Process and materials for production of glucosamine

Details Process and materials for production of glucosamine Process and materials for production of glucosamine

1998-07-15

2002-04-16

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S041000, C435S183000, C435S193000, C435S252330, C536S023100

Reexamination Certificate

active

06372457

ABSTRACT:

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
Amino sugars are usually found as monomer residues in complex oligosaccharides and polysaccharides. Glucosamine is an amino derivative of the simple sugar, glucose. Glucosamine and other amino sugars are important constituents of many natural polysaccharides. For example, polysaccharides containing amino sugars can form structural materials for cells, analogous to structural proteins.
Glucosamine is manufactured as a nutraceutical product with applications in the treatment of osteoarthritic conditions in animals and humans. The market for glucosamine is experiencing tremendous growth. Furthermore, 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-acetyl-D-glucosamine. Alternatively, glucosamine can also be produced by acid hydrolysis of variously acetylated chitosans. These processes suffer from poor product yields (in the range of 50% conversion of substrate to glucosamine). Moreover, the availability of raw material (i.e., a source of chitin, such as crab shells) is becoming increasingly limited. Therefore, there is a need in the industry for a cost-effective method for producing high yields of glucosamine for commercial sale and use.
SUMMARY OF THE INVENTION
One embodiment of the present invention relates to a method to produce glucosamine by fermentation of a microorganism. This method includes the steps of: (a) culturing in a fermentation medium a microorganism having a genetic modification in an amino sugar metabolic pathway; and (b) recovering a product produced from the step of culturing which is selected from the group of glucosamine-6-phosphate and glucosamine. Such an amino sugar metabolic pathway is selected from the group of a pathway for converting glucosamine-6-phosphate into another compound, a pathway for synthesizing glucosamine-6-phosphate, a pathway for transport of glucosamine or glucosamine-6-phosphate out of the microorganism, a pathway for transport of glucosamine into the microorganism, and a pathway which competes for substrates involved 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 a 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 further embodiments, the step of recovering can include purifying glucosamine from the fermentation medium, isolating glucosamine-6-phosphate from the microorganism, and/or dephosphorylating the glucosamine-6-phosphate to produce glucosamine. In one embodiment, at least about 1 g/L of product is recovered.
In yet another embodiment, the step of culturing 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 step of culturing is performed at a temperature of from about 28° C. to about 37° C. In yet another embodiment, the step of culturing is performed in a fermenter.
In one embodiment of the present invention, the microorganism has a modification in a gene which encodes a protein including, but not limited to, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetyl-glucosamine-specific enzyme II
Nag
, glucosamine-6-phosphate synthase, phosphoglucosamine mutase, glucosamine-1-phosphate acetyltransferase-N-acetylglucosamine-1-phosphate uridyltransferase, phosphofructokinase, enzyme II
Glc
of the PEP:glucose PTS, EIIM, P/III
Man
of the PEP:mannose PTS, and/or a phosphatase.
In another embodiment, the genetic modification includes a genetic modification which increases the action of glucosamine-6-phosphate synthase in the microorganism. Such a 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 operatively linked 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 which increases the action of the synthase. Such genetic modifications can result in reduced glucosamine-6-phosphate product inhibition of the glucosamine-6-phosphate synthase, for example.
In one embodiment, a recombinant nucleic acid molecule of the present invention which comprises a nucleic acid sequence encoding a glucosamine-6-phosphate synthase 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-28
2184
and nglmS-28
1830
.
Also included in the present invention are recombinant nucleic acid molecules encoding a glucosamine-6-phosphate synthase which comprises a genetic modification which increases the action of the glucosamine-6-phosphate synthase (i.e., a glucosamine-6-phosphate synthase homologue). Such a genetic modification can reduce glucosamine-6-phosphate product inhibition of the synthase, for example. In one embodiment, such a genetic modification in a recombinant nucleic acid molecule of the present invention which encodes a glucosamine-6-phosphate synthase results in at least one amino acid modification selected from the group of an addition, substitution, deletion, and/or derivatization of an amino acid residue of the glucosamine-6-phosphate synthase. In one embodiment, such an amino acid modification is in an amino acid sequence position in the modified protein (i.e., homologue) which corresponds to one or more of the following amino acid positions in amino acid sequence SEQ ID NO:16: Ile(4), Ile(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 yet another embodiment of the present invention, an amino acid modification as described above is preferably a substitution selected from the group of: Ile(4) to Thr, Ile(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 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: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 sel

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