Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-08-22
2003-03-25
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, C435S072000, C435S183000, C435S193000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06537786
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of microbial production of polysaccharides. More specifically, the invention pertains to nucleic acid molecules encoding enzymes involved in biosynthesis of exopolysaccharides from Methylomonas sp.
BACKGROUND OF THE INVENTION
Polysaccharides are sugar polymers that have been used widely as a thickener in food and non-food industries (Sanford et al.
Pure
&
Appl. Chem.
56: 879-892 (1984); Sutherland,
Trends Biotechnol,
16(1): 41-6 (1998)). They can be found in food products such as salad dressing, jam, frozen food, bakery products, canned food and dry food. Many other applications include suspending agents for pesticides, paints and other coating agents. They can act as flocculants, binders, film-formers, lubricants and friction reducers. Furthermore, exopolysaccharides are commonly used in oil field for oil recovery.
Traditionally, industrially useful polysaccharides have been derived from algal and plant sources. Over the past decade polysaccharides derived from microbes have been found increased usage (Sanford et al.
Pure
&
Appl. Chem.
56: 879-892 (1984)); Sutherland,
Trends Biotechnol,
16(1): 41-6 (1998)). One of the commercially well-known microbial exopolysaccharide is xanthan gum. Xanthan gum is a complex exopolysaccharide produced by a gram-negative bacterium
Xanthomonas campestris
pv.
Campestris
which is a pathogen of cruciferous plants. Xanthan consists of a &bgr;-1,4-linked D-glucose backbone with trisaccharides side chains composed of mannose-(&bgr;-1,4)-glucuronic acid-(&bgr;-1,2)-mannose attached to alternate glucose residues in the backbone by &agr;-1,3 linkages. The polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose 1-phosphate, glucose, mannose, glucuronic acid, and mannose on polyprenol phosphate carrier (Ielpi et al.,
J. Bacteriol.
175:2490-2500, 1993).
One of the most characterized pathways for the production of microbial exopolysaccharides is found in Xanthomonas. For example, the biosynthetic pathway of xanthan in
Xanthomonas campestris
comprises five stages: (i) conversion of simple sugars to nucleotidyl derivative precursors, (ii) assembly of pentasaccharide subunits attached to the inner membrane polyprenol phosphate carrier, (iii) addition of acetyl and pyruvate groups, (iv) polymerization of pentasaccharide repeat units, and (v) secretion of polymer.
Several enzymes or proteins involved in biosynthesis of xanthan and other exopolysaccharides are well known in the art. UDP-glucose pyrophosphorylase is the enzyme that catalyzes the reaction generating UDP-glucose (UTP+glucose-1-phosphate <--> UDP-glucose+Ppi) (Wei et al.,
Biochem Biophys Res Commun.
226:607-12 (1996)). UDP-glucose is the building blocks for many exopolysaccharides containing glucose.
A cluster of gum genes are found to be required for xanthan gum synthesis in
Xanthomonas campestris
(Katzen et al.
J. Bacteriol.
180:1607-1617 (1998); Chou, F. L., et al,
Biochem. Biophys. Res. Commun.
233 (1), 265-269 (1997)). For example, GumD, the glycosyltransferase, is responsible for the transfer of the first glucose to the lipid-linked intermediates in exopolysaccharide biosynthesis in
Xanthomonas campestris.
GumH is the protein involved in the transfer of the mannose to the lipid-linked intermediates in exopolysaccharide synthesis in
Xanthomonas campestris.
Many other genes involved in exopolysaccharide biosynthesis have been characterized or sequenced from other organisms. The epsB gene encodes the EpsB protein that is probably involved in polymerization and/or export of EPS, has been sequenced in
Ralstonia sola
(Huang et al,
Mol. Microbiol.
16: 977-989 (1995). The espM gene encoding EspM protein has been found in the esp gene cluster from
Streptococcus thermophilus
(Stingele et al,
J. Bacteiol.
178: 1680-1690 (1996)). Another putative polysaccharide export protein, WZA, is identified in
E. coli.
(Blattner et al.,
Science
277: 1453-1474 (1997)). Finally, the epsV gene encodes the EpsV protein, a transferase which transfers the sugar to polysaccharide intermediates, and it has also been sequence in
Streptococcus thermophilus
(Bourgoin et al.
Plasmid
40: 44-49 (1998); Bourgoin,F., et al.,
Gene
233:151-161 (1999).
In spite of the abundance of information regarding gene encoding microbial exopolysaccharides, no genes involved in this pathway have been isolated or characterized from C1 utilizing organisms, such as Methylomonas. As noted above, microbial exopolysaccharides have a variety of uses and it would be an advantage to synthesize this material from an abundance and inexpensive carbon source such as methane.
The problem to be solved therefore is to identify the genes relevant to exopolysaccharide synthesis in a C1 utilizing organism for the production of exopolysaccharides in both similar and unrelated microbes. Applicants have solved the stated problem by isolating and characterizing a complete enzymatic pathway for the synthesis of exopolysaccharide from a
Methylomonas sp.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid molecule encoding a
Methylomonas sp exopolysaccharide biosynthetic
enzyme, selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
Specifically the invention provides: 1) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 293 amino acids that has at least 58% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 2) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 473 amino acids that has at least 36% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:4, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 3) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 366 amino acids that has at least 36% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:6, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 4) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 779 amino acids that has at least 35% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:8, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 5) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 472 amino acids that has at least 23% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:10, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 6) an isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 272 amino acids that has at least 28% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:12, or a second nucleotide sequence comprising the complement of the first nucleotide sequence; 7) an isolated nucleic acid molecule comprising a f
Koffas Mattheos
Odom James M.
Wang Siqun
Wang Tao
Ye Rick W.
Fronda Christian L.
Nashed Nashaat T.
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