DNA segments and methods for increasing polysaccharide...

Details DNA segments and methods for increasing polysaccharide... DNA segments and methods for increasing polysaccharide...

1999-11-15

2001-09-04

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C536S023200, C536S023100

Reexamination Certificate

active

06284516

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to DNA sequences and fragments, thereof, which are involved in the biosynthetic production of sphingan polysaccharides of and isolated from Sphingomonas sp. The isolated DNA fragments may be inserted into the same or different strains of Sphingomonas sp. or related bacteria in multiple copies to increase polysaccharide, preferably sphingan production. The engineered bacteria containing exogenous DNA produce significantly greater amounts of polysaccharide compared to non-engineered bacteria under identical fermentation conditions. The present invention also relates to methods of engineering strains of Sphingomonas sp. and other related bacteria to be hyperproducers of polysaccharide as well as the engineered bacteria. Methods of identifying and isolating DNA sequences useful for increasing the production of sphingan polysaccharides in Sphingomonas sp. are also described.
BACKGROUND OF THE INVENTION
A number of microorganisms produce extracellular polysaccharides, also known as exopolysaccharides or EPS. Of the exopolysaccharides, xanthan gum and a group of polysaccharides known as “sphingans” are included. “Sphingans” are produced by gram-negative bacteria of the genus Sphingomonas.
Xanthomonas campestris
is a gram-negative bacterium which constitutively produces an exopolysaccharide, xanthan gum, in large amounts. Jeanes, et al.,
J. Appl. Polymer Sci
., 5, 519-526 (1961). The biosynthesis of xanthan gum has been studied in considerable detail because of its commercial importance. Recently, another bacterial exopolysaccharide, gellan, was developed as a gelling agent. Gellan is a member of a family of related polysaccharides which includes S-88 (See, Kang and Veeder, U.S. Pat. No. 4,535,153); welan (See, Kang and Veeder, U.S. Pat. No. 4,342,866); NW11 (See, Robison and Stipanovic, U.S. Pat. No. 4,874,044); rhamsan (See, Peik, et al., U.S. Pat. No. 4,401,760); S-198 (See, Peik, et al. U.S. Pat. No. 4,529,797); S-657 (See, Peik, et al., Eur. Patent Application 209277A1); and heteropolysaccharide-7 (See, Kang and McNeely, U.S. Pat. No. 4,342,866). This group of polysaccharides is referred to as “sphingans” because they are all produced by gram-negative bacteria belonging to the genus Sphingomonas.
The above documents include several patents which relate to sphingan polysaccharide compositions. None of the patents remotely relates to the subject matter of the instant invention.
Strain
Sphingan
Patent Number
ATCC 31461
gellan
4,326,053
S60
S60
ATCC31554
S-88 
4,535,153
S88
ATCC31853
S-198
4,529,797
S198
ATCC21423
S-7 
3,960,832
S7
ATCC31555
welan
4,342,866
S130
S-130
ATCC31961
rhamsan
4,401,760
S193
S-194
ATCC53159
S-657
EurApp 0209277
S-657
ATCC53272
NW-11
4,874,044
NW11
The chemical structures of the sphingan polysaccharides are all somewhat related. The main chain of each sphingan consists of a related sequence of four sugars-D-glucose, D-glucuronic acid, L-mannose and L-rhamnose. Polysaccharide members of the sphingan group are distinguishable from each other by virtue of the carbohydrates which comprise the polymer backbone (main chain) and the sidechains. The sphingan carbohydrates may contain carbohydrate side chains and acetyl or pyruvyl groups attached to carbohydrates on the polymer backbone.
Various sphingans are useful as specialty polymers and as additives in textile applications, foods, cosmetics, paper, paint, cements, e.g. as viscosity modifiers, in various other coating applications, and as adhesives and additives to petroleum products and specialty chemicals.
The focus of initial studies which culminated in the present invention was the first step in the biosynthesis of a representative sphingan polysaccharide, S-88. This sphingan is biosynthesized by Sphingomonas strain S88. Prior to the instant invention, it was known that some, but not all, bacterial polysaccharide biosynthesis of other than sphingans utilize an isoprenylphosphate carrier. For example, in the case of xanthan gum biosynthesis by
X. campestris
, since the main chain of xanthan gum contains only glucose, the first synthetic step is likely the transfer of glucose-phosphate from UDP-glucose to a C55-isoprenylphosphate (IP) carrier. With cell-free incorporation assays, Ielpi, et al.,
FEBS lett
., 130, 253 (1982) and
J. Bacteriol
., 175, 2490 ((1993), con-firmed that glucose, followed by a second glucose, and then mannose, glucuronic acid and mannose are added sequentially to carrier IP to assemble the repeating unit of xanthan gum. Quite similarly, the repeating subunit of colanic acid in
Eschenchia coli
is assembled by first transferring glucose-P to IP. Johnson and Wilson,
J. Bacteriol
., 129, 225 (1977). By contrast, in the case of the synthesis of succinoglycan polysaccharides by
Rhizobium meliloti
, a galactose-P is transferred first to IP. See, Tolnasky, et al.,
J. Biol. Chem
., 257, 6751 (1982). Isoprenyl carriers, however, are not involved in the synthesis of dextran or levan polysaccharides, and the role of isoprenyl carriers in alginate synthesis is unknown.
Prior to the investigation which led to the present invention, the importance of the role of the carrier in the complex kinetics of the biosynthesis of polysaccharides was not known. In addition, it was not known what role the isoprenylphosphate carrier might play in the overall synthesis of sphingan polysaccharides in Sphingomonas bacteria.
Previously, genetic complementation tests have shown that a special class of mutations in
X. campestris
which are simultaneously Bac
r
and Gum
−
(bacitracin-resistant and xanthan gum-negative) map within the gumD gene which is required for transferring glucose-P from UDP-Glc to IP to give Glc-PPI. Pollock, et al., 1994
, J. Bacteriol
, vol. 176, pp. 6229-6237, Vanderslice, et al., “Genetic Engineering of polysaccharide structure in
Xanthomonas campestris
”, p. 145-156, in V. Crescenzi, et al., Biomedical and Biotechnological Advances in Industrial Polysaccharides, Gordon and Breach Science Publishers, New York and N. E. Harding and Y. N. Patel, 1993
, Faseb Journal
, Vol. 7, Number 7. The latter reference discloses fragments of DNA that can restore synthesis of sphingan S-60 to non-producing mutants, but gives no indication of increased synthesis relative to the wild-type strain. Earlier experimentation also showed that the wild type gumD gene of
X. campestris
could restore synthesis of sphingans in analogous Bac
r
Sps
−
(sphingan polysaccharide-negative) mutants of Sphingomonas strains S88 and NW11. It was suggested that Bac
r
Sps
−
Sphingomonas mutants also appeared to be blocked in the transfer of glucose-P to IP.
OBJECT OF THE INVENTION
It is an object of the present invention to provide DNA segments which are isolated from Sphingomonas sp. and may be used to enhance the production of sphingan polysaccharide in a number of microorganisms, and in particular, a number of strains of Sphingomonas.
It is also an object of the present invention to provide hyproducer strains of microorganisms, and in particular, a number of strains of Sphingomonas which will produce significantly more sphingan polysaccharide than non-engineered strains.
It is a further object of the present invention to provide a method for producing strains of microorganisms, and in particular, strains of Sphingomonas sp. which are hyperproducers of sphingan polysaccharide.
It is an additional object of the present invention to provide a method for isolating DNA segments which may be inserted into Sphingomonas strains so that the resulting engineered microorganism becomes a hyperproducer of sphingan polysaccharide.
These and other objectives of the present invention may be readily gleaned from the description of the invention which follows.
SUMMARY OF THE INVENTION
In the present invention, sequences of DNA as segments or fragments are isolated from sphingan-producing bacteria, generally from Sphingomonas strains. The resulting genetic material is cloned, incorporated as multiple copies into sphingan-producing or non-producing mutants

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