U.S. patent application number 09/986803 was filed with the patent office on 2002-08-01 for genetic control of acetylation and pyruvylation of xanthan based polysaccharide polymers.
Invention is credited to Doherty, Daniel H., Ferber, Donna M., Hassler, Randal A., Marrelli, John D., Vanderslice, Rebecca W..
Application Number | 20020103370 09/986803 |
Document ID | / |
Family ID | 27534254 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020103370 |
Kind Code |
A1 |
Doherty, Daniel H. ; et
al. |
August 1, 2002 |
Genetic control of acetylation and pyruvylation of xanthan based
polysaccharide polymers
Abstract
Variant xanthan gums are provided which include a water-soluble
polysaccharide polymer comprising repeating pentamer units having a
D-glucose:D-mannose:D-glucuronic acid ratio of about 2:2:1, and a
water-soluble polysaccharide polymer comprising repeating tetramer
units having a D-glucose:D-mannose:D-glucuronic acid ratio of about
2:1:1. The D-glucose moieties are linked in a beta-[1,4]
configuration. The inner D-mannose moieties are linked in an
alpha-[1,3] configuration, generally to alternate glucose moieties.
The D-glucuronic acid moieties are linked in a beta-[1,2]
configuration to the inner mannose moieties. The outer mannose
moieties are linked to the glucuronic acid moieties in a beta-[1,4]
configuration. Processes for preparing the polysaccharide polymers
are also provided.
Inventors: |
Doherty, Daniel H.;
(Boulder, CO) ; Ferber, Donna M.; (Louisville,
CO) ; Marrelli, John D.; (Houston, TX) ;
Vanderslice, Rebecca W.; (Boulder, CO) ; Hassler,
Randal A.; (Lafayette, CO) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
27534254 |
Appl. No.: |
09/986803 |
Filed: |
November 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09986803 |
Nov 13, 2001 |
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08475823 |
Jun 7, 1995 |
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08475823 |
Jun 7, 1995 |
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08232416 |
Jan 25, 1994 |
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08232416 |
Jan 25, 1994 |
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07696732 |
May 7, 1991 |
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07696732 |
May 7, 1991 |
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07384621 |
Jul 25, 1989 |
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09986803 |
Nov 13, 2001 |
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07566875 |
Jun 11, 1990 |
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07566875 |
Jun 11, 1990 |
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07029090 |
Mar 23, 1987 |
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07029090 |
Mar 23, 1987 |
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06844435 |
Mar 26, 1986 |
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06844435 |
Mar 26, 1986 |
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06762878 |
Aug 6, 1985 |
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Current U.S.
Class: |
536/119 ;
536/123 |
Current CPC
Class: |
C12P 19/06 20130101;
C08B 37/0033 20130101; C09K 8/08 20130101; Y10S 435/822 20130101;
C12N 1/205 20210501; C12N 15/01 20130101; C12R 2001/64 20210501;
C12N 15/00 20130101 |
Class at
Publication: |
536/119 ;
536/123 |
International
Class: |
C07H 013/02 |
Claims
What is claimed is:
1. A water-soluble polysaccharide polymer comprising repeating
pentamer units having a D-glucose:D-mannose:D-glucuronic acid ratio
of about 2:2:1, wherein the D-glucose moieties are linked in a
beta-[1,4] configuration, inner D-mannose moieties are linked in an
alpha-[1,3] configuration primarily to alternate glucose moieties,
the D-glucuronic acid moieties are linked in a beta-[1,2]
configuration to said inner mannose moieties, and outer mannose
moieties are linked to said glucuronic acid moieties in a
beta-[1,4] configuration.
2. The polysaccharide polymer of claim 1, wherein said inner
mannose moieties are acetylated.
3. The polysaccharide polymer of claim 2, wherein said outer
mannose moieties are acetylated.
4. The polysaccharide polymer of claim 1, wherein a portion of said
outer mannose moieties are pyruvylated and the remainder are
acetylated.
5. The polysaccharide polymer of claim 2, wherein substantially all
of said outer mannose moieties are pyruvylated.
6. The polysaccharide polymer of claim 1, wherein said outer
mannose moieties are pyruvylated.
7. The polysaccharide polymer of claim 1, wherein said outer
mannose moities are acetylated.
8. A water-soluble polysaccharide polymer comprising repeated
tetramer units having a D-glucose:D-mannose:D-glucuronic acid ratio
of about 2:1:1 wherein (1) the D-glucose moieties are linked in a
beta-[1,4] configuration, (2) the D-mannose moieties, acetylated or
not acetylated at the 6-0 position, are linked in an alpha-[1,3]
configuration primarily to alternate glucose moieties, and (3) the
D-glucuronic acid moieties, and (3) the D-glucuronic acid moieties
are linked in a beta-[1,3] configuration to the mannose
moieties.
9. The polysaccharide polymer of claim 8, wherein the mannose
moieties of the polysaccharide polymer are acetylated at the 6-0
position.
10. The polysaccharide polymer of claim 8, wherein the mannose
moieties of the polysaccharide polymer are not acetylated at the
6-0 position.
11. The polysaccharide polymer of claim 8, wherein at least 90% of
the mannose moieties of the polysaccharide polymer are acetylated
at the 6-0 position.
12. A process for preparing the polysaccharide polymer of claim 1
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 1 that is
non-acetylated at the inner mannose and is non-acetylated and
non-pyruvylated at the outer mannose, and incubating said
inoculated growth medium at a suitable temperature, pH and
dissolved oxygen level for a time sufficient to produce the
polysaccharide polymer of claim 1.
13. A process for preparing the polysaccharide polymer of claim 2
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 2 that is
acetylated at the inner mannose and is non-acetylated and
non-pyruvylated at the outer mannose, and incubating said
inoculated growth medium at a suitable temperature, pH and
dissolved oxygen level for a time sufficient to produce the
polysaccharide polymer of claim 2.
14. A process for preparing the polysaccharide polymer of claim 3
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 3 that is
acetylated at the inner mannose and is acetylated and
non-pyruvylated at the outer mannose, and incubating said
inoculated growth medium at a suitable temperature, pH and
dissolved oxygen level for a time sufficient to produce the
polysaccharide polymer of claim 3.
15. A process for preparing the polysaccharide polymer of claim 4
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 4 that is
non-acetylated at the inner mannose and is acetylated and
pyruvylated at the outer mannose, and incubating said inoculated
growth medium at a suitable temperature, pH and dissolved oxygen
level for a time sufficient to produce the polysaccharide polymer
of claim 4.
16. A process for preparing the polysaccharide polymer of claim 5
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 5 that is
acetylated at the inner mannose and is pyruvylated but
non-acetylated at the outer mannose, and incubating said inoculated
growth medium at a suitable temperature, pH and dissolved oxygen
level for a time sufficient to produce the polysaccharide polymer
of claim 5.
17. A process for preparing the polysaccharide polymer of claim 6
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 6 that is
non-acetylated at the inner mannose and is pyruvylated but
non-acetylated at the outer mannose, and incubating said inoculated
growth medium at a suitable temperature, pH and dissolved oxygen
level for a time sufficient to produce the polysaccharide polymer
of claim 6.
18. A process for preparing the polysaccharide polymer of claim 7
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing the polysaccharide polymer of claim 7 that is
non-acetylated at the inner mannose and is acetylated but not
pyruvylated at the outer mannose, and incubating said inoculated
growth medium at a suitable temperature, pH and dissolved oxygen
level for a time sufficient to produce the polysaccharide polymer
of claim 7.
19. A process for preparing the polysaccharide polymer of claim 8
which comprises; inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing a polytetramer gum of claim 8, and incubating the
inoculated growth medium at suitable temperature, pH and dissolved
oxygen levels to produce a polysaccharide polymer having a
D-glucose:D-mannose:D-glucuronic acid ratio of about 2:1:1.
20. The process of claim 19 wherein the polysaccharide polmer is
recovered from the growth medium by precipitation or
ultrafiltration.
21. The process of claim 19 wherein the microorganism is of the
species Xanthomonas campestris.
22. The process of claim 19 wherein the microorganism is a
Transferase V deficient mutant of Xanthomonas campestris.
23. The process of claim 19 wherein the microorganism is a
Transferase V and Acetylase deficient mutant of Xanthomonas
campestris.
24. The process of claim 19 wherein the microorganism is a
Transferase V and acetyl coenzyme A deficient mutant of Xanthomonas
campestris.
25. The process of claim 19 wherein the microorganism is a
phosphoenolpyruvate deficient mutant of Xanthomonas campestris.
26. A process for preparing the polysaccharide polymer of claim 11
which comprises; inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which is capable of
synthesizing a polytetramer gum of claim 11, and incubating the
inoculated growth medium at suitable temperature, pH and dissovled
oxygen levels to produce a polysaccharide polymer having a
D-glucose:D-mannose:D-glucuronic acid ratio of about 2:1:1 wherein
said polymer is not acetylated.
27. The process of claim 12, wherein said microorganism is a
Ketalase, Acetylase I, and Acetylase II deficient mutant of
Xanthomonas campestris.
28. The process of claim 13, wherein said microorganism is a
Ketalase and Acetylase II deficient mutant of Xanthamonas
campestris.
29. The process of claim 14, wherein said microorganism is a
Ketalase deficient mutant of Xanthomonas campestris.
30. The process of claim 15, wherein said microorganism is an
Acetylase I deficient mutant of Xanthomonas campestris.
31. The process of claim 16, wherein said microorganism is an
Acetylase II deficient mutant of Xanthomonas campestris.
32. The process of claim 17, wherein said microorganism is an
Acetylase I and Acetylase II deficient mutant of X campestris.
33. The process of claim 18, wherein said microorganism is an
Acetylase I and Ketalase deficient mutant of X campestris.
34. The process of claim 26 wherein the microorganism is an
Acetylase deficient mutant of Xanthomonas campestris.
35. The process of claim 26 wherein the microorganism is ATCC No.
53473, an Acetylase deficient mutant of Xanthomonas campestris,
(X1006).
36. A process for preparing the polysaccharide polymer of claim 1
comprising: inoculating a suitable growth medium with a
microorganism of the genus Xanthomonas which contains a chromosomal
deletion mutation and is capable of synthesizing the polysaccharide
polymer of claim 1 that is non-acetylated at the inner mannose and
is non-acetylated at the outer mannose, and incubating said
inoculated growth medium at a suitable temperature, pH and
dissolved oxygen level for a time sufficient to produce the
polysaccharide polymer of claim 1.
37. The process of claim 36 wherein the microorganism is of the
species Xanthomonas campestris.
38. The process of claim 36 wherein the microorganism contains an
Acetylase I and II chromosomal deletion mutation.
39. The process of claim 36 wherein the microorganism is an
Acetylase I and II chromosomal deletion mutant of Xanthomonas
campestris (X1910).
Description
BACKGROUND OF THE INVENTION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 07/384,621, filed on Jul. 25, 1989 hereby
specifically incorporated herein by reference. This application is
also a continuation-in-part of U.S. patent application Ser. No.
07/566,875, filed Aug. 13, 1990, which is a continuation
application of U.S. patent application Ser. No. 07/029,090, filed
Mar. 23, 1987, which is a continuation-in-part application of U.S.
patent application Ser. No. 06/844,435, filed Mar. 26, 1986, all of
which are hereby specifically incorporated herein by reference.
[0002] This invention relates to polysaccharide polymers. In
particular, it relates to xanthan-based polysaccharide polymers,
defined herein as polymers structurally similar to xanthan gum and
produced by components of the xanthan biosynthetic pathway,
including those xanthan-based polymers modified so that the outer
mannose can be specifically acetylated but not pyruvylated,
pyruvylated but not acetylated, or unmodified while the inner
mannose can be independently controlled to be acetylated or
unmodified.
[0003] Xanthan gum is produced by bacteria of the genus
Xanthomonas, in particular by microorganisms of the species X.
campestris. Xanthan gum is a widely used product due to its unusual
physical properties, i.e., its extremely high specific viscosity
and its pseudoplasticity. It is commonly used in foods as a
thickening agent and in secondary or tertiary oil recovery as a
mobility control and profile modification agent, as well as in
petroleum drilling fluids.
[0004] Chemically, xanthan gum is an anionic heteropolysaccharide.
The repeating unit of the polymer is a pentamer composed of five
sugar moieties, specifically two glucose, one glucuronic acid and
two mannose moieties. These sugar residues are arranged such that
the glucose moieties form the backbone of the polymer chain, with
side chains of mannose-glucuronic acid-mannose residues generally
extending from alternate glucose moieties. Usually, this basic
structure is specifically acetylated and pyruvylated, as described,
for example, by Janson, P. E., Kenne, L., and Lindberg, B., in
Carbohydrate Research, 45:275-282 (1975) and Melton, L. D., Minot,
L., Rees, D. A., and Sanderson, G. R., in Carbohydrate Research,
46:245-257 (1976), each of which is specifically incorporated
herein by reference. The extent of acetylation and pyruvylation is
known to vary. The structure of xanthan gum is depicted in formula
I below: 1
[0005] In spite of the broad utility of naturally-occurring xanthan
gum, there are some situations where its physical properties become
limiting. In particular, in secondary or tertiary oil recovery it
is not uncommon for the temperature of the oil bearing reservoir
and the salt concentrations in the reservoir brine to be higher
than are optimal for xanthan solutions. When these conditions
occur, xanthan can precipitate, flocculate and/or lose its
viscosity. Therefore, new viscosifying products which perform well
at various conditions encountered during oil recovery, such as high
temperature and high salt concentrations would be desirable.
[0006] The present invention discloses a family of xanthan-based
polysaccharides having improved properties relative to
naturally-occurring xanthan gum. Modifications of xanthan gum have
been previously described. For example, Bradshaw et al.
(Carbohydrate Polymers, 3:23-38 (1983)) describe methods for
preparing chemically-modified xanthan gum which is deacetylated or
depyruvylated. Various means of chemically deacetylating xanthan
gum produced by Xanthomonas campestris also are described in U.S.
Pat. Nos. 3,000,790 and 3,054,689. To date, the predominant method
utilized for these deacetylation processes has been chemical
removal of the acetate moieties from normally acetylated xanthan
gum. It has been found that chemical processes for deacetylating
xanthan gums can result in a number of undesirable side effects and
may cause hydrolysis of the glycosidic backbone, resulting in an
irreversible change in the conformation of the molecule and lowered
molecular weight.
[0007] Some of the Theological properties of deacetylated xanthan
in aqueous media are known. See, e.g., Tako and Nakamura, Agric.
Biol. Chem. 48:2987-2993 (1984) and U.S. Pat. Nos. 3,000,790 and
3,054,689. Also, a method of increasing the viscosity of an aqueous
solution using a deacetylated polysaccharide is described in U.S.
Pat. No. 3,096,293. Thus, a method for obtaining non-acetylated
xanthan which does not cause untoward side effects has been
sought.
[0008] Xanthan gum can be chemically depyruvylated as well, as
described by Holzwarth and Ogletree in Carbo. Res. 76:277-280
(1979). This chemical method of depyruvylation also can alter the
xanthan polymeric unit and/or cause hydrolysis of the glycosidic
backbone. While a strain of X. campestris has been described in
U.S. Pat. No. 4,296,203 which produces non-pyruvylated xanthan gum,
this non-pyruvylated gum was either fully acetylated or
deacetylated using chemical means.
[0009] Additionally, the extent of acetylation of the internal
mannose on the xanthan side chain and the extent of the
pyruvylation of the terminal mannose may vary. The present
inventors believe that a fully acetylated and/or fully pyruvylated
xanthan will have improved Theological properties for certain oil
recovery purposes.
[0010] Moreover, the present inventors have identified
polysaccharides which are based on alterations of the normal
xanthan pentamer building block. These polymers exhibit improved
rheological properties over normal xanthan gum with respect to
shear rate, their ability to tolerate salinity and their response
to temperature as it affects their viscosifying properties. These
altered polysaccharides include the polytrimer which is depicted
below and the non-acetylated polytetramer. 2
[0011] These polysaccharides also include the acetylated and
non-acetylated polytrimer described by Vanderslice et al. in
copending U.S. patent application Ser. No. 762,878, entitled "A
Polysaccharide Polymer Made By Xanthomonas," filed Aug. 6, 1985,
which is specifically incorporated herein by reference.
[0012] An object of the present invention is to provide a family of
polysaccharide polymers which are better viscosifiers of water than
naturally-occurring xanthan gum. Another object of the present
invention is to provide a family of polysaccharide polymers having
improved Theological properties over naturally-occurring xanthan
gum at elevated temperatures and/or in the presence of salts and
which members possess other desired properties.
[0013] An object of the present invention is to provide a family of
polysaccharide xanthan polymers in which the inner mannose is
acetylated or unmodified while the outer mannose is acetylated,
pyruvylated or unmodified.
[0014] It is also an object of the present invention to provide an
in vitro method for obtaining these products and microorganisms
having the ability to produce members of this family of
polysaccharide polymers in vivo. A further object of the present
invention is to provide processes for preparing members of this
family of polysaccharides by aerobically fermenting microorganisms
having the ability to produce the various polysaccharide
polymers.
[0015] It is also an object of the present invention to provide a
process for preparing members of this family of polysaccharides by
aerobically fermenting microorganisms containing chromosomal
mutations which give these microorganisms the ability to produce
the various polysaccharide polymers.
[0016] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The objects and advantages may be realized and
attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
[0017] To achieve the objects and in accordance with the purposes
of the invention, as embodied and broadly described herein, there
is provided a composition comprising a polysaccharide polymer
having a D-glucose:D-mannose:D-glucuronic acid ratio of about
2:2:1, wherein the D-glucose moieties are linked in a beta-[1,4]
configuration, inner D-mannose moieties are linked in an
alpha-[1,3] configuration, generally to alternate glucose moieties,
the D-glucuronate moieties are linked in a beta-[1,2] configuration
to the inner mannose moieties and outer mannose moieties are linked
to the D-glucuronate moieties in a beta-[1,4] configuration.
[0018] To further achieve the objects and in accordance with the
purposes of the present invention, there is provided a composition
comprising xanthan gum wherein the inner mannose moieties are
acetylated at the 6-0 position. Another structure contemplates both
the inner and outer mannose moieties being acetylated. A further
structure is provided having a portion of the outer mannose
moieties pyruvylated at the 4-6 position and a portion acetylated.
Another structure is provided with the outer mannose moieties
pyruvylated at the 4-6 position and the inner mannose moieties
acetylated at the 6-0 position. Two additional structures are
provided, one having the outer mannose moieties pyruvylated at the
4-6 position and the other having the outer mannose moieties
acetylated.
[0019] To further achieve the objects and in accordance with the
purposes of the invention, as embodied and broadly described
herein, there is provided a composition comprising a polysaccharide
polymer having a D-glucose:D-mannose:D-glucuronic acid ratio of
about 2:1:1, wherein the D-glucose moieties are linked in a
beta-[1,4] configuration, the D-mannose moieties are linked in an
alpha-[1,3] configuration, generally to alternate glucose moieties,
and the D-glucuronate moieties are linked in a beta-[1,2]
configuration to the mannose moieties. This polysaccharide polymer
is herein termed "polytetramer" because it consists of a repeating
tetramer unit: glucose-glucose-mannose-glucuronic acid. There is
also provided a polytetramer composition, as described above,
wherein at least 90%, preferably 95% and most preferably 100% of
the mannose moieties are acetylated at the 6-0 position as well as
a polytetramer which is non-acetylated.
[0020] Also, the present invention relates to a xanthan gum herein
referred to as "fully acetylated xanthan gum," wherein at least 90%
of the internal mannose moieties are acetylated, preferably 195%
and more preferably 100%, are acetylated. Also, the present
invention relates to a xanthan gum wherein at least 90% of the
terminal mannose moieties, preferably 95% and more preferably 100%,
are pyruvylated, herein referred to as "fully-pyruvylated xanthan
gum."
[0021] This invention also contemplates processes for the
production of the polysaccharide polymers described above. The
polysaccharide polymers of this invention can be made generally by
genetic manipulations of the microbial biosynthetic pathways which
lead to the production of polysaccharides. In particular, microbial
pathways for the production of xanthan gum may be manipulated to
create an in vivo or in vitro system for the production of an
altered polymeric unit. Thus, systems can be created through the
use of mutated Acetylase I, Acetylase II and Ketalase genes, in
particular, to create polysaccharides which are acetylated or
pyruvylated to varying degrees. For example, it is contemplated
that xanthan gum which is 10%, 20%, 30%, 40%, or 50% can be
synthesized as well as xanthan which is 10%, 20%, 30%, 40%, 50%,
60%, 70%, or 80% pyruvylated. Microorganisms which produce the
present polysaccharide polymers in vivo and methods of using these
polysaccharide polymers are also disclosed.
[0022] The inventors also describe an in vivo system for the
production of an altered polymeric unit where the mutated genes are
incorporated into the chromosome of the microorganism rather than
in a recombinant plasmid. This chromosomal deletion mutation is
advantageous because it eliminates potential problems with plasmid
maintenance, and also could contribute to strain stability.
[0023] It is understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention, as
claimed. The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate various
embodiments of the invention and, together with the description,
serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1a shows the BamHI restriction map of a 16 kb region of
the chromosome of X. campestris that contains a cluster of twelve
genes required for biosynthesis of xanthan and also shows the
approximate locations of these twelve genes relative to the BamHI
restriction map.
[0025] FIG. 1b shows some restriction sites in and around genes F
and G and the DNA sequence at the junction of genes F and G.
[0026] FIG. 2 depicts the construction of a deletion mutation
(delCla) within gene gumF of the gum gene cluster.
[0027] FIG. 3a shows the structure of plasmid p13delCla derived
from pRK290-H336 by in vivo insertion of transposon TnK12 into the
vector portion of pRK290-H336 in the approximate location shown in
the figure and the subsequent deletion as described in FIG. 2 of
the 660 base pair ClaI DNA fragment within gene gumF.
[0028] FIG. 3b shows the structure of plasmid pH336KBmldelCla which
is similar in structure to p13delCla, containing the same 660 base
pair deletion within gene gumF. This plasmid contains an insertion
mutation (KBm1) at the BamHI site within gene gumG. The DNA
fragment inserted there is a BamHI restriction fragment carrying
the kanamycin-resistance gene of plasmid pUC4-K.
[0029] FIG. 4 depicts the chemical structure, and a schematic
representation, of the repeating unit of the polytetramer variant
of xanthan gum.
[0030] FIG. 5a shows the structure of plasmid pHA3KBm2delCla
derived from pRK290-HA3 which is identical to pRX290-H336 except
that it does not contain the 1.4 kb and 1.5 kb BamHI fragments of
the X. campestris gum biosynthetic operon DNA and therefore lacks
genes B and C but contains genes D through M. pHA3KBm2delCla
contains the gene gumF deletion mutation described in FIG. 2 and an
insertion mutation in the BamHI site of gene gumI. The inserted DNA
is again a BamHI restriction fragment of pUC4-K which carries a
gene conferring kanamycin resistance.
[0031] FIG. 5b shows the extent of a chromosomal deletion mutation
present in X. campestris strain X1106 with genes D through M being
deleted, while B and C are intact and functional in the
chromosome.
[0032] FIG. 6 depicts schematic representations of the structures
of repeating units of the polysaccharides produced by wild-type X.
campestris and mutants defective in genes F, G, or L and all
possible combinations of mutations in genes F, G, and L.
Abbreviations used are: G=glucose; M=mannose; GA=glucuronic acid;
Ac=acetate; Pyr=pyruvate.
[0033] FIG. 7a shows a map of a 4.3Kb Bg1II fragment of the gum
gene cluster that contains the gum F and gum G genes.
[0034] FIG. 7b shows the deletion mutation of the Cla I fragment
internal to the gum F gene.
[0035] FIG. 7c shows deletion of the second Cla I fragment which
creates an in-frame fusion of the proximal portion of the gum F
gene with the distal portion of the gum G gene.
[0036] FIG. 8 shows the two-step recombination procedure as
described in Example 5.
[0037] FIG. 9 depicts a viscosity comparison between wild-type and
non-pyruvylated gums.
[0038] FIG. 10 depicts a viscosity comparison between nonacetylated
and chemically-deacetylated gums.
[0039] FIG. 11 depicts the approximate physical location of 3 TnK12
insertion mutations within the cloned gum gene cluster DNA of
recombinant plasmid pRK290-H336. This figure also shows the
approximate locations of SpeI restriction endonuclease cleavage
sites in pRK290-H336.
[0040] FIG. 12 depicts the presumed pathway of xanthan gum
biosynthesis. Abbreviations used are: Glu=Glucose; GluA=Glucuronic
acid; Man-Mannose; Glu-Glu=Cellobiose; P=Phosphate;
PP=Pyrophosphate; C55=Isoprenoid Lipid Carrier;
PEP=Phosphoenolpyruvate; AcCoA=Acetylcoenzyme A; I-IV
=Glycosyltransferases; UDP=Uridine 5'diphosphate; and GDP=Guanosine
5'-Diphosphate.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Reference will now be made in detail to the presently
preferred embodiments of the present invention which, together with
the following examples, serve to explain the principles of the
invention. All references referred to herein are hereby
specifically incorporated herein by reference.
[0042] The polysaccharide polymers of the present invention have
been described in detail above. These polysaccharide polymers can
be produced in vitro with a cell-free enzyme system or can be
produced in vivo by growing cells of an appropriate mutant strain.
Other means of preparing the polysaccharide polymers are also
described below.
[0043] In Vitro Polysaccharide Synthesis
[0044] The basic method relating to the use of a cell-free system
to make non-variant xanthan gum is described by Ielpi, L., Couso,
R. O., and Dankert, M. A. in FEBS Letters 130:253-256 (1981),
specifically incorporated herein by reference. It has been found
that a modified version of this method may be employed to create
the variant polysaccharides of this invention.
[0045] For this novel, modified method, the in vitro cell-free
system is prepared generally by lysing cells of a microorganism of
the genus Xanthomonas, preferably Xanthomonas campestris, in the
presence of a suitable buffer, preferably including EDTA, and
obtaining the appropriate biosynthetic enzymes which are able to
subsequently process exogenously added substrates. This general
method for this in vitro system in described in U.S. Pat. No.
4,713,449 of Vanderslice et al., specifically incorporated herein
by reference. Alternate means of lysis may be used, including but
not limited to sonication, French Pressure cell, detergent
treatment, enzyme treatment and combinations thereof.
[0046] Generally, to produce the variant polysaccharides of the
present invention, a lysate of a microorganism possessing the
enzymes required to assemble the desired polysaccharide is
incubated with the appropriate substrates, which, depending on the
gum desired, may include UDP-glucose, GDP-mannose, UDP-glucuronic
acid, acetyl-CoA and phosphoenolpyruvate. The choice of substrates
is dependent on the polysaccharide which it is desired to produce.
For example, a non-acetylated polysaccharide is obtained by
eliminating acetyl-CoA as a substrate. Similarly, a non-pyruvylated
gum is obtained by eliminating phosphoenol-pyruvate as a substrate.
Chemical and/or enzymatic treatment of the cell lysates in order to
deplete endogeneous substrates will be evident to one skilled in
the art.
[0047] In addition, cell-free systems may be created from mutant
organisms deficient in one or more of the enzymes of the xanthan
biosynthetic pathway (for example, the pathway set forth in FIG.
12). Such mutant-derived cell lysates would produce the variant
gums described herein, either due solely to the mutation or due to
the mutation in combination with a withheld substrate. For example,
a cell-free system created from a mutant culture lacking
Transferase V would produce polytetramer while the same cell-free
system, when no acetyl-CoA was present, would produce
non-acetylated polytetramer.
[0048] The biosynthetic process may, in one embodiment, be
monitored by the incorporation of radiolabeled substrates into the
polymeric units. Other methods may also be used to allow
identification of the biosynthetic intermediates that are known to
those of ordinary skill in the art. In particular, chromatographic
methods have been developed to separate and to identify the
oligosaccharide intermediates. These include thin layer
chromatography and high-performance liquid chromatography.
[0049] The cell-free biosynthesis of xanthan has been found to be a
time-dependent, sequential process that is dependent on the
addition of all three specific nucleotides. The background of
non-specific incorporation of labeled substrate is minimal and does
not interfere with the detection of the xanthan-specific polymer in
the gum fraction.
[0050] The involvement of lipid carriers, specifically isoprenoid
pyrophosphate, has been shown in several polysaccharide
biosynthetic pathways. Additionally, the involvement of
pyrophosphoryl-linked lipid carrier in xanthan biosynthesis has
been demonstrated. Thus, the xanthan biosynthetic intermediates
have been found to be recoverable in the organic soluble fraction
with these carrier lipids. The recovered oligosaccharide can
subsequently be freed from the carrier lipid by mild acid
hydrolysis, for example, pH 2 for 20 minutes at 90.degree. C. and
dephosphorylated with alkaline phosphatase for analysis.
[0051] Using these methods for recovery of intermediate products,
it has been discovered that, under in vitro conditions, certain
lysates of X. campestris mutants will produce non-acetylated or
non-pyruvylated xanthan gum even in the presence of all substrates
required for non-variant gum synthesis. In light of the teachings
herein, these methods will enable one skilled in the art to
identify cell lysates which produce other altered
polysaccharides.
[0052] In Vivo Polysaccharide Synthesis
[0053] The development of the cell-free synthesis process for the
polysaccharides described above demonstrates that various
Xanthomonas campestris cells have all the enzymes necessary to
synthesize xanthan-based polymers that have the mannose residues
acetylated, pyruvylated or unmodified. However, to use whole cells
to synthesize polytetramer in vivo, a means for blocking xanthan
gum synthesis at Reaction V (see FIG. 12) would be required.
Moreover, in order for the whole cells to synthesize non-acetylated
polytetramer, means for blocking the acetylation reaction (see FIG.
12) as well as reaction V would be required.
[0054] Furthermore, for whole cells to synthesize non-acetylated
xanthan gum, a means of blocking the acetylation of either the
inner or outer mannose during xanthan gum synthesis would be
required. Additionally, for the whole cells to synthesize
non-acetylated, non-pyruvylated xanthan gum, a means of blocking
xanthan gum synthesis at both the acetylation and pyruvylation
steps would be required. In one embodiment of the present
invention, mutagenesis was employed to alter some of the genes
responsible for these various reactions.
[0055] Transposons, including but not limited to Tn10, TnK12 (Tn10
del16del17KanR), and Tn903, can be used to mutagenize Xanthomonas
campestris. These transposons, in one embodiment, confer resistance
to tetracycline or kanamycin. Transposons have the ability to
insert themselves into genes wherein they cause mutations by
interrupting the coding sequence. The transposons can be introduced
into Xanthamonas campestris on various vectors, including on
so-called suicide vectors, such as pRK2013. Vector pRK2013, as
described by Ditta, G., Corbin, D. and Helinski, D. R. in Proc.
Natl. Acad. Sci. U.S.A., 77:7347-7351 (1980), specifically
incorporated herein by reference, has the ability to transfer
itself into non-enteric bacteria, such as Xanthomonas campestris,
but cannot replicate in that host. Thus, if the suicide vector is
introduced into a population of Xanthomonas campestris cells and
that population is subsequently challenged with either tetracycline
or kanamycin, the individuals which survive are those in which one
of the transposons has inserted into the genome of Xanthonomas
campestris. Survivors of such a challenge can be screened for those
which have lost the ability to make xanthan gum. Such mutants may
appear less mucoid than mold-type Xanthonomas campestris.
[0056] In other embodiments of the invention, other means of
mutagenesis can be employed to generate mutants that do not
acetylate and/or pyruvylate the gums they produce. Such means will
readily occur to one skilled in the art, and include, without
limitation, irradiation, recombinant DNA technology (in particular,
as described in U.S. patent application Ser. No. 07/333,868 of
Capage et al., entitled "Recombinant-DNA Mediated Production of
Xanthan Gum," filed Apr. 3, 1989, and incorporated specifically
herein by reference, and in Example 1 below) and chemical mutagen
treatment. Examples of such mutagenesis procedures have been
described by Miller, J. H. in Experiments in Molecular Genetics
(1972); Davis, R. W., Bostein, D. and Roth, J. R. in Advanced
Bacterial Genetics (1980); and Maniatis, T., Fritsch, E. F. and
Sambrook, J. in Molecular Cloning (1982), Cold Spring Harbor.
[0057] Although mutants can first be chosen which appear less
mucoid than wild-type organisms, those desired generally retain the
ability to make some polysaccharide. Cell-free extracts of each of
the xanthan mutants can be prepared and tested as noted above by
the addition of different combinations of substrates and analysis
of the resultant products.
[0058] Alternatively, appropriate mutants can be detected by
assaying the culture broth of each mutant for the presence of the
desired polysaccharide, e.g., xanthan gum which has the outer
mannose acetylated but not pyruvylated, pyruvylated but not
acetylated, both pyruvylated and acetylated or unmodified while the
inner mannose is acetylated or unmodified. Thus, mutants can be
found which appear to be blocked at various positions of the
xanthan gum pathway. Mutants of Xanthomonas campestris which
produce xanthan gum that is acetylated at the inner mannose and
acetylated at the outer mannose (X1397), acetylated at the inner
mannose and pyruvylated at the outer mannose (X1398), acetylated at
the inner mannose and unmodified at the outer mannose (X1399),
unmodified at the inner mannose with a portion of the outer mannose
moieties pyruvylated and a portion acetylated (X1400), unmodified
at the inner mannose and acetylated at the outer mannose (X1401),
and unmodified at the inner mannose and pyruvylated at the outer
mannose (1402), and unmodified at the inner mannose and unmodified
at the outer mannose (X1403), have each been placed on deposit at
the American Type Culture Collection, Rockville, Md., under
Accession Nos. 68033, 68034, 68035, 68036, 68037, 68038 and 68039,
respectively.
[0059] A mutant of Xanthomonas campestris which produces
non-acetylated xanthan gum (X1006), a mutant which produces
non-pyruvylated xanthan gum (X921), a mutant which produces xanthan
gum containing low levels of pyruvylation (less than 5% of the
terminal mannose X934), and a mutant which produces non-acetylated,
non-pyruvylated xanthan gum [X1231(p41KS22)] have each been placed
on deposit at the American Type Culture Collection, Rockville, Md.,
under Accession Nos. 53472, 53473, 53474 and 67344
respectively.
[0060] Mutants of Xanthomonas campestris which produce variants of
xanthan gum and which contain chromosomal mutations have also been
produced, by way of example only, X1910 is described in Example 5
which produces polymer unmodified at the inner mannose and
pyruvylated at the outer mannose.
[0061] It is not beyond the scope of the invention to employ enzyme
inhibitors of Acetylase I, Acetylase II, Transferase V, and
Ketalase to arrive at the same products. Still other alternatives
for producing this family of polysaccharides are contemplated,
including enzymatic and chemical degradation of natural xanthan
gum.
[0062] The mutants can be grown under conditions generally known in
the art for growth of wild-type Xanthomonas campestris. For
example, they can be grown on suitable assimilable carbon sources
such as glucose, sucrose, maltose, starch, complex carbohydrates
such as molasses or corn syrup, various organic acids and the like.
Mixtures of carbon sources can also be employed. The concentration
of carbon source supplied is often between 10 and 60 grams per
liter. Also necessary for growth are an assimilable source of
organic or inorganic nitrogen, generally between about 0.1 and 10.0
grams per liter, and minerals, the choice of which are easily
within the skill of the art. Examples of suitable nitrogen sources
are ammonium salts, nitrate, urea, yeast extract, peptone, or other
hydrolyzed proteinaceous materials or mixtures thereof. Examples of
suitable minerals include phosphorus, sulfur, potassium, sodium,
iron, magnesium; these are often added with a chelating agent such
as EDTA or citric acid.
[0063] Optimal temperatures for growth of Xanthomonas campestris
generally are between 18.degree. and 35.degree. C., preferably
between about 27.degree. and 30.degree. C. Xanthomonas campestris
cells are grown aerobically by supplying air or oxygen so that an
adequate level of dissolved oxygen is maintained, for example,
above about 10% of saturation. Preferably, the level is kept above
about 20%. The pH often is maintained at about 6.0 to 8.0,
preferably at about 6.5 to 7.5.
[0064] The polysaccharides of the present invention can be
recovered from fermentation broths by a suitable means.
Precipitation with isopropanol, ethanol, or other suitable alcohol
readily yields the polysaccharides of this invention. Generally,
alcohols are added to a concentration of about 50 to 75%, on the
basis of volume, preferably in the presence of potassium chloride,
sodium chloride or other salt. Alternatively, the polymers can be
recovered from the broth by ultra-filtration.
[0065] Mobility control solutions for use in enhanced oil recovery
may also be prepared from the variant polysaccharide polymers
disclosed herein. Solutions of the polysaccharide polymers at
concentrations of from about 50 to about 3000 ppm are appropriate
for such mobility control solutions. Other known additives may also
be used in combination with these solutions to further enhance oil
recovery. Such additives include, for example, surfactants,
alkaline agents or metal or organic crosslinking agents.
[0066] The polysaccharide polymers, like xanthan gum, can also be
used as thickening agents in foods, cosmetics, medicinal
formulations, paper sizing, drilling muds, printing inks, and the
like and as a gelling agent. In addition, they can be used to
reduce frictional drag of fluid flow in pipes.
EXAMPLES
[0067] The following examples illustrate certain of the preferred
embodiments of the present invention. All United States patent
applications and other references cited in these Examples are
specifically incorporated herein by reference.
EXAMPLE 1
[0068] This example demonstrates that there are two X. campestris
genes which encode enzymes that catalyze acetylation of xanthan
gum.
[0069] Capage et al., U.S. patent application Ser. No. 07/333,868,
described the nucleotide sequence of a 16 kb segment of X.
campestris DNA that contains a gene cluster required for xanthan
gum biosynthesis. Mutations were isolated that inactivated each of
the genes identified by the DNA sequence. The phenotypes of mutant
strains carrying these mutations were determined. Mutations in gene
gumF (see FIG. 1a), caused by transposon insertion, resulted in
production of xanthan gum that contained no detectable acetate.
Insertion mutations in gene gumG did not result in any obvious
defect in xanthan gum biosynthesis. Mutants with gene gumG defects
produced high levels of xanthan gum, and this gum contained all of
the normal constituents of xanthan in approximately normal molar
ratios. On the basis of these initial results, it was concluded
that gene gumF encoded an enzyme that catalyzed the known
acetylation of the inner mannose of xanthan, while the activity of
the gene gumG protein remained unknown.
[0070] However, when the DNA sequence was used to predict the
amino-acid sequences of the products of genes F and G (gpF and
gpG), these proteins were found to have extensive homology to one
another. This finding indicated that the functions of gpF and gpG
might be similar. The phenotypes of mutants defective in gene gumG
were subsequently reexamined, and the compositions of xanthans
produced by these mutants were precisely quantitated. These data
showed a small (5%-10%) but significant decrease in the acetate
content of gum produced by G mutants as compared to wild-type X.
campestris. Therefore further experiments were performed to
determine what role gpG might have in acetylation of xanthan.
[0071] The hypothesis that gpG normally directs 10% of the
acetylation of xanthan gum was seemingly contradicted by the
observation that transposon insertion mutations in gene gumF
resulted in elimination of acetylation. Clearly, these mutant gums
did not retain 10% of the normal acetate content. However, it was
possible that insertions in gene gumF reduced or eliminated the
expression of gene gumG as a result of so-called "polar" effects.
Insertions of Tn10 generally reduce expression of genes located
downstream, in terms of transcription, from the insertion site as
reported by Kleckner, N. et al., in J. Mol. Bio. 97:561-575 (1975).
Moreover, the reduction can be quite severe in instances where the
downstream gene is "translationally coupled" to the gene containing
the insertion mutation as reported by Oppenheim, D. S. and
Yanofsky, C., in Genet. 95:785-795 (1980). Translational coupling
is a phenomenon wherein the translational stop signal of one gene
overlaps the translational start signal of an adjacent downstream
gene. In some cases where such coupling occurs, the initiation of
translation of the downstream gene is largely or entirely dependent
on termination of translation of the upstream gene occurring at the
coupler. Thus, insertions in the upstream member of the coupled
genes can dramatically reduce, or even eliminate, expression of the
downstream gene because these inserts invariably cause frame
shifting and premature termination of translation of the upstream
gene.
[0072] The DNA sequence of the gum gene cluster revealed that the
translational stop of gene gumF does overlap the translational
start of gene gumG, i.e., the two are "coupled" (see FIG. 1b).
Moreover, the sequence of the translational initiation signal for
gene gumG is not particularly strong, which suggests that the
translational coupling might play a significant role in gene gumG
expression. To test this hypothesis, a deletion mutation (as shown
in FIG. 2) was constructed within the coding sequence of gene gumF.
This deletion eliminated 660 base pairs between the ClaI sites
within gene gumF. The deleted DNA falls entirely within the coding
sequence of gene gumF, and no foreign DNA is inserted. Thus, the
deletion removes a large portion (approximately 60%) of the gene
but does not alter the reading frame since the number of base pairs
deleted is evenly divisible by 3. The mutant gpF produced by this
deletion mutation (gpFdel) is missing 220 amino acids out of a
total of 364, but the translational start of gene gumF and the gene
gumF translational stop, coupled to the start of gene gumG, remain
unaltered. The elimination of two-thirds of the amino acid residues
of gpF is very likely, although not certain, to result in
elimination of all protein activity. Thus, any residual acetylase
activity from this mutant is most apt to be due to activity of
gpG.
[0073] This ClaI deletion mutation was constructed on plasmid
pRK290-H336.13 (FIG. 3a) which carries an otherwise wild-type gum
gene cluster and an insertion of transposon Tn10 del16 del17 KanR
described by Way et al., in Gene 32:369-379 (1984) and here termed
TnK12, located within the vector position of the plasmid. The TnK12
insertion provides convenient drug resistances for selection of
plasmid transfer. The deleted plasmid, termed p13delCla, was
transferred into the X. campestris Gum deletion strain X1231, which
is missing genes B-M, and polysaccharide produced by the resulting
strain X1231(p13delCla) was analyzed. This gum contained a low but
significant amount of acetate; roughly (10-15)% the amount normally
found in wild-type xanthan. This result indicated that both gpF and
gpG are acetylases and that the bulk of acetylation of xanthan is
catalyzed by gpF with a minor component of xanthan acetylation
being catalyzed by gpG. However, it remained a possibility that the
low level acetylation observed in the mutant X1231 (p13delCla)
resulted not from the activity of gpG, but from a residual activity
of gpFdel. To address this issue, a double mutant derivative of
plasmid pRK290-H336 was constructed. As shown in FIG. 3b, this
double mutant combined the gene gumF ClaI deletion mutation and an
insertion mutation (KRm1) in gene gumG. The double mutant plasmid
pH336 BmldelCla was transferred into strain X1231, and the
polysaccharide produced was analyzed. If the low level acetylation
observed in gum produced by X1231(p13delCla) results from the
activity of gpG, then the double mutant X1231(pH336KBmldelCla)
should eliminate gpG activity by virtue of the insertional mutation
in gene gumG, and no acetylation should be observed. If, however,
the real source of acetylating activity in X1231(p13delCla) is the
mutant gpFdel, the addition of the gene gumG insertion should not
affect acetylation, and the same 10% level observed in
X1231(p13delCla) should be seen in gum produced by the double
mutant strain. The polysaccharide produced by strain
x1231(pH336KBmldelCla) was found to contain no acetate. This proved
that gpG does catalyze acetylation of xanthan and that, in
wild-type strains, gpG is responsible for roughly 10% of the total
acetylation that is observed.
EXAMPLE 2
[0074] This example demonstrates that the target residue for
acetylation by gpG (but not gpF) is the outer mannose of the
xanthan repeating unit and that this acetylation is enhanced when
pyruvylation of the outer mannose is blocked.
[0075] Mutations in gene gumL (FIG. 1a) of the xanthan biosynthetic
gene cluster were previously shown to inactivate the ketalase
enzyme which catalyzes pyruvylation of the outer mannose. Mutants
lacking gpL activity produce xanthan gum devoid of pyruvate.
However, initial-studies of such mutants revealed that these
non-pyruvylated polymers contained unusually high levels of
acetate, generally>0.8 acetate/mannose. Thus, the outer mannose
can be efficiently acetylated when pyruvylation is genetically
blocked and further studies have shown that this acetylation is
catalyzed by gpG and not gpF.
[0076] In order to examine the interaction of the two acetylase
genes with the ketalase gene and each other, a set of eight mutant
strains comprising all combinations of mutations in gene gumF
(Acetylase I), gene gumG (Acetylase II), and gene gumL (Ketalase)
were constructed. The various combinations of mutations were
constructed on plasmid pRK290-H336 which contains the entire gum
gene cluster.
[0077] The gene gumF mutation employed in these constructions is
the in-frame deletion within this gene. As described above, this
deletion eliminates 660 base pairs between the ClaI sites located
within gene gumF. The deleted DNA falls entirely within the coding
sequence of gene gumF, and no foreign DNA is inserted. Thus, the
deletion removes a large portion (approximately 66%) of the gene
but does not alter the reading frame since the number of base pairs
deleted is evenly divisible by 3. The mutant gpF produced by this
deletion mutation (gpFdel) is missing 220 amino acids out of a
total of 364, but the translational start of F and its
translational stop coupled to the start of G remain unaltered. The
elimination of two-thirds of the amino acid residues of gpF was
shown above to eliminate gpF activity.
[0078] The gene gumG mutation used in these mutants is an insertion
(KBm1) within gene gumg at a BamHI site that interrupts the coding
sequence of gene gumG. The inserted DNA is a restriction fragment
containing the 1.3 kb Kan.sup.r DNA segment of plasmid pUC4-K as
described by Vieira, J. and Messing, J., in Gene 19:259-268 (1982),
which is ultimately derived from the kanamycin resistance gene of
transposon Tn903.
[0079] The gene gumL mutations used were of two types. One is an
insertion of transposon TnK12 within the coding region of gene
gumL. The second type is derived from this insertion by deletion of
a 3 kb HindIII fragment of TnK12 which carries the genes encoding
resistance to kanamycin and streptomycin. In this TnK12 deletion
mutation, an insert of 1 kb of TnK12 DNA still remains within the
gene gumL coding sequence and this results in insertional
inactivation of the gene gumL product.
[0080] The various combinations of these mutations were constructed
on plasmid pRK290-H336 using in vitro recombinant DNA technology.
The eight mutant plasmids obtained were then conjugally transferred
from E. coli into X. campestris strain X1231 which contains the
deletion mutation that eliminates the entire gum gene cluster from
the chromosome. The 8 resulting strains X1396-X1403 (Table 1) were
then analyzed for polymer production.
1 TABLE 1 Genotype Strain Acetylase I Acetylase II Ketalase
X1396.sup.a + + + X1397 + + -.sup.b X1398 + -.sup.c + X1399 +
-.sup.c -.sup.d X1400 -.sup.e + + X1401 -.sup.e + -.sup.b X1402
-.sup.e -.sup.c + X1403 -.sup.e -.sup.c -.sup.d .sup.awild-type,
carries TnK12 insertion within pRK290 portion of the plasmid
.sup.bTnK12 insertion mutation .sup.cKan.sup.r fragment insertion
mutation .sup.dTnK12 deletion derivative insertion mutation
.sup.ein-frame, non-polar deletion mutation
[0081] All strains were grown in 50 ml each FXC-RAH-1 medium at pH
7.0 that contained:
[0082] 3.2 g/l N-Z-amine AS
[0083] 1.7 g/l MgSo.sub.4.7H.sub.2O
[0084] 0.7 g/l KH.sub.2PO.sub.4
[0085] 40 g/l glucose
[0086] 19.5 g/l (2-(N-morpholino) ethane sulfonic acid)
[0087] 5-10 mg/l kanamycin
[0088] 1 mg/l Tetracycline (where applicable) in 300 ml baffled
shake flasks. Temperature was maintained at 30'C. After
approximately 60 hours of incubation, the culture broths were
diluted with two to four volumes of distilled H.sub.2 0 and the
cells removed by centrifugation at 14,000-18,000.times.g for 30
minutes at 10' C. Gums were precipitated from the supernatants by
the addition of 2-3 volumes of 2-propanol and collected by
centrifugation using the conditions described previously. The
precipitates were then rehydrated in 100-300 ml of 20 mM NaCl and
the precipitations repeated. The gums were finally rehydrated in
100 ml distilled H.sub.2O each. Samples of each were subsequently
dialyzed against 4 l of distilled H.sub.2O for four days with daily
H.sub.2O changes in 12,000-14,000 MW cutoff cellulose tubing.
[0089] Triplicate samples of each purified gum were then
concentrated 3-4-fold by vacuum drying and hydrolyzed in 2 M
trifluoroacetic acid at 120' C. for 2-1/2hours. After
neutralization with 1.2 M Na.sub.2CO.sub.3, the hydrolysates were
filtered through 0.45 mim filters and ready for analysis by
high-performance liquid chromatography (HPLC).
[0090] The analyses were performed using a Beckman HPLC equipped
with an Aminex HPX-87H ion exclusion column (300.times.7.8 mm).
Organic acids were detected by ultraviolet absorbance at 214 nm.
Refractive index was used to detect neutral sugars. The column was
run isocratically with 0.01 N H.sub.2So.sub.4 as the mobile phase
at a flow rate of 0.6 ml/minute at 45' C.
[0091] The molar ratios of the components in each hydrolysate were
calculated using a series of calibration curves based on peak areas
for each sugar and organic acid.
[0092] The molar ratios of acetate and pyruvate to mannose are
shown in Table 2.
2TABLE 2 Molar Ratios of Acetate and Pyruvate to Mannose Acetate/
Pyruvate Strain Acetylase I Acetylase II Ketalase Mannose Mannose
X1396 + + + 0.66 0.43 X1397 + + - 1.01 0.00 X1398 + - + 0.63 0.36
X1399 + - - 0.51 0.00 X1400 - + + 0.10 0.39 X1401 - + - 0.47 0.00
X1402 - - + 0.00 0.37 X1403 - - - 0.00 0.00
[0093] The following key observations about the data presented in
Table 2 can be made.
[0094] 1. The 660 bp deletion in gene gumF inactivated the gene F
protein (Acetylase I). See X1402 vs X1398.
[0095] 2. The gene gumg protein (Acetylase II) acetylated xanthan
and at a much reduced level compared to wild-type when Ketalase was
active. See X1400 vs X1396, described in Ex. 4.
[0096] 3. If Ketalase was inactivated, acetylation by Acetylase II
increased dramatically (X1400 vs.
[0097] X1401), described in Ex. 4.
[0098] 4. The extent of acetylation by Acetylase I did not increase
in response to the inactivation of Ketalase. See X1398 vs X1399,
described in Ex. 4.
[0099] 5. Pyruvylation did not vary significantly regardless of the
extent of acetylation. See X1396, X1398, X1400, and X1402,
described in Ex. 4.
[0100] These data indicate that the gene gumG protein (Acetylase
II) catalyzes the acetylation of the external mannose of xanthan.
This appears to occur to a limited extent when Ketalase is active,
but increases dramatically in Ketalase mutants. These data indicate
that pyruvylation blocks acetylation, but the converse is not true
since pyruvylation didn't change significantly regardless of the
level of acetylation. The gene gumF protein (Acetylase I) catalyzes
the acetylation of the internal mannose only, and previous data for
polytrimer and polytetramer variants of xanthan U.S. Pat. No.
4,713,449 and U.S. application Ser. No. 844,335 have shown that
Ketalase catalyzes the pyruvylation of the external mannose
only.
EXAMPLE 3
[0101] This example demonstrates that gpG (Acetylase II) does not
catalyze acetylation of the inner mannose of the xanthan repeating
unit.
[0102] Gene I of the gum gene cluster encodes Transferase V (FIG.
1), the enzyme that adds mannose to the lipid-linked
tetrasaccharide intermediate in xanthan biosynthesis. This system
(is described in U.S. Pat. No. 4,713,449. Mutations that inactivate
gene gumI lead to the synthesis of a lipid-linked tetrasaccharide.
This tetrasaccharide repeating unit is polymerized to yield
polytetramer gum, which contains the internal mannose in its normal
linkages but lacks the outer mannose normally found on xanthan gum
(FIG. 4). A double mutant plasmid, pKBm2delCla, was constructed
which contains an insertion mutation within gene gumI and the ClaI
deletion mutation within gene gumF (see FIG. 5). The double mutant
plasmid pKBm2delCla was transferred into the X. campestris deletion
strain X1106 which contains only gum genes B and C in its
chromosome. Genes B and C are provided by the chromosome since the
mutant plasmid, derived from pRK290-HA3, does not carry B or C but
contains all the remaining gum genes, D through M. The resulting
strain, X1106(pKBm2delCla) or X1419, was analyzed for polymer
composition twice. Both analyses failed to detect acetate in the
polymer. This result shows that Acetylase II cannot acetylate the
internal mannose of the polytetramer to any significant degree. In
this mutant strain, Acetylase II is active because gene gumg is not
mutated and the gene gumF mutation is the non-polar ClaI deletion
which has been shown above not to affect the expression of gene
gumG.
EXAMPLE 4
[0103] This example describes the repeating units that comprise the
polysaccharide family that can be produced by genetic control of
acetylation and pyruvylation of the pentasaccharide repeating unit
of xanthan gum. The structures of these repeating units are shown
in schematic form in FIG. 6.
[0104] (a) Wild-type (X1396); Acetylase I.sup.+, Acetylase
II.sup.+, Ketalase.sup.+.
[0105] Normal xanthan is extensively acetylated at the inner
mannose residue and is frequently pyruvylated on the outer mannose
residue. Contrary to general belief, a significant percentage
(10-20) of the outer mannose residues of normal xanthan are
acetylated. Thus, normal xanthan repeating units are heterogeneous
with respect to modifications of the outer mannose, containing
either pyruvate or acetate.
[0106] (b) L.sup.-(X1397); Acetylase I.sup.+, Acetylase II .sup.+,
Ketalase.sup.-.
[0107] This polymer contains no pyruvate and as a result is
extensively acetylated at the outer mannose residue. The inner
mannose residue is highly acetylated as in wild type.
[0108] (c) G.sup.-(X1398); Acetylase I.sup.+, Acetylase II.sup.-,
Ketalase.sup.+.
[0109] This polymer is heavily acetylated on the-inner mannose as
in wild type, and the outer mannose is pyruvylated in the wild-type
fashion. However, there is no acetylation of the outer mannose.
[0110] (d) G.sup.-, L.sup.-(X1399); Acetylase I.sup.+, Acetylase
II.sup.-, Ketalase.sup.-.
[0111] This polymer has the high level wild-type acetylation of the
inner mannose, but the outer mannose is unmodified.
[0112] (e) F.sup.-(X1400); Acetylase I.sup.-, Acetylase II.sup.+,
Ketalase.sup.+.
[0113] The inner mannose of this polymer is unmodified, while the
outer mannose is modified as in wild-type. That is, the outer
mannose in generally pyruvylated, but a significant fraction of the
outer mannose residues are acetylated instead.
[0114] (f) F.sup.-, L.sup.-(X1401); Acetylase I.sup.-, Acetylase
II.sup.+, Ketalase.sup.-.
[0115] This polymer contains an unmodified inner mannose. The outer
mannose is not pyruvylated but is heavily acetylated.
[0116] (g) F.sup.-, G.sup.-(X1402); Acetylase I.sup.-, Acetylase
II.sup.-, Ketalase.sup.+.
[0117] This polymer is not acetylated at either the inner or outer
mannose residues. Pyruvylation of the outer mannose occurs normally
as in wild-type.
[0118] (h) F.sup.-, G.sup.-, L.sup.-(X1403); Acetylase I.sup.-,
Acetylase II.sup.-, Ketalase.sup.-.
[0119] This polymer contains no acetate or pyruvate. Neither the
inner nor the outer mannose residues are modified.
EXAMPLE 5
[0120] This example describes the construction of a chromosomal
deletion mutation defective in both acetylase genes gumF and
gumG.
[0121] The variant xanthans described in Example 4 are produced by
mutant strains of X. campestris in which the gum gene cluster has
been deleted from the chromosome and is present in the cell on a
recombinant plasmid. In some instances it might be desirable to
have the gum gene cluster located in the X. campestris chromosome
as this would eliminate the need for plasmid maintenance, and thus
should improve strain stability. A chromosomal deletion mutation
defective in genes gumF and gumG, which encode Acetylase I and II,
respectively, was constructed as described below. This mutant
produces non-acetylated xanthan gum.
[0122] FIG. 7a shows a map of a 4.3 Kb Bg1II fragment of the gum
gene cluster that contains the gumF and gumG genes. This Bg1II
fragment is cloned into the Bg1II site of the plasmid vector pS1 to
generate a recombinant plasmid termed pS1Bg1. The pS1 vector is a
derivative of pRK290 in which the origin of vegetative, but not
conjugal, DNA replication is replaced by the replication origin of
pBR322. The pS1 replication origin does not function in X.
campestris. Therefore, pS1 and its derivatives, such as pS1Bg1 are
"suicide" plasmids, i.e., they can be conjugally transferred into
X. campestris but cannot replicate in X. campestris.
[0123] The three ClaI sites shown in FIG. 7a were used in
construction of the deletion mutation. Deletion of the ClaI
fragment internal to the gumF gene was accomplished as described in
Example 1 (by digesting pS1Bg1 with ClaI and religating at low DNA
concentration) to produce pS1Bg1.DELTA.Cla shown in FIG. 2b. In
those experiments digestion at the third ClaI site within gene gumG
was not observed, although the DNA sequence at this site is the
ClaI recognition site, ATCGAT.
[0124] However, digestion by ClaI is known to be sensitive to
methylation of the first adenine (A) residue of the recognition
sequence. If that residue is methylated, the enzyme will not cleave
the sequence. In E. coli, the sequence GATC is a substrate for the
dam methylase which methylates the A residue of this
tetranucleotide sequence. The ClaI recognition site in gene Gumg is
preceded by a G residue. Thus, in that sequence, GATCGAT, the
underlined A is methylated because it occurs within the GATC
tetranucleotide sequence; and therefore, the ClaI site is
refractory to ClaI digestion.
[0125] In order to digest the ClaI site within gene gumG, it was
necessary to produce unmethylated DNA by propagating
pS1Bg1.DELTA.Cla in an E. coli dam strain which is defective for
the relevant methylase activity. When unmethylated pS1Bg1.DELTA.Cla
DNA was prepared, digestion at the gene gumG ClaI was readily
observed. Using this plasmid DNA as a substrate, the second ClaI
fragment was deleted by digesting pS1Bg1.DELTA.Cla with ClaI and
religating at low DNA concentration to produce pS1Bg1.DELTA.Cla2
which is shown in FIG. 7c. The resultant deletion of both ClaI
fragments creates an in-frame fusion of the proximal portion of the
gumF gene with the distal portion of the gumG gene. Therefore,
transcription and translation through this region, and of distally
located genes H through M (FIG. 1), should proceed normally.
[0126] The plasmid pS1Bg1.DELTA.Cla2 including the deletion
mutation was transferred into the X. campestris chromosome via gene
replacement. This two-step recombination procedure is diagrammed in
a general fashion in FIG. 8. In general, in the first step,
homologous recombination between the plasmid and the chromosome
results in integration of the whole plasmid into the chromosome.
This event is selected by selection for maintenance of an
antibiotic resistance marker carried within the vector portion of
the plasmid. In the second step, a subsequent recombination results
in loss of the plasmid and retention of the constructed deletion
mutation. This event is detected by the screening for loss of the
antibiotic-resistance determinant and Southern blot hybridization
analysis of chromosomal DNA structure.
[0127] In the present example, pS1Bg1.DELTA.Cla2 was conjugally
transferred into wild type X. campestris strain X77 by standard
triparental matings and tetracycline-resistant transconjugants were
selected. These tetracycline-resistant colonies were either light
or dark yellow in appearance. DNA was prepared from four of each
(light and dark type), digested with BamHI (pS1g1.DELTA.Cla2
contains no BamHI sites), separated by agarose gel electrophoresis
and Southern blot hybridization was performed. Probing with labeled
gum gene cluster DNA showed a wild type pattern in DNA from the
light yellow isolates. The hybridization pattern for DNAs from the
dark yellow isolates indicated that, in these isolates,
pS1Bg1.DELTA.Cla2 had recombined into the chromosome.
[0128] Two of the bonafide pS1Bg1.DELTA.Cla2 insertion isolates
were then grown in the absence of tetracycline to screen for the
subsequent recombination in the second step of the gene replacement
process. In these experiments, liquid cultures are grown to
saturation in the absence of tetracycline, diluted 1:50 in fresh
growth medium, and again grown to saturation. In several
experiments of this type, the number of subculturing steps varied
from 2 to 6.
[0129] Following the liquid subculturing steps, the cells were
diluted and plated, in the absence of tetracycline, to yield
isolated colonies. These colonies were tested for
tetracyclineresistance. In different experiments, the frequency of
tetracycline-sensitive colonies varied widely, from .about.0.5%
to>50%. The frequency of tetracycline-sensitive isolates
increased with the number of generations of growth (i.e. subculture
steps) in the absence of tetracycline.
[0130] Individual tetracycline-sensitive isolates were grown and
their chromosomal DNAs analyzed by Southern blot hybridization to
identify those isolates which contained the deletion mutation in
the chromosome and had lost the plasmid sequences as a result of
homologous recombination. One such isolate, termed X1910, was
characterized for polysaccharide polymer production in vivo by the
procedures detailed in Example 2.
[0131] The composition of the polysaccharide polymer produced by
X1910 was found to be indistinguishable from wild type xanthan with
respect to glucose, mannose, glucuronic acid and pyruvate; whereas,
in contrast, no acetate was detected in the gum made by X1910. Thus
this mutant produces non-acetylated xanthan having a composition
equivalent to that produced by X1402 (Example 4), but does so as a
result of the chromosomal deletion mutation. No recombinant plasmid
or any foreign DNA is present in this strain.
[0132] The general approach and strategy described above could
readily be applied to create analogous chromosomal deletion mutants
that would produce any of the variant xanthans described in this
application or in Vanderslice and Shannon U.S. Pat. No.
4,713,449.
EXAMPLE 6
[0133] This example shows the methods of mutagenesis and screening
employed to generate X. campestris mutant strains having defects in
Acetylase or Ketalase activity.
[0134] The genes encoding the enzymes of xanthan gum biosynthesis
have been shown to comprise a set of clustered genes on the X.
campestris chromosome. This "gum gene cluster" has been described
in detail by Capage et al. Segments of gum gene DNA have been
cloned on plasmid vectors such as pMW79 as detailed in Capage et
al.
[0135] Regionally-directed mutagenesis was performed upon subcloned
portions of the gum DNA carried in plasmid pMW79. These cloned DNA
segments were mutagenized in vivo with transposons and in vitro, by
using recombinant DNA technology to generate insertion, deletion,
and substitution mutations within the cloned X. campestris DNA. In
order to study the phenotypes conferred by these mutations, the
plasmids carrying the mutations were transferred back into X.
campestris and subsequently recombinants were identified in which
the plasmid-borne, mutated gene had been inserted in the chromosome
via homologous recombination. The tetracycline resistance encoded
by Tn10 affords a convenient selective system for movement of
mutations from a plasmid into the chromosome.
[0136] One such mutant strain (X1006) carried a Tn10 insertion that
was found to cause inactivation of the Acetylase activity. This
mutant strain was characterized as described in Examples 7 and 8,
and found to produce a polysaccharide that was non-acetylated. A
second mutant strain was constructed by the in vitro insertion of a
fragment of DNA containing the tetracycline resistance gene of Tn10
into a restriction site within the gum gene cluster. This mutant
strain (X921) was found to be defective in the Ketalase activity.
As found by the methods of Examples 7 and 8, this mutant produced
xanthan that was non-pyruvylated.
[0137] A third mutant strain (X934) was also found that greatly
reduces the ketalase activity. This mutant strain produces xanthan
gum that has a very low level of pyruvylation: 1-5% of the level of
pyruvylation found in normal xanthan.
[0138] The mutant strain X934 was found as described below. In
preliminary experiments designed to study recombination between
plasmid-borne X. campestris DNA and the X. campestris PstI fragment
cloned in plasmid RSF1010. This insertion of Tn10 causes the
Gum-defect in the mutant strain X655 as described by Capage et al.
and Vanderslice et al. The experiment was to mobilize PTX655 with
plasmid pRK2013 and transfer it from E. coli into X. campestris by
selecting for transfer of the tetracycline resistance encoded by
Tn10. The initial results of this mating were anomalous and
suggested that Tn10 did not express tetracycline resistance
efficiently in X. campestris when carried on the plasmid, but that
the drug resistance was more efficiently expressed when Tn10 was
carried in the chromosome of X. campestris. This phenomenon has
also been described for Tn10 in E. coli. There, it has been shown
that strains carrying one copy of Tn10 inserted in the chromosome
are resistant to significantly higher concentrations of
tetracycline than are strains carrying Tn10 on a multicopy plasmid.
The selection of Tet.sup.r X. campestris out of the above mating
resulted in a high frequency (0.5 per recipient) of progeny which
grew very poorly (i.e., only small, watery colonies) on
tetracycline. After prolonged incubation, a large fraction of the
colonies (25%) produced sectors of more vigorously growing cells.
More than 50% of these sectors appeared to be Gum-in morphology.
These probably result from recombination between the plasmid-borne
DNA containing the Tn10 insertion and the chromosomal wild type
DNA. when the Tn10 is recombined into the chromosomal, high-level
Tet.sup.r is obtained and the vigorously growing sector is
observed. When these Gum-, Tet.sub.r sectors were picked and
restreaked on tetracycline, they grew well and displayed a
characteristic Gum-morphology.
[0139] Gum+, Tet.sup.r isolates were also characterized. Some of
these strains (in particular X934) were found to contain the entire
plasmid pTX655 inserted into the chromosome of X. campestris via
homologous recombination. The chromosomal structure of the X934
strain was determined by Southern blot hybridization of the
chromosomal DNA which shows that the plasmid sequences exist in a
chromosomally integrated form.
EXAMPLE 7
[0140] This example shows how the altered polysaccharides of the
present invention can be prepared in vitro. For instance, it shows
how non-acetylated and/or non-pyruvylated xanthan gum was prepared
in vitro.
[0141] Preparation of Lysates
[0142] Xanthomonas campestris B1459 S4-L or S4-L mutants described
in Examples 6 and 10 were grown in YM (yeast-malt medium)
supplemented with 2% (w/v) glucose as described by Jeanes, A. et
!;al. in U.S. Department of Agriculture, ARS-NC-51, pp. 14 (1976),
specifically incorporated hereby by reference. Cultures were grown
to late log phase at 30.degree. C. The cells were harvested by
centrifugation and washed with cold Tris-HCl, 70mM, pH 8.2 with 10
mM EDTA and were freeze-thawed three times by a procedure similar
to Garcia, R. C. et al. described in European Journal of
Biochemistry 43:93-105 (1974), specifically incorporated hereby by
reference. This procedure ruptured the cells, as was evidenced by
the increased viscosity of the suspensions and the complete loss of
cell viability (one of 10.sup.6 survivors) after this treatment.
The freeze-thawed lysates were frozen in aliquots at -80.degree.C.
Protein concentration was determined with BIO RAD assay (BIO RAD
Laboratories, Richmond, Calif.) and was found to be 5 to 7 mg cell
protein per ml of lysate.
[0143] Biosynthetic Assay Procedure
[0144] As described by Ielpi, L. Couso, R. O., and Dankert, M. A.
in FEBS Letters 130:253-256 (1981), specifically incorporated
herein by reference, an aliquot of freeze-thawed lysate (equivalent
to 300 to 400 ug protein), DNAase I (10 ug/ml), and MgCl.sub.2 (8
mM) were preincubated at 20.degree. C. for twenty minutes. An equal
volume of 70 mM Tris-HCl, pH 8.2, with the desired radiolabled
sugar nucleotides (UDP-glucose, GDP-mannose and UDP-glucuronic
acid) were added and incubated at 20.degree. C. Radiolabeled
phosphoenol pyruvate and acetyl coenzyme A were added when desired
as described in Ielpi et al., supra, and Ielpi, L. Couso, R. O.,
and Dankert, M. A. Biochem. Biophys. Res. Comm. 102:1400-1408
(1981) and Ielpi, L., Couso, R. O., and Dan Kert, M. A., Biochem.
Intern. 6:323-333 (1983), both of which are specifically
incorporated herein by reference. At various times, the reactions
were stopped by dilution with 4.degree. C. buffer. The samples were
centrifuged and the pellets were washed two times with buffer. The
supernatants were combined, carrier xanthan (100 ug) was added, and
the xanthan plus synthesized polymer was precipitated with ethanol
(60%)-KCl (0.8%). The precipitated polymer was resuspended in water
and reprecipitated two more times to remove unincorporated label.
Radioactivity incorporated into the precipitate (termed the gum
fraction) was determined in d liquid scintillation counter and the
data were processed to obtain incorporation in terms of picomoles
of the radiolabeled components.
[0145] Cell lysates of X1006 did not incorporate carbon-14 acetate
from [.sup.14C] acetyl CoA into the gum fraction of the in vitro
system. Cell lysates of S4-L did produce in vitro gum radiolabeled
with [.sup.14C] acetate. Similarly, cell lysates of X921 did not
incorporate [.sup.14C] pyruvate into the gum fraction while S4-L
cell lysates did incorporate radiolabeled pyruvate from phosphoenol
[.sup.14C] pyruvate into the gum fraction of the in vitro system.
Thus, X1006 was identified as a mutant strain with a defect in the
gene for Acetylase and X921 as a mutant strain with a defect in the
gene for Ketalase. Lysates of these strains produced non-acetylated
xanthan and non-pyruvylated xanthan, respectively, in vitro.
[0146] It has also been shown that, by withholding substrates, X.
campestris B1459 S4-L lysates produce altered polysaccharides in
vitro. For example, cell lysates of S4-L did produce
non-acetylated, non-pyruvylated xanthan gum in vitro when the
endogenous acetyl-CoA and phosphoenolpyruvate were depleted.
[0147] A mutation in the gene for Transferase V would result in the
production of polytetramer. This phenotype would be demonstrated by
the in vitro method described above. The gum fraction would reveal
a polysaccharide composed of glucose, mannose and glucuronic acid
in molar ratios of 2:1:1. The tetrameric intermediate hydrolyzed
from the lipid carrier would also be detected by its mobility on
TLC and its molar ratio of sugars.
EXAMPLE 8
[0148] This example demonstrates the use of the Acetylase-deficient
strain, X1006, to produced non-acetylated gum in vivo. This example
also demonstrates the in vivo production of the non-pyruvylated gum
from the Ketalase-minus strain, X921, and xanthan gum with a
reduced level of pyruvylation from the strain X934.
[0149] The three mutant strains described above and S4-L were grown
overnight in broth with two percent glucose at 30.degree. C. The
gum was harvested by removing cells by centrifugation,
precipitating the gum from the supernatant by addition of
2-propanol or ethanol with 0.5 to 1% potassium chloride. The gum
precipitate was recovered by centrifugation and resuspended. The
procedure was repeated. The resuspended gum was dialysed against
water. A sample of each polysaccharide was acid hydrolyzed and
analyzed by HPLC using a BIO RAD HPX-87H column. Xanthan components
were quantitated by the injection of standards of known
concentration.
[0150] The HPLC analysis of the hydrolyzed gums showed that X921
produced xanthan gum without pyruvate. Strain X1006 produced
xanthan gum with no acetate. Strain X934 produced a gum that
contained pyruvate at a level of 1-5% that of S4-L gum.
EXAMPLE 9
[0151] This example describes methods and strategies that could be
employed to construct mutants of X. campestris that will produce
xanthan that is both non-acetylated and non-pyruvylated.
[0152] Mutant strains of X. campestris lacking Ketalase (X921) or
Acetylase (X1006) activity have-been described. One could use
microbial genetics and recombinant DNA methods as described by
Vanderslice et al. and Capage et al. to introduce a Ketalase defect
and an Acetylase defect into a single strain of X. campestris. This
double-mutant strain would produce xanthan gum that was both
non-acetylated and non-pyruvylated.
[0153] Methods for producing insertion mutations into cloned gum
gene DNA carried on plasmid vectors have been described by Capage
et al. One could envision using a plasmid carrying the insertion
mutation that gave rise to mutant strain X921 as a substrate for a
second round of mutagenesis in vivo or in vitro. This second round
of mutagenesis could employ any of a number of transposable
elements that would readily occur to one skilled in the art or any
of an equally obvious number of DNA restriction fragments
containing selectable markers. A set of plasmids carrying two
insertions mutations could be used in the gene replacement
technique of Capage et al. to transfer the plasmid-borne mutations
into the chromosome of X. campestris. Phenotypes of the resultant
strains can be analyzed by the in vivo and in vitro techniques
described by Vanderslice et al. and Capage et al., supra, in
Example 7. These analyses will reveal doubly mutant strains that
are blocked in both the Ketalase and Acetylase activity. These
double mutant strains will produce xanthan that is simultaneously
non-pyruvylated and non-acetylated.
EXAMPLE 10
[0154] This example describes procedures to obtain a X. campestris
mutant which produces polytetramer.
[0155] As describe above and in Vanderslice et al., supra, a
xanthan-related polysaccharide with a truncated side chain,
polytrimer, has already been obtained. This mutant was
characterized by in vivo and in vitro methods described herein.
Using the methods described herein and in the referenced patent
applications, one versed in the art can create mutant strains of X.
campestris which make polytetramer gum described above.
[0156] Identification of these mutant strains can be achieved using
in vitro methods or by analysis of the polysaccharides produced in
vivo, as described herein and in Capage et al., supra.
[0157] Once the polytetramer producing mutant has been obtained,
additional mutational steps, such as those described in Examples 6
and 10 and generally known to those versed in the art, can be
carried out to generate mutant strains which produce nonacetylated
polytetramer.
EXAMPLE 11
[0158] This example discusses cloning the acetylase gene and the
ketalase gene onto vectors to ensure that the polysaccharides
described herein are fully acetylated, fully pyruvylated, or
both.
[0159] The X. campestris strains X1006 and X921 have mutations in
the genes for acetylase and ketalase, respectively, as described in
Examples 6 and 7. These mutants were created employing the methods
described in Example 6. It is possible for one skilled in the art
to employ methods described in Betlach et al., supra, to recover
native DNA restriction fragments containing the Acetylase or
Ketalase genes. That is, plasmids with DNA restriction fragments
containing the Acetylase or Ketalase genes interrupted by a drug
resistance marker can be used to probe lambda genomic libraries to
obtain native DNA sequences for the Acetylase or Ketalase gene. In
another embodiment, the genes for the acetylase and ketalase
enzymes have already been cloned onto the plasmid pRK290-H336, ATCC
Accession No. 67049 as described by Capage et al., supra, and other
plasmids described therein. It is a simple matter for those skilled
in the art to subclone the Acetylase and Ketalase genes themselves
from these plasmids.
[0160] The native DNA sequences obtained by either method can then
be inserted onto plasmids capable of replication in X. campestris,
for instance pMW79, using the methods revealed in Betlach et al.
Expression of the Acetylase and/or Ketalase gene can be controlled
by modifying the existing DNA sequence or inserting regulatable
promoters upstream from the gene. Such techniques are well known to
those versed in molecular biology and genetics. Similarly, the
plasmids onto which the genes are inserted can be high copy number
plasmids. As revealed in Capage et al., the xanthan biosynthetic
enzymes are present in low amounts in X. campestris. Insertion of
the plasmids described above into X. campestris, and growth of
cultures under appropriate conditions for expression of the
plasmid-borne genes, will result in synthesis of much greater
numbers of the Acetylase and/or Ketalase enzymes than the other
xanthan biosynthetic enzymes. Overexpression of the Acetylase
should cause the xanthan polysaccharide to be fully acetylated,
i.e., all internal mannose residues acetylated. Similarly,
overexpression of the Ketalase should cause xanthan polysaccharide
to be fully pyruvylated on the terminal mannose.
[0161] It should be evident to one skilled in the art that full
acetylation and full pyruvylation of xanthan can be achieved by the
methods described above. Furthermore, full acetylation of
polytetramer, and full acetylation of polytrimer (described in
Vanderslice et al., supra) can be achieved employing the methods
described herein.
EXAMPLE 12
[0162] This example demonstrates the economic and technical
advantages of a non-pyruvylated polysaccharide produced by a
genetically modified Xanthomonas campestris for viscosifying water
at high temperatures.
[0163] Xanthan gum, a natural product of Xanthomonas campestris, is
an effective viscosifier of water for use in, for example, enhanced
recovery of petroleum. These viscosity applications frequently
necessitate xanthan gum to be applied at high temperature and in
saline brines. Xanthan gums are effective viscosifiers even at high
temperature (for example, 75.degree. C. to 100.degree. C.),
although their viscosity is substantially reduced over that at
lower temperature (for example, 25.degree. to 60.degree. C.).
[0164] The inventors have discovered a new and novel polysaccharide
produced by a genetically modified strain of Xanthomonas campestris
(strain X921) that produced a viscosity at high temperature equal
to that of wild-type xanthan gum produced from the S4-L parent
strain (strain X237). This xanthan gum has the normal pentameric
xanthan structure but, because of the genetic modification,
contains no pyruvate moiety on the terminal mannose. This novel
polysaccharide has low viscosity at fermentation temperature near
30.degree. C. which will result in substantial cost savings and
processing conveniences. The cost to produce pyruvylated xanthan
gum is high primarily because the polymer's high viscosity requires
great energy input for agitation, aeration and cooling.
[0165] FIG. 9 shows a viscosity comparison of the novel
non-pyruvylated xanthan gum to a wild-type xanthan gum produced by
the unmodified parent. The wild-type gum shows high viscosity at
low temperature but the viscosity decreases rapidly with increasing
temperature. The non-pyruvylated gum, on the other hand, has lower
viscosity at low temperature, but retains a viscosity at high
temperature essentially equivalent to the wild-type xanthan gum.
The viscosities reported in FIG. 9 were recorded at a shear rate of
8 s.sup.-1 in a concentric cylinder viscometer on solutions of 1000
ppm active polymer solids in 5000 ppm NaCl brine.
EXAMPLE 13
[0166] This example demonstrates the advantages of a non-acetylated
xanthan produced by a genetically modified Xanthomonas campestris
for use as a viscosifying agent for aqueous solutions.
[0167] FIG. 10 compares the viscosities of chemically deacetylated
commercial xanthan and its parent compound with those of a
non-acetylated xanthan polysaccharide made from a genetically
manipulated Xanthomonas campestris (strain X1006) and of the
xanthan gum made from the wild-type X. campestris parent (strain
X237). The viscosities were obtained at a shear rate of 8 s.sup.-1
for 1000 ppm polymer concentration in 50,000 ppm NaCl brine.
Viscosities were measured over the temperature range of 250 to
about 80.degree. C.
[0168] FIG. 10 demonstrates that chemical deacetylation of xanthan
results in a loss of viscosifying power over the entire temperature
range. However, elimination of acetylation by genetic means results
in substantially increased viscosity compared to the wild-type
xanthan gum. Thus, non-acetylated xanthan gum produced by a mutant
strain of X. campestris is an improved polysaccharide compared to
the xanthan itself and compared to deactetylated xanthan gum
produced by chemical methods.
EXAMPLE 14
[0169] This example describes the methods used to construct a
double mutant strain of X. campestris that produces non-acetylated,
non-pyruvylated xanthan.
[0170] Capage et al. have described the cloning of a gene cluster
from X. campestris that contains genes that direct the biosynthesis
of xanthan gum. They also described the isolation of chromosomal
deletion mutations in X. campestris that eliminate all or varying
portions of this gene cluster. One such deletion mutant, strain
X1231, lacks all of the X. campestris DNA that is carried on the
recombinant plasmid pRK290-H336. Thus, strain X1231 does not
synthesize xanthan. When pRK290-H336 is transferred into strain
X1231, the ability to synthesize xanthan is restored. Capage et al.
also described methods of isolation and characterizing insertion
mutations of transporon TnK12 within the cloned gum gene DNA
carried on pRK290-8336. Two such mutant plasmids are pRK290-H336.22
and pRK290-H336.41. The approximate locations of the TnK12 inserts
on each of these plasmids is shown in FIG. 11. When pRK290-H336.22
is transferred into X1231, the resultant strain produces
non-acetylated xanthan. When pRK290-H336.41 is transferred into
X1231, non-pyruvylated xanthan is produced. These two plasmid
mutants have been used to construct, by means on in vitro
recombination, a double mutant plasmid that directs synthesis of
non-acetylated, non-pyruvylated xanthan when carried in the
deletion strain X1231.
[0171] The in vitro strategy for generating a non-acetylated,
non-pyruvylated double mutant plasmid is as follows. A
kanamycin-sensitive (Kan ) derivative of pRK290-H336.41 was
generated by doing a partial HindIII digestion of the plasmid,
ligating the digestion products at very low DNA concentrations (to
promote intramolecular ligation), and then screening
tetracycline-resistant (Tet.sup.r) transformants for kanamycin
sensitivity. Plasmid DNAs were then prepared from Tet.sup.r,
Kan.sup.s isolates and analyzed by restriction endonuclease
digestion and agarose gel electrophoresis. There are only three
HindIII sites in pRK290-H336.41, and two of these occur within
TnK12 and bracket the Kan gene. Thus, a high proportion of the
deletions generated in the partial digestion were deleted for the
Kan gene whereas the rest of the plasmid was retained intact. The
Kan.sup.s plasmid still carries an insertion (1 kb) in the ketalase
gene and thus was expected to yield non-pyruvylated gum. This
plasmid is termed p41KS. The next step was to clone the large SpeI
fragment of the non-acetylated mutant plasmid pRK290-H336.22 into
p41KS. As shown in FIG. 11, each plasmid contains three SpeI sites
at positions 758, 771, and 11,716 within the DNA sequence of Capage
et al. The 10.9 kb SpeI fragment carries the Tnk12 insertion of
pRK290-H336.22. The small (13 bp) SpeI fragment lies entirely
within a tRNA gene which is nonessential for X. campestris growth
and xanthan production. Thus, deletion of this small SpeI segment
in the process of the double mutant construction ought not to
affect xanthan biosynthesis. Plasmids p41KS and pRK290-H336.22 were
purified and digested to completion with SpeI and a ligation was
performed. In this ligation, p41KS/SpeI was ligated in 10.times.
molar excess with H336.22/SpeI. Thus, when recombinants containing
the Kan SpeI fragment of H336.22 were selected, they should most
often be associated with the SpeI vector fragment of p41KS. We
performed transformations with these ligations and obtained
Kan.sup.r transformants. The plasmids carried by these
transformants were analyzed to identify the recombinants of
interest. The desired recombinant plasmid was readily identified
among the Kan.sup.r transformants. This recombinant plasmid, termed
p41KS22, contains the p41KS-derived insertion in the ketalase gene
and the H336.22-derived TnK12 insertion within the acetylase gene.
Appropriate restriction digestion analysis confirmed the presence
of both insertion mutations and, furthermore, showed that the SpeI
fragment containing the TnK12 mutation had been inserted in the
correct orientation.
[0172] Plasmid p41KS22 was subsequently transferred into a series
of X. campestris strains via conjugation. The large Gum.sup.-
deletion strain X1231 was among the recipients. This deletion lacks
all of the gum gene DNA carried on p41KS22; therefore, X1231
carrying p41KS22 should produce non-acetylated, non-pyruvylated
xanthan. The plasmid transferred efficiently into X1231, and the
resultant phenotype was clearly mucoid but significantly less so
than a wild-type control. Polysaccharide produced by X1231 carrying
p41KS22 was prepared and analyzed. This polymer contained glucose,
mannose, and glucuronic acid but no detectable acetate or pyruvate,
demonstrating that X1231 (p41KS22) does produce the expected
non-acetylated, non-pyruvylated gum.
EXAMPLE 15
[0173] This example describes the construction and properties of a
double mutant plasmid that combines an Acetylase mutation and a
Transferase IV mutation.
[0174] Capage et al. described methods for isolating and
characterizing transposor TnK12 insertions within the cloned gum
gene DNA carried by plasmid pRK290-H336. One mutant plasmid
carrying such an insertion is pRK290-H336.6. The approximate
location of the TnK12 insertion in this plasmid is shown in FIG.
11. When present in the deletion strain X1231, this plasmid directs
the synthesis of polytrimer gum as a result of the insertional
inactivation of Transderase IV. Using procedures analogous to those
described in Example 14, a double mutant plasmid was constructed
that combines this Transferase IV defect with the Acetylase
mutation carried in pRK290-H336.22. A kanamycin-sensitive
derivative of pRK290-H336.6 was derived by deletion of the HindIII
fragment of TnK12. This plasmid, p6KS, is analogous to the
Kan.sup.s plasmid 41KS and still retains 1 kb of TnK12 inserted
within the Transferase IV gene. Subsequently, the large
TnK12-containing SpeI fragment of pRK290. H336.22 was ligated into
SpeI-digested p6KS plasmid DNA as described in Example 9 and the
double mutant plasmid p6KS22 was obtained. This plasmid carries
insertion mutations in Transferase IV and the Acetylase. When
transferred into deletion strain X1231, it ought to direct the
synthesis on non-acetylated polytrimer. However, in several
independent plasmid transfer experiments, no transfer of p6KS22
into strain X1231 was detected, although the plasmid was
successfully transferred at high frequency into other recipients,
including both Gum.sup.- and Gum.sup.+ strains. This result
suggests that the presence of p6KS22 in strain X1231 is lethal,
probably as a direct result of production of non-acetylated
polytrimer gum. Capage et al. described three other lethal
mutations within the gum gene cluster and concluded that these
lethal mutations cause the accumulation of a toxic intermediate in
xanthan biosynthesis. Accumulation of non-acetylated polytrimer
could potentially be toxic if this polysaccharide cannot be
secreted by the transport system that normally secretes
xanthan.
[0175] It will be apparent to those skilled in the art that various
modifications and variations can be made in the processes and
products of the present invention. Thus, it is intended that the
present invention cover the modifications and variations of this
invention provided they come within the scope of the appended
claims and their equivalents.
* * * * *