Genes encoding exopolysaccharide production

Abstract

Genes have been isolated from a Methylomonas sp encoding elements of the exopolysaccharide biosynthetic pathway. The genes and gene products are the first isolated from an organisms capable of utilizing single carbon (C1) substrates as energy sources. The genes are useful for engineering other C1 utilizing microorganisms to make altered levels of exopolysaccharide which is used in a variety of commercial applications.

What is claimed is:  
     
         1 . 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).    
     
     
         2 . The isolated nucleic acid molecule of  claim 1  selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17.  
     
     
         3 . A polypeptide encoded by the isolated nucleic acid molecule of  claim 1 .  
     
     
         4 . The polypeptide of  claim 3  selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18.  
     
     
         5 . 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.  
     
     
         6 . 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.  
     
     
         7 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 36% 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.  
     
     
         8 . 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.  
     
     
         9 . 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.  
     
     
         10 . 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.  
     
     
         11 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 284 amino acids that has at least 21% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:14, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         12 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 398 amino acids that has at least 26% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:16, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         13 . An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a polypeptide of at least 317 amino acids that has at least 51% identity based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:18, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.  
     
     
         14 . A chimeric gene comprising the isolated nucleic acid molecule of any one of claims  1  or  5 - 13  operably linked to suitable regulatory sequences.  
     
     
         15 . A transformed host cell comprising the chimeric gene of  claim 14 .  
     
     
         16 . The transformed host cell of  claim 15  wherein the host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, and green plants.  
     
     
         17 . The transformed host cell of  claim 16  wherein the host cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.  
     
     
         18 . The transformed host cell of  claim 16  wherein the host cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, Arabidopsis, cruciferous vegetables, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses.  
     
     
         19 . A method of obtaining a nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme comprising: 
 (a) probing a genomic library with the nucleic acid molecule of any one of claims  1  or  5 - 13 ;    (b) identifying a DNA clone that hybridizes with the nucleic acid molecule of any one of claims  1  or  5 - 13 ; and    (c) sequencing the genomic fragment that comprises the clone identified in step (b),    wherein the sequenced genomic fragment encodes a Methylomonas sp exopolysaccharide biosynthetic enzyme.    
     
     
         20 . A method of obtaining a nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme comprising: 
 (a) synthesizing an at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and    (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a); wherein the amplified insert encodes a portion of an amino acid sequence encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme.    
     
     
         21 . The product of the method of claims  19  or  20 .  
     
     
         22 . A method for the production of exopolysaccharide comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of a carbon source whereby exopolysaccharide is produced, said transformed host cell comprising a set of nucleic acid molecules encoding SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; under the control of suitable regulatory sequences.  
     
     
         23 . A method according to  claim 22  wherein the transformed host cell is selected form the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.  
     
     
         24 . A method according to  claim 22  wherein said methanotrophic bacteria: 
 (a) grows on a Cl carbon substrate selected from the group consisting of methane and methanol; and  
 (b) comprises a functional Embden-Meyerhof carbon pathway, said pathway comprising a gene encoding a pyrophosphate dependent phosphofructokinase enzyme.  
 
     
     
         25 . A method according to  claim 24  wherein said methanotrophic bacteria is methylomonas 16a ATCC PTA 2402.  
     
     
         26 . A method according to  claim 22  wherein the transformed host cell is selected form the group consisting of: soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, Arabidopsis, cruciferous vegetables, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses.  
     
     
         27 . A method according to  claim 22  wherein the carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, carbon dioxide, methanol, methane, formaldehyde, formate, and carbon-containing amines.  
     
     
         28 . A method according to  claim 22  wherein the transformed host is selected from the group consisting of Methylomonas, Methylobacter and Methanobacterium and the carbon source is selected from the group consisting of methane and methanol.  
     
     
         29 . A method of regulating exopolysaccharide biosynthesis in an organism comprising, over-expressing at least one isoprenoid gene selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, and 17 in an organism such that the exopolysaccharide biosynthesis is altered in the organism.  
     
     
         30 . A method according to  claim 29  wherein said exopolysaccharide gene is over-expressed on a multicopy plasmid.  
     
     
         31 . A method according to  claim 29  wherein said exopolysaccharide gene is operably linked to an inducible or regulated promoter.  
     
     
         32 . A method according to  claim 29  wherein said exopolysaccharide gene is expressed in antisense orientation.  
     
     
         33 . A method according to  claim 29  wherein said exopolysaccharide gene is disrupted by insertion of foreign DNA into the coding region.  
     
     
         34 . A mutated nucleic acid molecule encoding a Methylomonas sp exopolysaccharide biosynthetic enzyme having an altered biological activity produced by a method comprising the steps of: 
 (i) digesting a mixture of nucleotide sequences of any one of claims  1  or  5 - 13  with restriction endonucleases wherein said mixture comprises: 
 a) a native microbial gene;  
 b) a first population of nucleotide fragments which will hybridize to said native microbial sequence;  
 c) a second population of nucleotide fragments which will not hybridize to said native microbial sequence;  
   wherein a mixture of restriction fragments are produced;    (ii) denaturing said mixture of restriction fragments;    (iii) incubating the denatured said mixture of restriction fragments of step (ii) with a polymerase;    (iv) repeating steps (ii) and (iii) wherein a mutated microbial gene is produced encoding a protein having an altered biological activity.