Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn Zle Glu Glu Val Arg Wing Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Wing Asp Gln Asp Wing Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His Zle Zle Glu Met Pro His Val Glu Glu Val He Wing Pro Zle Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Zle Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 Glu _W¡i _ ++ _- * - máÉ m Miill-áiimttiü-i! J I - - * - ** • * "

Claims

CLAIMS 1. A method for producing glucosamine by fermentation, comprising: (a) growing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a microorganism having a genetic modification in a metabolic pathway of amino sugar, said metabolic pathway of amino acid selected from the group consisting of a path to convert glucosamine-6-phosphate into another compound, a path to synthesize glucosamine-6-phosphate, a path to transport glucosamine or glucosamine-6-phosphate outside the microorganism, a path to transport glucosamine to the microorganism, and a path that competes with substrates included in the production of glucosamine-6-phosphate; wherein said cultivation step produces a product selected from the group consisting of intracellular glucosamine-6-phosphate and glucosamine of said microorganism; and (b) recovering said product.
2. The method according to claim 1, characterized in that said glucosamine-6-phosphate is intracellular and said glucosamine is extracellular, wherein said recovery step comprises a recovery step selected from the group consisting of recovering said glucosamine-6-phosphate from said microorganism, said glucosamine being recovered from said fermentation medium, and a combination thereof.
3. The method according to claim 1, characterized in that said product is glucosamine which is segregated in said fermentation medium by said microorganism and wherein said recovery step comprises the purification of said glucosamine from said fermentation medium.
The method according to claim 1, characterized in that said product is intracellular glucosamine-6-phosphate and said recovery step comprises isolating said glucosamine-6-phosphate from said microorganism.
5. The method according to claim 1, characterized in that said product is intracellular glucosamine-6-phosphate and said recovery step further comprises dephosphorylating said glucosamine-6-phosphate to produce glucosamine.
The method according to claim 1, characterized in that said cultivation step comprises maintaining said carbon source at a concentration of from about 0.5% to about 5% in said fermentation medium.
The method according to claim 1, characterized in that said cultivation step is carried out at a temperature 20 from about 28 ° C to about 37 ° C.
The method according to claim 1, characterized in that said cultivation step is carried out in a fermentor.
The method according to claim 1, characterized in that at least about 1 g / L of said product is 25 recover. ^^^ ¡^^^^^^^^^^^^^^^ g ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
10. The method according to claim 1, characterized in that said genetic modification is a modification in a nucleic acid molecule that encodes a protein selected from the group consisting of deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase, enzyme llNa9 specific for? / - acetyl-glucosamine, glucosamine-6-phosphate synthase, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C of PEP: glucose PTS, IEM, P / lllMan of PEP: mannose PTS, and a phosphatase. eleven . The method according to claim 1, characterized in that said microorganism has a genetic modification that increases the action of glucosamine-6-phosphate synthase. The method according to claim 1, characterized in that said genetic modification results in overexpression of glucosamine-6-phosphate synthase by said microorganism. The method according to claim 1, characterized in that said genetic modification comprises the transformation of said microorganism with a recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase having glucosamine-6-phosphate synthase enzymatic activity , wherein said recombinant nucleic acid molecule is operably linked to a transcription control sequence. The method according to claim 13, characterized in that said recombinant nucleic acid molecule comprises - f «*" '- «-" *' a nucleic acid sequence encoding a glucosamine-6-phosphate synthase homolog. 15. The method according to claim 1, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence SEQ ID NO.16. 16. The method according to claim 1, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 1 5. 17 The method according to claim 1, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of pKLN23-28, nglms-282184 and nglmS-28183o. The method according to claim 1, characterized in that said recombinant nucleic acid molecule is integrated into the genome of said microorganism. 19. The method according to claim 13, characterized in that said recombinant ribonucleic acid molecule encoding the glucosamine-6-phosphate synthase comprises a genetic modification that increases the action of said glucose synthase mi na-6-f osf ato. The method according to claim 19, characterized in that said recombinant nucleic acid molecule encoding the glucosamine-6-phosphate synthase comprises a genetic modification that reduces the inhibition of the glucosamine-6-phosphate product of said glucosamine-6 synthase. -phosphate. The method according to claim 19, characterized in that said genetic modification results in at least one amino acid modification selected from the group consisting of elimination, insertion, inversion, substitution and derivation of at least one amino acid residue of said glucosamine synthase. -6- phosphate, said amino acid modification resulting in increased glucosamine-6-phosphate synthase action. 22. The method according to claim 21, characterized in that said at least one amino acid modification is at a position of the amino acid sequence, which corresponds to the amino acid sequence SEQ ID IMO: 16, selected from the group consisting of (4), lle (272), Ser (450); Wing (39), Arg (250), Gly (472), 15 Leu (469) and combinations thereof. 23. The method according to claim 22, characterized in that said amino acid modification is a substitution selected from the group consisting of: (a) an amino acid residue having an aliphatic hydroxyl side group for lle (4); (b) an amino acid residue having an aliphatic hydroxyl side group for Ile (272); (c) an amino acid residue having an aliphatic side group for Ser (450); 25 (d) an amino acid residue having a side group * j »« ¿alíiilm! ***. t a, .. .. t A L .. Jau ......,. .,.,., .. * * t.u, t * M ?. »«,. ,; _1J_ _ ^ 1_1 'aliphatic hydroxyl for Ala (39); (e) an amino acid residue having an aliphatic hydroxyl side group for Arg (250); (f) an amino acid residue having an aliphatic hydroxyl side group for Gly (472); (g) an amino acid residue having an aliphatic side group for Leu (469); (h) and combinations of (a) - (g). The method according to claim 22, characterized in that said amino acid modification is a substitution selected from the group consisting of: lle (4) in Thr, lle (272) in Thr, Ser (450) in Pro, Ala (39) in Thr, Arg (250) in Cys, Gly (472) in Ser, Leu (469) in Pro and combinations thereof. 25. The method according to claim 22, characterized in that said amino acid modification is a substitution of a proline residue of a leucine residue at the position of the amino acid sequence Leu (469). The method according to claim 22, characterized in that said amino acid modification is a substitution of an amino acid residue selected from the group consisting of: (a) a threonine residue having an alanine residue at the Ala (39) position; (b) a cysteine residue for an arginine residue in the Arg (250) position; (c) a serine residue for a glycine residue in the «• '* **. Gly position (472); and any combination 27. The method according to claim 22, characterized in that said amino acid modification is a substitution selected from the group consisting of: (a) a threonine residue for an isoleucine residue in the lle (4) position; (b) a threonine residue for an isoleucine residue in the lle position (272); (c) a proline residue for a serine residue in the Ser (450) position; and (d) any combination of (a), (b) or (c). The method according to claim 19, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a glucosamine-6-phosphate synthase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19 SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28 and SEQ ID NO: 31. The method according to claim 19, characterized in that said nucleic acid molecule recombines, and comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 7, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 30. 30. The method according to claim 19, characterized in that said recombinant nucleic acid molecule comprises i .ti .i .i i -i,. , i fí .. J »» = i a recombinant nucleic acid molecule from the group consisting of pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149, pKLN23-1 51, nglmS-492? 84, nglmS -49? 83o, nglmS-542184, nglmS-54? 83o, nglmS-1242184, nglmS-124183o, nglmS-1492184, nglmS-149? 83o, nglmS-1 512? 8 l nglmS-151 183o. 31 The method according to claim 1, characterized in that said recombinant nucleic acid molecule is integrated into the genome of said microorganism. The method according to claim 1, characterized in that said microorganism has at least one additional genetic modification in a gene encoding a protein selected from the group consisting of deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase , γ-acetyl-glucosamine specific llNag enzyme, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosam i na-1-phosphate uridyltransferase, phosphofructokinase, PEP Enzyme IIG | C: glucose PTS, IEM, P / lllMan of PEP: mannose PTS, where said genetic modification reduces the action of said protein. The method according to claim 1, characterized in that said microorganism has at least one additional genetic modification encoding a phosphatase, wherein said genetic modification increases the action of said phosphatase. 34. The method according to claim 1, characterized in that said microorganism has additional modifications in genes encoding the following proteins: deacetylase N- a .. í '? », ^ 1.4 1 1 acetylglucosamine-6-phosphate, deaminase glucosamine-6-phosphate and enzyme lNaQ specific for? / - acetyl-glucosamine. wherein said genetic modification reduces the action of said proteins. 35. The method according to claim 34, characterized in that said genetic modification is a deletion of at least a portion of said genes. 36. The method according to claim 1, characterized in that said microorganism is selected from the group consisting of bacteria and yeast. 37. The method according to claim 1, characterized in that said microorganism is a bacterium of the genus Escherichia. 38. The method according to claim 1, characterized in that said microorganism is Escherichia coli. 39. The method according to claim 38, characterized in that said genetic modification is a mutation in an Escherichia coli gene selected from the group consisting of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmil, glmS, ptsG and a phosphatase gene. A method for producing glucosamine by fermentation, comprising: (a) growing in a fermentation medium comprising assimilable ces of carbon, nitrogen and phosphate, an Escherichia coli transformed with a recombinant nucleic acid molecule encoding a glucosamine-6-phosphate, wherein said ? tí ^^^^ asa? ií-ii- - • •• * * • 'h¿? £ i. - '• * ° »••' * * < - nucleic acid molecule "t t? ant" increases the action of glucosamine-6-phosphate synthase in said Escherichia coli, and wherein said recombinant nucleic acid molecule is operatively linked to a transcription control sequence; wherein said cultivation step produces a product selected from the group consisting of glucosamine-6-phosphate and glucosamine of said Escherichia coli; and (b) recovering said product. 41 The method according to claim 40, characterized in that said Escherichia coli has at least one additional genetic modification in at least one gene selected from the group consisting of nagA, nagB, nagC, nagD, nagE, manXYZ, glmM, pfkA, pfkB, glmU, glmS, ptsG and a phosphatase gene. 42. The method according to claim 40, characterized in that said at least one additional genetic modification comprises a removal of nagA, nagB, nagC, nagD, nagE, and a mutation in manXYZ, said modification resulting in reduced action of deacetylase? / -acetylglucosamine -6-phosphate, deaminase glucosamine-6-phosphate and enzyme lNa9 specific for? / - acetyl-glucosamine. 43 A microorganism for producing glucosamine by a biosynthetic process, said microorganism being transformed with a recombinant nucleic acid molecule encoding glucosamine-6-phosphate synthase, said recombinant nucleic acid molecule operably linked to a transcription control sequence and comprising a i & i-i *, i genetic modification that increases the action of said glucosamine-6-phosphate synthase; wherein the expression of said recombinant nucleic acid molecule increases the production of glucosamine by said microorganism. 44. The microorganism according to claim 43, characterized in that said microorganism has at least one additional genetic modification in a gene encoding a protein selected from the group consisting of deacetylase N-acetylglucosamine-6-phosphate, deaminase glucosamine-6-phosphate, enzyme lNa9 specific for? / - acetyl-glucosamine, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C of PEP: glucose PTS, IEM, P / lllMan of PEP : mannose PTS, where said genetic modification reduces the action of said protein. 45. The microorganism according to claim 43, characterized in that said microorganism has at least one additional genetic modification in a gene encoding a phosphatase, wherein said genetic modification increases the action of said phosphatase. 46. The microorganism according to claim 43, characterized in that said microorganism is Escherichia coli having at least one additional genetic modification in a gel selected from the group consisting of nagA, nagB, nagC, nagD, * n í 'm A ^ a. &, ». *. a ^ Í i ** nagE, manXYZ, glmM, pfkA, pfkB, glmU, glmS and ptsG, wherein said modification reduces the genetic modification the action of a protein encoded by said gene. 47. The microorganism according to claim 43, characterized in that said microorganism is Escherichia coli that has a deletion of nag regu- tion genes. 48. The microorganism according to claim 43, characterized in that said microorganism is Escherichia coli which has a deletion of the nag regu- tion genes and a genetic modification in manXYZ genes in such a way that the proteins encoded by said manXYZ genes have reduced the action. 49. The microorganism according to claim 43, characterized in that said microorganism produces at least about 1 g / L of glucosamine when cultured from about 1 0 to about 60 hours at from about 28 ° C to about 37 ° C at a cell density of at least about 8 g / L per dry cell weight in a fermentation medium of pH 7.0 comprising: 14 g / L of K2HPO4, 16 g / L of KH2PO4, 1 g / L of Na3Citrate-2H2O, 5 g / L L (NH 4) 2 SO 4, 20 g / L of glucose, 10 mM of MgSO 4, 1 mM of CaCl 2, and from about 0.2 mM to about 1 mM of IPTG. 50. A microorganism for producing glucosamine by a biosynthetic process, said microorganism comprising: (a) a recombinant nucleic acid molecule encoding glucosamine-6-phosphate, said nucleic acid molecule l i * • t J -? -? Hl. J < , m..t > . Recombinant tt i t t i t t t by recombinantly binding a transcription control sequence, wherein the expression of said recombinant nucleic acid molecule increases the action of glucosamine-6-phosphate synthase by said microorganism; and 5 (b) at least one genetic modification in a gene encoding a protein selected from the group consisting of deacetylase? / - acetylglucosamine-6-phosphate, glucosamine-6-phosphate deaminase, llNaa enzyme specific for / V-acetyl-glucosamine , phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase -? / - 10 acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, Enzyme IIG | C of PEP: glucose PTS, IEM, P / lllMan of PE P: mannose PTS, wherein said genetic modification reduces the action of said protein. 51 The microorganism according to claim 50, characterized in that said microorganism has at least one additional genetic modification in a gene encoding a phosphatase, wherein said genetic modification increases the action of said phosphatase. 52. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a glucosamine-6-phosphate synthase having a genetic modification resulting in increased glucosamine-6-phosphate synthase action. 53. The recombinant nucleic acid molecule according to claim 52, characterized in that said genetic modification 25 results in at least one amino acid modification selected from "* •" ** "-" í At * t- »* •«. jt l: group consisting of elimination, insertion, inversion, substitution and derivation of at least one amino acid residue of said glucosamine-6-phosphate synthase, said at least one amino acid modification resulting in glucosamine-6 synthase action -phosphate increased. 54. The recombinant nucleic acid molecule according to claim 52, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid sequence encoding a glucosamine-6-phosphate synthase comprising an amino acid sequence selected from the group consisting of SEQ. ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28 and SEQ ID NO: 31. 55. The recombinant nucleic acid molecule according to claim 52, characterized in that said recombinant nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 30. 56. A glucosamine-6-phosphate synthase having glucosamine-6-phosphate synthase action, said synthase being encoded by a nucleic acid sequence having a genetic modification resulting in increased glucosamine-6-phosphate synthase action. 57. The glucosamine-6-phosphate synthase according to claim 56, characterized in that said synthase comprises «*« & »^^^^ ¡i ^^^ &? minus one amino acid modification selected from the group consisting of elimination, insertion, inversion, substitution and derivation of at least one amino acid residue. 58. The glucosamine-6-phosphate synthase according to claim 56, characterized in that said synthase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 30. 59. The glucosamine-6-phosphate synthase according to claim 56, characterized in that said synthase comprises an amino acid sequence selected from the group consisting of SEQ ID NO.J 9, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 31. 60. A method for producing glucosamine by fermentation, comprising: (a) growing in a fermentation medium comprising assimilable sources of carbon, nitrogen and phosphate, a genetically modified microorganism having increased glucosamine-6-phosphate synthase action, wherein said genetically modified microorganism is produced by a process comprising the steps of: (1) generating modifications in an isolated asylated nucleic acid molecule comprising a nucleic acid sequence encoding the ii i I * I I? glucosamine-6-phosphate synthase to create a plurality of genetically modified microorganisms; (2) transforming microorganisms with said modified nucleic acid sequences to produce genetically modified microorganisms; (3) classifying said genetically modified microorganism by the action of glucosamine-6-phosphate synthase; and (4) selecting said genetically modified microorganisms having increased glucosamine-6-phosphate synthase action; wherein said cultivation step produces a product selected from the gruó consisting of glucosamine-6-phosphate and glucosamine of said microorganism; and (b) recovering said product. SUMMARY The present invention relates to a method and materials for producing glucosamine by fermenting a genetically modified microorganism. Included in the present invention are the genetically modified microorganisms useful in the present method for producing glucosamine, as well as recombinant nucleic acid molecules and the proteins produced by such recombinant nucleic acid molecules.