DNA segments and methods for increasing polysaccharide production
AssigneesShin-Etsu Cio, Inc., Shin-Etsu Chemical Co., Ltd.
InventorsThomas J. Pollock, Motohide Yamazaki, Linda Thorne, Marcia Mikolajczak, Richard W. Armentrout
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Abstract
The present invention relates to DNA segments isolated from Sphingomonas sp. and involved in the biosynthetic production of sphingan polysaccharides to increase the production of the polysaccharide in engineered microorganisms. The present invention also relates to methods of engineering strains of Sphingomonas to produce bacteria which are hyperproducers of sphingan, methods of identifying and utilizing DNA fragments useful to enhance production of sphingan in bacteria and the hyperproducer bacteria.
Claims31
An isolated DNA sequence isolated from DNA of sphingan-producing bacteria selected from the group consisting of Sphingomonas sp., with the exception of ATCC 31461 ATCC 31554 and ATCC 53272, said DNA sequence when incorporated into a recipient Sphingomonas sp. bacterium in multiple copies produces a hyperproducer of sphingan polysaccharide relative to said recipient bacterium wherein said sphingan polysaccharide has the general formula:
- ##STR2## wherein Glc is glucose;
- GlcA is glucuronic acid;
- Rha is rhamnose;
- Man is mannose:
- X is Rha or Man;
- Z is attached to Glc residue 2 and is selected from the group consisting of .alpha.-L-Rha-(1-6-.alpha.-L-Rha, .alpha.-L-Man and .alpha.-L-Rha;
- W is at5ached to Glc residue number 1 and is .beta.-D-Glc-(1-6)-.alpha.-D-Glc or .alpha.-L-Rha;
- subscripts v and y may be 0, 0.33, 0.5, 0.67 or 1:
- and wherein the reducing end of the polymer is toward the X residue of the backbone, such that the backbone excludes W and Z when v and y are equal to 0.
A method of engineering a bacterium derived from a strain of Sphingomonas sp. to be a hyperproducer of sphingan polysaccharide comprising:
- isolating a DNA sequence from DNA of donor sphingan-producing bacteria selected from the group consisting of Sphingomonas sp., with the exception of ATCC 31461, ATCC 31554 and ATCC 53272;
- and
- inserting said DNA sequence into a recipient sphingomonas sp. bacterium in multiple copies to produce a bacterium which hyperproduces sphingan polysaccharide relative to said recipient bacterium under identical fermentation conditions wherein said sphingan polysaccbaride has the general formula:
- ##STR3## wherein Glc is glucose:
- GlcA is glucuronic acid:
- Rha is rhamnose:
- Man is mannose:
- X is Rha or Man Z is attached to Glc residue 2 and is selected from the group consisting of .alpha.-L-Rha-1-6)-.alpha.-L-Rha, .alpha.-L-Man and .alpha.-L-Rha, W is attached to Glc residue number 1 and is .beta.-D-Glc-(1-6)-.alpha.-D-Glc or .alpha.-L-Rha:
- subscripts v and y may be 0, 0.33, 0.5, 0.67 or 1:
- and wherein the reducing end of the polymer is toward the X residue of the backbone, such that the backbone exclude W and Z when v and y are equal to 0.
A bacterium derived from a strain of Sphingonmonas sp., said bacterium containing multiple copies of a DNA sequence isolated from DNA of donor sphingan-producing bacteria selected from the group consisting of Sphingomonas sp. with the exception of ATCC31461, ATCC31554 and ATCC 53272, said DNA sequence, when inserted in multiple copies into a recipient Sphingomonas sp. bacterium, results (resulting) in said recipient bacterium becoming a hyperproducer of sphingan polysaccharide.
The bacterium according to 13 wherein said DNA sequence contains a gene which codes a glycosyl transferase enzyme.
A method of enhancing the production of sphingan in a Sphingomonas bacterium comprising:
- 1) incorporating a DNA sequence isolated from a strain of donor sphingan-producing Sphingomonas bacteria selected from the group consisting of Sphingomonas sp., with the exception of ATCC 31461, ATCC 31554 and ATCC 53272, into a recipient Sphingomonas bacterium in multiple copies to produce a bacterium which hyperproduces sphingan polysaccharide relative to said recipient bacterium;
- 2) culturing the bacterium from step 1 in a fermentation broth to produce sphingan;
- and
- 3) isolating the sphingan from step 2 wherein said sphingan polysaccharide has the general formula:
- ##STR4## wherein Glc is glucose:
- GlcA is glucuronic acid;
- Rha is rhamnose;
- Man is mannose;
- X is Rha or Man;
- Z is attached to Glc residue 2 and is selected from the group consisting of .alpha.-L-Rha-(1-6)-.alpha.-L-Rha, .alpha.-L-Man and .alpha.-L-Rha;
- W is attached to Glc residue number 1 and is .beta.-D-Glc-(1-6)-.alpha.-D-Gla or .alpha.-L-Rha:
- subscripts v and y may be 0, 0.33, 0.5, 0.67 or 1:
- and wherein the reducing end of the polymer is toward the X residue of the backbone such that the backbone excludes W and Z when v and y are equal to 0.
Dependencies are inferred from the claim text.