JP2996975B2 - Method for synthesizing sugar nucleotides using recombinant DNA method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention This is a US Patent Application N filed March 24, 1986.
It is a continuation-in-part application of o.843,349. The present invention relates to recombination for the production of various sugar nucleotides.
With respect to the DNA method, this sugar component is then again converted by the recombinant DNA method.
Introduced into microbially produced polysaccharides
You. Various polysacchara industrially useful for certain microorganisms
It has long been known that Id can be produced
You. Prior to the biosynthesis of polysaccharides,
Is a sugar nucleotide that constitutes a polysaccharide
Must have a source. Produce xanthan gum
Xanthomonas campest, a microorganism that can
Squirrel (Xanthomonas campestris) with sugar nucleo
Pide UDP-glucose, GDP-mannose and UDP
-Glucuronic acid is a direct precursor in the biosynthetic pathway
Everything has been found. That is, xantha
In describing the biosynthetic pathway of
Sugar nucleoti in campestris (X. campestris)
Find the DNA sequence responsible for the synthesis of
In Monas (Xanthomonas) sp. And alternative hosts
Recombinant DNA methods to produce these sugar nucleotides
developed. Xanth can produce xanthan gum naturally
Increased sugar nucleotide production in Tomonas sp.
The development of these methods for
Should allow for increased production of this polysaccharide
It is. Some of these microorganisms have detectable levels.
Bell sugar nucleotide precursors are present but their levels
The bell was found to be low. Furthermore, polysaccharide
Mutation exists in the part of the biosynthetic pathway responsible for assembling
In Xanthomonas organisms, these sugar nucleotides
It is also noted that the intracellular amount of the precursor increases by 2 to 7 times.
Was. Therefore, the rate-limiting step in natural xanthan production is sugar
The nucleotide precursor concentration, and the sugar nucleotide
With the introduction of additional DNA sequences that can direct production
Can increase intracellular sugar nucleotide concentration
I believe it. In addition, the recombinant DNA method for the production of xanthan gum
Found to be a good alternative host for use in
Some microorganisms generally require the required sugar nucleosides.
Does not produce tide precursors or can produce xanthan
May not produce them in an effective amount to
Was found. Recently, such recombinant DNA method and
And the alternate host, filed on March 26, 1986,
combinant DNA-Mediated Production of Xanthan Gum "
U.S. Patent Application No. 844,332 to Michael A. Capage et al.
No. Therefore, in these methods
Expresses the xanthan biosynthesis gene,
To produce the required sugar nucleotide precursors
It is necessary to induce a replacement host. The invention is partly
Is provided. Furthermore, the method of the present invention provides a
Sugar nuts when it is intended to introduce sugars into kalido
It can be used to induce reotide production.
For example, the sugar nucleotide of the present invention as a biosynthesis precursor
Xanthan variants requiring some or all
Various other gums have been identified. These gums
Applied for “A Polysaccharide P, filed on August 6, 1985.
olymer Made by Xanthomonas "
It is described in pending U.S. Patent Application No. 762,878. Furthermore, for the production of the sugar nucleotides disclosed herein
Recombinant DNA method is also of interest as a pharmaceutical preparation
Also used for the production of polysaccharides of different species
There is expected. For example, produced by E. coli.
Polysaccharide colonic acid (colonic acid)
This is expected as a potentially useful antigen for vaccination purposes.
Colonic acid production is a microorganism with increased precursor nucleotides
Is believed to be initiated or augmented by strategic production. The usual regulatory mechanisms that regulate expression and activity are foreign DNA or
Is not effective for enzymes, so DNs to other microbial species
The introduction of the A sequence is a sugar nucleus dictated by these sequences.
Ocide production could be enhanced. Further
In addition, many antibiotics such as Streptomyces
omyces) of macrolide antibiotics produced by seeds
A common class is sugar components derived from sugar nucleotide precursors.
Have. Recombinant DNA method for producing sugar nucleotides
Applied to such microorganisms for the biosynthesis of antibiotics
The utilization of essential precursors can be enhanced. SUMMARY OF THE INVENTION One object of the present invention is to provide a recombinant DAN for sugar nucleotides.
It is to provide a manufacturing method by the method. These sugar nu
Nucleotide is an in vivo synthesis of various polysaccharides.
In particular, Xanthan and March 26, 1986
The “Family of Xanthan-Based Polysaccharide”
polymers Including Non-Acetylated and / or Non-Pyr
uvylated Gum and Acetylated or Non-Acetylated Pol
et al., US Patent Application No. 84 entitled "ytetramer Gum"
Structurally described in xanthan, well described in 4,435
Used in the synthesis of new polysaccharides related to
Expect to be able to. Promotes synthesis of these sugar nucleotides by recombinant DNA
Another object of the present invention is to make these portable arrays
Is to provide a vector containing These
Microbial production using recombinant
Sugars in an amount sufficient to produce
It can produce nucleotides. Additional objects and advantages of the invention will be set forth in part in the description.
Or will be known by the practice of this invention.
These objects and advantages are set forth in the appended claims.
Realized by means and combinations specifically pointed out,
And will be achieved. To achieve these objectives, and to
Accordingly, a method for producing a sugar nucleotide is described. this
Sugar component of such sugar nucleotide filed on March 26, 1986
“Recombinant-DNA Mediated Production of Xa
US Patent Application No. 844,332 to Capage et al., entitled "Nthan Gum"
Xantha using the method and vector described in
Will be introduced into the polysaccharide. Portable DNA sequences can be synthetic or restricted ("natural").
DAN sequence). In a preferred embodiment, the porter
DNA sequences are available from the X. campestris library
Release and transfer to an alternative host, at least
The production of one sugar nucleotide can be directed. Furthermore, in order to achieve the above object,
According to the purpose, using the portable DNA sequence described above,
For the production of santa gum and other polysaccharides
Production of sugar nucleotides that can be used for
Is disclosed. This recombinant DAN method comprises: a) producing at least one sugar nucleotide;
At least one that can direct an alternative host microorganism to
Preparing a portable DNA sequence of the same; b) capable of transferring to a host microorganism and replicating therein.
Vector that can be produced
Factor for expression of the portable DNA sequence
Cloning the portable DNA sequence into a vector containing
C) sugar nucleosides under the direction of said portable DNA sequence
An inn that can produce biosynthetic enzymes for tide synthesis
The vector containing the portable DNA sequence
D) maintaining the vector and synthesizing the sugar nucleotide
Culturing said host microorganism under conditions suitable for
And optionally, e) harvesting said sugar nucleotide. To further achieve the above objectives and to provide
Therefore, the portable DNA sequences described above
Provided with a series of plasmids, each containing one
Is done. In particular, plasmids pAS7, pAS9 and pTS13 are disclosed.
It is. In addition, X. campez lacking phosphoglucomutase
A mutant strain of Tris X872 is provided. Xanthomonas mosquito
Impestris X872 strain was acquired on March 21, 1986 by American T
ype Culture Collection (ATCC), Rocoville, Maryland
Was deposited as Accession No.53471. Plasmid pA
E. coli LE392 (pAS9) carrying S9, plasmid pAS7
Carrying the carrying E. coli LE392 (pAS7) and plasmid pTS13
The carrying E. coli LE392 (pTS13), on March 21, 1986,
No.67050, No.67048 and No.67047 respectively
Entrusted. Both the foregoing general description and the following detailed description are merely
It is intended to illustrate and explain
It should be understood that the present invention is not limited by the box.
It is. The accompanying drawings are incorporated in this specification and
It constitutes a part thereof, and includes various components of the present invention.
Illustrative examples, and in conjunction with this description,
It is useful for explaining the reasoning. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows UDP-glucose, UDP-glucuronic acid, and
And a biosynthetic pathway for the synthesis of GDP and mannose.
It is. FIG. 2 shows UDP-glucose, GDP-matrix in the standard mixture.
Northose, UDP-galacturonic acid and UDP-glucuron
3 shows the separation of acids by high performance liquid chromatography. each
Ultraviolet spectrum from 240 to 300 nm is shown for the compound
ing. FIG. 3 shows UDP-glucose, UDP-glucuronic acid,
And a mutant lacking UDP-galacturonic acid (class 1)
Chromatogram of cell extract from representative strain X872
Show. Spectrum of peak eluting in the region of interest
Are shown for comparison with that of FIG. FIG. 4 shows mutants lacking GDP-mannose (class
Chromatography of cell extract from X869 strain, representative of 2)
Show ram. The spectrum of the peak eluted in the region of interest
The vector is shown for comparison with that of FIG. FIG. 5 shows UDP-glucuronic acid and UDP-galacturo.
From the X871 strain, which is a representative of the mutant strain lacking acid (Class 3)
The chromatogram of the cell extract of is shown. Featured
The spectrum of the peak eluted in the region is shown in FIG.
Shown to compare with it. FIG. 6 shows UDP-glucose, GDP-mannose, UDP
-Mutations lacking glucuronic acid and UDP-galacturonic acid
Of the cell extract from the X866 strain, which is representative of the strain (Class 4)
3 shows a chromatogram. The eluting peak in the region of interest
The spectrum of the peak is shown for comparison with that of the second
ing. FIG. 7 shows UDP-G in mutant X649 (class 1).
Deficiency preventing the synthesis of Lucose and UDP-glucuronic acid
5 shows the stage of construction of plasmid pTS13, which complements E. coli. FIG. 8 shows UDP-G in mutant X736 (class 3).
A plasmid that complements the defect that prevents the synthesis of lucuronic acid
The stage of construction of pAS9 is shown. FIG. 9 shows UDP-G in mutant X652 (class 4).
Of glucose, GDP-mannose and UDP-glucuronic acid
Deficiency, and mutants X711 and X712 (class
The deficiency that prevents the synthesis of GDP-mannose in 2)
The stage of construction of the complementing plasmid pAS7 is shown. Fig. 10 shows the results for Paracoccus denitrificans
chromatograph of cell extracts from coccus denitrificans)
Gram, where UDP-glucose and GDP-mannose are
Present but not UDP-glucuronic acid
Is shown. Fig. 11 shows the paracoccus denitrif shown in Fig. 10.
UDP-glucose and other bacteria in cell extracts
3 shows a spectrum confirming a lysine-containing compound. Fig. 12 shows the Paracoccus denitrif shown in Fig. 10.
Confirm the identification of GDP-mannose in cell extracts
And the spectra observed in this figure.
Compound eluted later is an adenosine-containing compound
Indicates that DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention generally relates to various sugars using recombinant DNA methods.
Related to nucleotide synthesis. The following specific description is
For example, for the production of xantham gum and its variants
Also useful UDP-glucose, GDP-mannose and
The description of the production of UDP-glucuronic acid is used. The sugar nucleotides UDP-glucose, GDP-mannose and
And UDP-glucuronic acid are biosynthetic for xanthan gum
It is a direct precursor of the pathway. Vanderslice et al., Supra, (
Incorporated herein by reference).
To direct in vitro biosynthesis of xanthan
Requires all of these compounds. Sugar trans
Due to the specificity of ferase, such as ADP-glucose
Other sugar nucleotides serve as xanthan precursors
Can not do. X. Three types of campestris
The essential sugar nucleotide synthesis pathway is shown in FIG.
You. Xanthomonas campestris is a detectable concentration
Without UDP-galactose, and therefore like E. coli
Glucose-1-phosphate observed in other bacteria
-UDP-Galactose, Uridyl, Transfer
No Rose reaction can occur. X. unable to produce xanthan gum in vivo.
The campestris mutant (Gum-strain) is UDP-glucose.
, GDP-mannose and UDP-glucuronic acid
Cell lysis to synthesize xanthan gum in vitro
They can be divided into two classes based on their ability.
Mutants that cannot produce xanthan in vitro
Is the xanthan biosynthesis mechanism (sugar transferase and
Limerase). When exogenous substrate is supplied
Strain capable of producing xanthan in vitro
Lacks the ability to produce the necessary precursors in vivo.
Some of this second class of mutants have glucose transport
Transport and its metabolism are deficient.
The rate of xanthan synthesis is very low. Others below
One or more sugar nucleotides having normal complement of assimilase
Lacks the enzymes needed to synthesize leotide itself
I have. Such a mutant is a transposome of X. campestris.
Gum-colonies were picked after mutagenesis and added.
UDP-glucose, GDP-mannose and UDP-glucose
In vitro biosynthesis of xanthan in the presence of
It was obtained by precipitation. Specific sugar nucleotide deficiency
Extracts sugar nucleotides and extracts the high-speed liquid
Analysis can be performed using chromatographic methods.
Identified in vivo. Four classes of mutant strains were identified
Was. These include 1) UDP-glucose, UDP-glucuro
Acid and other sugar nucleotides derived from these compounds
Lacking the synthesis of: 2) GDP-mannose and related compounds
3) UDP-glucuronic acid and related compounds
Lacks product synthesis, but not UDP-glucose.
And 4) UDP-glucose, UDP-glucuron
Lack of synthesis of acids, GDP-mannose and related compounds
Is included. UDP-glucose to UDP-glucuronic acid
Variant class 3 is evident since conversion is a one-step process
Lack UDP-glucose dehydrogenase. other
Of the mutant classes required for sugar nucleotide biosynthesis
It was characterized by analysis of Botro enzyme activity. Glucose-6-phosphate and lactose-6
-Phosphate is a key intermediate in microbial metabolism
You. Fructose-6-phosphate to UDP-N-A
The route to cetyl glucosamine is gram-negative bacteria and
Common to bacterial positive bacteria. UDP-N-acetylglucosa
Min is an essential precursor of peptidoglycan in the cell wall
You. Two types of pyrophos, indicated as 4 and 12 in FIG.
Sphorylase is a derivative of UDP-glucose and GDP-mannose.
Committed steps in biosynthesis
In other organisms, glucose-6-phosphate
Reacts with other nucleotide triphosphates to form other
Sugar nucleotides such as ADP-glucose and TDP-glu
A course can be generated. In the UDP-glucose pathway, the UDP-glucose pipe
The gene for lophosphatase is as described in Example 4.
Isolated. The mutation in this gene is UDP-glucose
Not only other sugar nucleotides derived from this,
Synthesis of UDP-glucuronic acid and UDP-galacturonic acid
To prevent UDP-glucose dehydrogenase (yeast
The gene of element 5) was also isolated as described in Example 5.
Was. Mutations in this gene are UDP-glucuronic acid and
Other sugar nucleotides derived therefrom, namely UDP-GA
Prevents synthesis of lacturonic acid. Such an extreme effect (po
lar effects) followed by X. campestris
Disrupts lipopolysaccharide synthesis as well as tan biosynthesis
I do. This provides a supply of sugar nucleotides for the process.
By destroying. In addition, mutants deficient in GDP-mannose synthesis
Was found. These mutants grow normally on mannose.
Is an enzyme that multiplies, and therefore is commonly found in bacteria
Certain enzyme 2, phosphomannose isomerase
Have. Thus, these are enzymes 11 as described in Example 9.
And / or 12 must be missing. Cross high
Hybridization mapping and restriction maps
Mutation of at least another in the chromosome of X. campestris
Indicates that it exists in two places. Insert into these mutants
Plus triggers the synthesis of GDP-mannose when
Mido was developed. Details of this plasmid are shown in Example 6.
You. In addition, this plasmid contains phosphoglucomutase
(Pgm) and GDP-mannose cannot be produced
Complements the Gum-deficiency of the mutant. This mutation causes pgm expression
Within the restriction gene to regulate or pgm structural inheritance
Will be in the child itself. Other mutant X872 lacking only phosphoglucomutase found
Was issued. By the method described in this specification, those skilled in the art
Given the current state of applicable science, the wild type of this gene
A plasmid carrying the copy can be constructed. That is, a residue for the synthesis of direct xanthan precursors.
The gene has been identified by the present inventors. These genes
Are UDP-glucose, GDP-mannose and UDP-glucose
Genetics for enzymes required for the synthesis of clonic acid
Includes children. Wild nature of the genes encoding these enzymes
Plasmid containing type copy was constructed in phage lambda
Obtained from a gene library. By transposon
Chromosome DN from Sugar Nucleotide Deficient Mutants Induced
Vector carrying the cloned DNA segment of A
RSF1010 recombinant plasmid ³² P-labeling plus
Mid DNA was identified. The cloned fragment is
Contains zon and flanking chromosomal DNA. this
Such recombinants are described in Capage et al., Supra.
Thus, it can be easily isolated. 3 types of multi-copy
-Broad host range plasmids are described in more detail in the following examples.
Created using standard techniques. These plasmies
PTS13, pAS7 and pAS9 are respectively the above-mentioned mutation class 1,
Contains DNA that complements two and three strains. In addition, plastic
Smid pAS7 complements the class 4 mutant. These programs
Rasmid is a xanthan biosynthesis gene itself
Hybridization with DNA in or across the region containing
Do not phage cross-hybridization,
Restriction fragment analysis and genetic complementation result in two different loci
Identified in plasmid pAS7. Of these plasmids
Construction and additional information is shown in FIGS. 7-9, and
This will be discussed in more detail in the examples below. Each plasmid is inserted into a mutant in the appropriate complementing group.
When introduced, by the appearance of mucus in the resulting colony
As shown, the ability of the mutant to produce xanthan gum
Restore strength. In addition,
Complemented strains to confirm that they have regained growth potential
The sugar nucleotides in extracts from were tested. plus
Mido pTS13 is a family of UDP-glucose pyrophosphorylase
Most likely to carry a gene. Because the mutation
Bacterium X649 cannot produce UDP-glucose,
Because it has a wild type amount of phosphoglucomutase
is there. Furthermore, plasmid pTS13 is a mutant strain derived from X649.
When inserted into UDP-glucose pyrophosphorilla
Give the ability to produce sesase (Example 8). This strain is a guru
Proliferates normally on the course and therefore grows
Glucose-6-pho which is a key intermediate in the use of
It does not lack phosphate synthesis. As mentioned above, bacteria synthesize xanthan gum
Include UDP-glucose, GDP-mannose and UDP-glucose
Chronic acid is absolutely essential. Each as a precursor
Acetyl-Coenzyme A and phosphoenolpyruvine
Axane by acetylation and pyruvation requiring acid
Modification of the tongue affects the rheological properties of the gum,
Acetylation and pyruvation are essential for its biosynthesis.
There is no. In addition, both of these latter precursors are bacterial
It is an essential component of Xie. UDP-glucose, GDP-mannau
And UDP-glucuronic acid are common in some bacteria
Sugar nucleotides, but all bacteria these
Or do not have xanthan synthesis
They do not have these in sufficient amounts to support.
Xanthan biosynthesis in organisms other than X. campestris
Expression of the synthetic pathway suggests that sugar nucleotide precursors are such alternatives
In the host, preferably supporting economical production of xanthan gum
Need to be synthesized fast enough to hold
You. Young with little or no UDP-glucuronic acid
An alternative host of dried was identified (Example 7). Xantha at high speed
In order to obtain glucose synthesis, UDP-glucose was added to such a strain.
・ Insert DNA carrying dehydrogenase gene
It will be essential. Expression of sugar nucleotide biosynthesis genes in alternative hosts
The basic requirements for this are similar to those in X. campestris
Similar. One or more required genes are grouped into chromosomes
Stably maintained by transfection or maintenance on plasmid
They must be inserted into the host in such a way as to obtain
Absent. Genetic constructs allow the host to synthesize and
To translate it into a functional protein, which is preferably
Must be relatively stable in the host
Absent. In addition, the host is responsible for the synthesis of the sugar nucleotide itself.
Cannot provide substrates and cofactors for
I have to. High copy number plasmid and low copy with broad host range
Both of several plasmids are present. Appropriate for plasmid
Insertion of sugar nucleotide biosynthesis genes has already taken place
You. Plasmid from E. coli to X. campestris mutant
Such a plasmid was transformed in the same manner as described above.
Transformation for introduction into alternative hosts
Alternatively, conjugation can be used. this
Their broad host range plasmids generally infect Gram-negative bacteria
Will do. Optionally, the X. campestris gene
Shuttle bed that can transfer to Gram-positive bacteria
There is a doctor. Plasmid insertion and maintenance is standard
It can be confirmed by the method. Monitor gene expression in alternative hosts in several ways
-Can be. That is, transcribed from the gene
mRNA can be detected by hybridization.
The protein itself is detected by immunoassay or functional measurement.
Sugars that can be
Identify nucleotides by established chromatographic methods
Can be Minutes from one or several of these techniques
Analysis, if desired, to construct genes to optimize expression
It should allow adjustment of things. When a similar external environment is maintained, such as pH and temperature
In particular, the intracellular environment of the alternative host is X.
Probably similar to that of Stris. Kisa
The biosynthesis of lanthanum extends over a wide temperature range,
At a rate between 27 ° C and 37 ° C.
The best is known from in vivo studies. clear
In addition, the enzyme responsible for the synthesis of sugar nucleotide precursors
Must be functional in scope. The first analysis
It will be performed at 27-30 ° C. Priming for the production of specific sugar nucleotides in various hosts
The widespread application of the techniques described herein has been extended to the teachings contained herein.
In the light of what is possible with the art.
Should. Therefore, the following examples are merely illustrative.
It is not intended to limit the invention as claimed.
Absent. Example 1. This example demonstrates various X. mosquitoes that are Gum- in vivo.
Specific Sugar Nucleotides Identified in Impestris Strains
Consider the defect. Initial yield of mutants lacking sugar nucleotide synthesis
Make the collection as described in Capage et al., Supra.
Xantha in vivo after transposon mutagenesis
Cannot be produced. X. Campestris strain
I got it. Some Gum-strains contain the sugar nucleotide UDP-glu.
Course, supplied by GDP-mannose and UDP-glucuronic acid
Can produce xanthan in vitro if
did it. These mutants are synthesized from xanthan itself.
Rather than deficient in sugar nucleotide synthesis.
Was deficient. Next, these mutants are
It was found to be sensitive to gin blue. other
Gum- strains for toluidine blue sensitivity
Additional sugar nucleotide variants
Was done. These strains are wild-type X. campestris and
Kills Gum-mutants deficient in tantan biosynthesis itself
Resistant to several virulent phages
It was proved that. Gum + in vitro but G in vivo
Identify specific sugar nucleotide deficiencies in strains that are um-
The following method was used to do this. Isolated from each strain
Picked up a colony, and 125m Erlenmeyer
-YM medium containing 2% glucose in a flask (yeast
3g, malt extract 3g and peptone 5g /), 10m
Seeded. Culture at 30 ° C., 250 rpm for 24 hours, or
Incubated until out. 5% inoculum in YM medium
Modified PACE medium containing glucose as a carbon source (5 g
KH _Two PO _Four , 5g K _Two HPO _Four 0.2 g MgSO _Four ・ 7H _Two O, 0.53g
(NH _Four ) _Two SO ^Four , 0.006g H _Three BO _Three , 0.006 ZnC ₂ , 0.003g
FeC ₃ ・ 6H _Two O and 0.002 CaC _Two /] And then
And grown at 30 ° C. for 24 hours. Centrifuge the culture
And washed twice with PACE salt and absorbance at 600 nm
Was resuspended in a sufficient volume of PACE to give 100. Sa
1.5 ml of sample and a sufficient amount of glue to bring the concentration to 20 mM.
Put in a 50m Erlenmeyer flask containing the course
Was. Each sample was placed in a 30 ° C water bath at 400 rpm for 10 minutes.
Cubate, then remove 1m and eppend
Added to 0.1 m 11N formic acid in a Ruff centrifuge tube. This switch
Cap the tube and stir the contents in a vortexer for 1 second
Mix and then dry the tubes in a dry ice-alcohol bath
I put it inside. After processing all samples, the tube
Remove from dry ice, thaw at room temperature, and
Centrifuged for 5 minutes to pellet cell debris. Remove the supernatant
Place in a pre-cooled 15m conical centrifuge tube
And frozen in a dry ice-alcohol bath.
Place the frozen sample in a freeze dryer and dry.
Was. Transfer the contents of each tube to triethylamine (Aldo
Rich), 40 mM phosphoric acid adjusted to pH 6.5
Dissolved in 0.2 m of HPLC buffer. 0.45μm sample
Filter through filter into micro sample vial
And injected onto a 4.6 mm x 250 mm C18 reversed-phase ion-pair column.
The analysis was performed at 40 ° C. at a flow rate of 0.8 m / min.
Compare retention times with standards run under the same conditions
And the spectrum of compounds eluting in the region of interest
The sugar nucleotides were identified by examining the sugar nucleotides. Use the following methods for four classes of sugar nucleotide variants
Identified. (1) Synthesize UDP-glucose and UDP-glucuronic acid
(2) Mutants that cannot synthesize GDP-mannose
(3) UDP-glucose can be synthesized, but
However, mutations that cannot synthesize UDP-glucuronic acid
Strains; and (4) UDP-glucose, GDP-mannose and UDP-glucose
Mutants that cannot synthesize lucuronic acid. Representative chromatograms of each mutant class and standard
Various spectra are shown in FIGS. Class 1,3, and
And 4 mutants are also lipopolysaccharide of X. campestris.
Synthesis of UDP-galacturonic acid, a halide precursor
Did not. Based on these data, X. Campest
Squirrel transforms UDP-glucuronic acid into UDP-galacturonic acid
Must be synthesized. The extract from the wild type strain of X. campestris was Xantha.
Possesses all of the sugar nucleotides necessary for biosynthesis of
Was. As shown in Table 1, the biosynthesis of xanthan
Gum-mutants with defects in the tract itself are wild-type cells
Had a higher concentration of precursor sugar nucleotides. These data indicate that xantha in wild-type cultures
Synthesis rate is limited by the supply of precursor sugar nucleotides
It is shown that it is done. Example 2. This example demonstrates that X. campestris lack sugar nucleotide synthesis.
E in the mutants induced by the transposon
Shoglucomutase activity is described. Was previously found to lack sugar nucleotide synthesis
Cytoplasmic and membrane fractions from transposon mutants
Was prepared. X. campez to serve as positive control
An extract of Tris S4-L (NRRL B1459) was also prepared. culture
1% glue from the isolated colonies on the plate
Transfer to 5m YM medium containing course, and tube roller
And grown to stationary phase. Contains 1% glucose
YT medium (8 g tryptone, 5 g yeast extract and 5 g
2 in 500m flask containing 100m of NaC /)
m of culture and incubator at 30 ° C
It was placed on a medium shaker at 300 rpm. After 24 hours, remove the culture
And then centrifuged. Cell pellet 100m
Wash twice in phosphate buffered saline (pH 7.2), then 10m
M MgC ₂ 20w in 50mM MOPS buffer (pH 7.2) containing
Resuspended to / v%. Remove residual media from cells
This method was used to do this. French at 15,000psi
Suspended cells were broken by passing the press twice. Fine
Reduce viscosity by treating cell lysate with DNAase
And then centrifuged at 2,500 xg to remove unbroken cells and
And cell debris were removed. Using a Pasteur pipette
Carefully remove the clarification and then to an average centrifugal force of 130,000 xg
By centrifugation in a rotating bucket rotor.
It was separated into a cytoplasmic fraction and a membrane fraction. Contains cytoplasmic components
The supernatant was decanted and frozen at -70 ° C. Including each membrane
1.0m MOPS MgC with pellets ₂ Resuspend in buffer,
And also frozen at -70 ° C. Sugar nucleotide synthesis
The enzymes required for are normally found in cytoplasmic components.
Separation of cytoplasmic components from cell membranes is coupled to NADP reduction
Facilitates enzymatic measurements. NADPH oxidase is membrane bound
Enzyme, and unless removed or inactivated
Pyridine nucleotide (this accumulation tracks the reaction
Can be rapidly reoxidized)
You. The protein concentration in each cytoplasmic extract was determined as
Using clear albumin, Lowry et al., J. Biol. Chem. 193 : 265
−275 (1951), which is specifically incorporated herein by reference.
). Glucose-6 in each extract
The activity of phosphate dehydrogenase is glucose
During oxidation of -6-phosphate to 6-phosphogluconic acid
Of Absorbance at 340nm Due to the Accumulation of NADPH Produced in Rice
Was determined by tracking. This yeast is X. Campe
A key enzyme in the metabolism of glucose by stris
And serve as an internal control. Reaction conditions are shown in Table 2.
Write in footnotes. Glucose-6-phosphate is converted to UDP-glucose
Conversion to the direct precursor glucose-1-phosphate
Phosphoglucomutase is also measured in the cytoplasmic fraction
did. Since reversible mutase activity,
Lucose-1-phosphate was used. In addition, Sigma
-Purified glucose-6 purchased from Chemicalne
-Phosphate dehydrogenase in excess in the reaction mixture
added. The rate of NADPH formation depends on the amount of host present in the reactants.
Glucose-6-phosphate by hoglucomutase
Indicates the speed at which the is generated. Reaction conditions and assay
Table 2 summarizes the results of (a). (A) 0.05 m cytoplasmic fraction (about 10 mg / m protein)
The reaction was started by the addition. 40 mM reaction mixture
Tris-HC (pH 8.6), 5 mM glucose-6-phosphate
Plate, 1.6 mM NADP and 15 mM MgC ₂ The total volume of 1.0m
Contained in the amount. (B) Reaction conditions are the same as for G6PD,
Glucose-1-phosphate instead of s-6-phosphate
Used. The reaction mixture was further diluted with 1 mM dithiole
Itol, 0.2 mM glucose-1,6-diphosphate, and
And 10 units of G6PD manufactured by Sigma Chemical Co., Ltd.
Was. (C) Deposited as ATCC No. 53471. All extracts range from 97-268 nmo / min / mg protein
Significant activity of glucose-6-phosphate aldehyde
It had a logase. Synthesize UDP-glucose
All extracts prepared from
And phosphoglucomutase activity was 191 nmo / min / mg protein.
More than white matter. Can synthesize UDP-glucose
Can not. All mutants except X649 (X828, X652, X86
6, and the extract from X872) contains little or no phospho
It did not have glucomutase activity. Such a defect is UD
Sufficient to prevent the synthesis of P-glucose. X649 strain produces a UDP-glucose strain of sugar nucleotides
Cannot produce. Phenotypically, this is Tn903
It is similar to the mutant strain X872. However, X649
It has normal phosphoglucomutase activity, but X872 does not
The enzyme is deficient. X649 is UDP-glucose pyro
The phosphorylase itself must have a defect. The X652 strain is a sugar nucleotide UDP-glucose strain and G
The DP-mannose line cannot be produced. these
X828 and X86 are Tn903 mutants lacking any sugar nucleotides
As with 6, X652 has almost no phosphoglucomutase activity.
No or no. Deficient in GDP-mannose synthesis only
Mutant strains X657, X711, X712 and X869… are normal
It has high phosphoglucomutase activity. X736, X826 and X
Strain 871 is unable to produce UDP-glucuronic acid.
These also have normal phosphoglucomutase activity. Example 3. This example demonstrates the sugar nucleotides in a Tn10-induced mutant.
This section describes the method of mapping the defect. Tn10-induced mutants deficient in sugar nucleotide metabolism
For all of the above, the Tn10 + periphery (flanki
ng) Plasmid by cloning chromosomal sequence
Probe was obtained. By probing the λ bank
Hybridize to each probe, but with X.
Λ Recombination Carrying a Segment of Wild-Type DNA of Pastris
I got a body. These phages are plaque purified and
It is used to contain Tn10 and flanking DNA.
Sugar by cross-hybridization to a plasmid probe
Nucleotide mutations were mapped. Both sugar nucleotide probes are synthesized with xanthan
Map within the DNA region containing the genes of the pathway itself
Did not. Contains wild-type DNA for mutant X649
The phage will harbor any other sugar nucleotide probe.
Did not hybridize. Similarly, the T in the mutant X736 DNA
Phage containing wild-type DNA from the n10 insertion region
It did not hybridize to the sugar nucleotide probe.
Therefore, the defective gene in these strains is not compatible with other sugar nucleotides.
Not linked to the gene. Plasmids pTX652, pTX657, pTX711 and pTX712 were cloned
Recombinant lambda chromosome containing ligated X. campestris DNA
Hybridize to overlapping sets of images
Was. Plasmid pTX711 is inserted into pTX652, pTX657 and pTX712.
Some, but not all, of the hybridizing λ phages
Crab hybridized. X652 also contains UDP-glucose or UDP-glucuronic acid.
Mutations in the X652 strain and GDP-
The association with mutations that prevent mannose synthesis is unexpected.
Was. Mutations in X652 are UDP-glucose and GDP-manno
Transactin, which affects the synthesis of
g) in regulatory genes or for UDP-glucose synthesis
Encoding an essential enzyme but GDP-mannose inheritance
Structural remains that exert a polar effect on the expression of offspring
It will be in the messenger. Example 4. This example demonstrates the mutation of the mutation in strain X649 by plasmid pTS13.
Explain the complement. Transposon mutation of all sugar nucleotide variants
Obtained by induction. Lambda of genomic DNA of X. campestris
The library was prepared using UDP-glucose and UDP-gluconic acid.
Transposon Tn from mutant X649 with defective synthesis.
Cloning 10+ flanking chromosomal sequences
(As described in Capacity, etc., supra)
Probed with plasmid pTX649 induced by
Was. 10 λ recombinants that hybridize to the pTX649 probe
Was identified. Restriction digestion and gay swimming of DNA of each λ clone
Movement. In these digestion patterns,
In the wild-type fragment of mutated X. campestris DNA
Identified. This 6.5 kb Pst I fragment is used for preparative agarose
And purified by electroelution from the gel. This Pst I fragment is pBR
Cloned into the PstI site of 322 to create plasmid pTf6.5
Had made. Digest pTf6.5 and plasmid RSF1010 with EcoRI
And ligating both plasmids together
Was constructed. After transformation, strain E. coli
Confer resistance to putomycin and tetracycline
Hybrid plasmids were selected according to their ability. This texture
Plasmid pTS13 contains a 6.5 kb Pst I fragment (No. 7).
Figure). This plasmid is then directed by pRK2013
Tripalent Conjugal Transfer (tripar
ental conjugal transfer) by X. campestris X64
The number has been shifted to nine. The cloned 6.5 kb fragment carried by pTS13 is a mutant
Complements X649. Mate of pTS13 for migration to X649
Plate the mating mixture on Rif and Strep
If all of the colonies are Gum +,
Could not be distinguished from the type. 3 Gum + Kiso
Analyze exoconjugates, and these
It was shown to contain a sumid. In addition, plasmids
A "curing" experiment was performed. In this experiment
Promotes X649 (pTS13) loss of plasmid
Grow under conditions and then select a plasmid.
Plated in the absence of any possible drug.
In some experiments, significant frequencies were found in these plates.
Gum-colonies were observed in degrees (10-50%). Like this
Three Gum-isolates were tested and plasmid pTS13 was lost.
It was shown that. However, from this experiment
All three Gum + isolates were found to carry pTS13
Was done. Correlation between plasmid presence and Gum + phenotype
The relationship is that the nature of the first Gum + is not due to recombination.
Rather, due to the expression of the gene carried on the plasmid
Claims to be something. Preparation of extract from X649 strain and X649 strain containing pTS13
did. The extract from X649 with plasmid was
To be determined by the
Had a course. The extract from X649 alone is not
Did not. Extract from X649 has normal amounts of phosphoglucomutase
(Example 1) prevents UDP-glucose synthesis
Deficient enzyme is UDP-glucose pyrophosphoryler
Must be located in the gene encoding ze. This
Is contained in plasmid pTS13. This
Since Rasmid is a chimera of RSF1010, X. Campest
Can transfer to Gram-negative bacteria other than squirrels, and
The ability of the bacterium to produce glucose-1-phosphate
If you have the power to produce it UDP-glucose
Ability can be given. Example 5 This example demonstrates the complementation of a mutation in strain X736 by plasmid pAS9.
Explain the end. X. campestris chromosome from the region of the Tn10 insertion in X736
One set of five lambda phage containing DNA was isolated. These phages were hybridized to plasmid pTX736.
Screening for recombinant phage
And isolated from the λ bank gene. Plasmid pT
X736 is a mutant containing a Tn10 insertion that gives rise to a mutant phenotype
Pst I fragment of chromosomal DNA from X736 is a plasmid vector
It is cloned into RSF1010. These λ736 (+) phages were
Screening by hybridization
A relatively large DNA fragment containing the wild-type Pst fragment was identified.
Was. DNA from lambda recombinant to some restriction endonucleases
And digested with the same set of enzymes.
Swim on agarose gel with wild-type chromosomal DNA control
I moved it. This digest was then radiolabeled.
Probed with pTX736 plasmid DNA. Some differences
The SalI digest of the λ736 (+) isolate
A 9 kb fragment was hybridized. Wild type staining
9 kb Sal I digest of body DNA also anneals to this probe
Band was generated. λ736 recombinant has relatively small number of Sal I
Since a fragment was generated, shotgunk from λ to pMW79
Loan was performed. The plasmid vector pMW79
A unique Sal I located within the tracyclin resistance gene
Contains a site. Therefore, pMW79 and λ736 (+) DNA
Digest both with Sal I and combine digestion products together
Connected. The ligation reaction is used to transform E. coli and
Ampicillin resistant transformants were selected and their 65
0 were tested for sensitivity to tetracycline
To identify the recombinant plasmid. 10 Amps ^r Tet ^s Isolate
Was found. Plasmid DNA from these transformants
And restriction endonuclease digestion and agar
Analysis was performed by Rose gel electrophoresis. Notable recombinants are seen
Was issued. Contains the cloned 9 kb SalI fragment.
And the plasmid was named pAS9 (FIG. 8). X736 Gum-This plasmid was transformed with X.
I switched to Campestris. Plasmid pAS9 to X736
Rifampicin-resistant inducer (designated X1017) and Rif
^r Mobilized to wild-type (Gum +) strain X77. this
Their mobilization is a standard tripa mandated by pRK2013.
Rental conjugal transfer (triparenta
l conjugal transfer). Rifampicin
[For E. coli donors and mobilizer strains]
And streptomycin (presence of pAS9)
By selecting on)
X. campestris conjugate carrying the plasmid of interest
(Conjugant) was selected. X1017 to pAS9 mating
Rif of mating ^r And Strep ^r The descendants are exclusively Gum +
there were. Select 3 Gum + derived strains and add plasmid
Selected about All three are clearly plasmids
containing pAS9 and restriction endonuclease
This plasmid has some distinction when determined by digestion
Found that they have not undergone any serious rearrangement
Was. In addition, a plasmid “curing” experiment was performed.
Was. In this experiment, X1017 (pAS9) was
Growth under conditions that promote the loss of
In the absence of any drug that would select Sumid
Plated in a state. Gu in this plate with significant frequency
m-colonies were observed. 3 such Gum-isolates
And that plasmid pAS9 has been lost
Was found. However, three Gum from this experiment
+ All isolates were found to maintain pAS9.
The correlation between the presence of the plasmid and the Gum + phenotype is
The nature of um + is not due to recombination but rather plasmid
Claims that this is due to the expression of the gene carried on
are doing. Mutant X736 is capable of producing UDP-glucose
However, UDP-glucuronic acid cannot be produced.
Therefore, conversion of UDP-glucose to UDP-glucuronic acid
Single enzyme UDP-glucose dehydrogena
It is most likely that the enzyme is defective. For this enzyme
Is contained on plasmid pAS9. Example 6. This example demonstrates the strains X652, X711 and X712 with the plasmid pAS7.
The complementation of the mutations inside is explained. Transposon Tn10 from sugar nucleotide mutant X652
+ Flanking chromosomal sequence in plasmid RSF1010
Plasmid pT derived by cloning
X652, λ live of genomic DNA of X. campestris
Rally was probed. Mutant X652 is UDP-glucose
, GDP-mannose or UDP-glucuronic acid
No. Recombinant phage that hybridizes to pTX652
Purified. These files probed by pTX652
Southern blot of the restriction digest
9 kb BamH I fragment containing the wild sequence corresponding to the site of insertion
Pieces were identified. This 9 kb BamHI fragment was purified and plasmid pMW79
At the BamHI site. E. coli using the ligation mixture
Transform and select for ampicillin resistant transformants
did. These are screened for tetracycline sensitivity.
Learning. For insertion of foreign DNA into BamHI site of pMW79
Genes encoding more resistance to tetracycline
Will be inactivated. Contains a 9 kb BamHI fragment
Plasmid pAS7 was obtained by this method (FIG. 9). Next, the tripalental core commanded by pRK2013
Plasmids were transferred to E. coli by Njugal transfer.
Transferred from Li to X. campestris X1043. pTX652 to X.
Impestris strain X77 (X. campestris NRRL-B1459 S4
-Lifampicin resistant mutant obtained from -L)
And imposing a tetracycline selection
Therefore, homologous recombination allows X. campestri
X1043 was obtained. Different antibiotic resistance of X1043 from X652
Promotes subsequent reverse selection for E. coli donors
did. After transfer of plasmid pAS7, X1043 has a Gum + phenotype
Has recovered. This phenotype is Gum if the strain has lost pAS7
-, Gum + phenotype is transposon in strain X652
Due to the plasmid of the chromosomal DNA inactivated by insertion
This is due to the expression of the carried copy
Was. As shown in Example 3, of the X652, X711 and X712 strains
Are clustered on the X. campestris chromosome. Step
Rasmid pAS7 in tripa rental mating
After transfer to mutants X711 and X712 by conjugation, Gum + table
Restored the current model. If the complemented strain has lost the plasmid
It became Gum-. The X711 and X712 strains have GDP-mannose similarly to the X652 strain.
Cannot be synthesized. Plasmid pAS7 has this ability.
To recover, and thus to the synthesis of GDP-mannose
Contains genes encoding necessary enzymes. X652 also has a UDP-glucose deficiency. This lack
Loss is due to a lack of phosphoglucomutase in strain X652 (eg
It matches with 2). GDP-an enzyme required for mannose biosynthesis
The same linkage containing the gene encoding
Structural gene of glucomutase or phosphoglucomutase
It also contains a control gene that regulates the expression of. Example 7. This example illustrates the accumulation of sugar nucleotides in alternative hosts.
I will tell. Paracoccus denitrificans
nitrificans) (ATCC 17741), Pseudomonas strike
Tuzzelli (Pseudomonas stutzeri) (ATCC 17588),
And Pseudomonas Perfect Marina
s perfectomarina) (ATCC 14405)
Using the method developed for X. campestris
And analyzed. 2% glucose as a carbon source
The cells were grown for 12 hours. Collect and wash cells
And resuspended so that the absorbance at 600 nm is 100.
Was. This cell pellet is required for anaerobic growth
Typical of denitrifying bacteria that have derepressed cytochrome synthesis
Pink color (typical for oxygen limitation during growth)
reaction). Next, 25 mM nitrate was used as an additional electron acceptor.
Included in the incubation mixture. Cell suspension 20 mM
5 minutes with or without nitrate with or without nitrate
Incubate and ants as described in Example 1.
Extracted with acid. The extract is freeze-dried and TEA-phosphate
Dissolved in buffer and analyzed by HPLC. Featured
When confirming by the spectrum of the peak in the region,
Coccus denitrificans is composed of UDP-glucose and G
It had DP-mannose, but UDP-glucuronic acid
The amount was undetectable (FIGS. 10-12). Similarly, shoe
Domonas Perfect Marina and Pseudomonas Sus
Tozzelli has UDP-glucose and GDP-mannose
Was. UDP-Glue in extracts from any organism
Chronic acid was not detected. As described in Example 5, tripalental
Plasmid pAS9 was transformed into E. coli by conjugation in mating.
From the E. coli donor into all three bacteria
So that UDP-glucuronic acid can be synthesized
It is intended to modify these strains. Example 8. This example demonstrates X. cans with a deficiency in sugar nucleotide synthesis.
UDP- in a transposon-induced mutant of Pestris
The glucose pyrophosphorylase activity is described. Can the X649 strain produce UDP-glucose?
(Example 1), but with phosphoglucomutase activity
(Example 2). The experiment below shows that the mutation in this strain is UD
Influences P-glucose pyrophosphorylase activity
And UDP-glucose pyrophosphorylase
Indicates that the gene is contained on plasmid pTS13. Analysis of UDP-glucose pyrophosphorylase activity
For ease, a strep carrying the Tn10 insertion of strain X649
3 tomycin-sensitive rifampicin-resistant strains were constructed
Was. One such strain, X1023, is described in Capage et al., Supra.
The gene was constructed by the gene replacement method described above. other
Two strains X1024 and X1025 are chromosome mobilized (mobilizatio
n). Sugar nucleotide of X649 (Example 4)
Plasmids pTS13 to complement the deletion were replaced with X1023, X1024 and X102
X101, X1039 and X1040, respectively.
Furthermore, the plasmid pTS13 was replaced with a Gum + rifampicin resistant strain.
Insertion into X77 gave rise to strain X1052. These strains and X. campest serve as a positive control
The cytoplasmic fraction was prepared from squirrel X77 as described in Example 2.
Made. UDP-glucose pyrophosphorylase
Course-1-phosphate and UTP with UDP-glucose
Converted to pyrophosphate. This reaction is reversible
is there. Lieberman et al., Proc. Natl. Acad. Sci. USA 65 : 625−6
32 (1970), which is hereby incorporated by reference.
As described, phosphoglucomutase and phosphoglucomutase
And glucose-6-phosphate dehydrogenase
Addition from UDP-glucose to the reduction of NAD or NADP
Coupling the formation of glucose-1-phosphate
UDP-glucose pyrophosphorylase activity
The properties were measured. In these measurements, Tris buffer
Use HEPES buffer instead and remove from the reaction mixture.
Sodium acid was omitted. Sodium fluoride (5mM)
Added to inhibit pyrophosphatase activity. In experiments using NAD, the X1023 strain was 5.4 nmo / mg protein.
UDP-glucose pyrophosphorylase activity in white matter / minute
Which is the UDP-glucose of the wild-type strain X77.
・ Pyrophosphorylase activity 72.2 nmo / mg protein / min / 10
%. 340 nm absorbance in X1023 reaction mixture
Almost all of the increase in
Competitive reaction not due to holylase activity itself
It was due. X1039 strain is 18.7 nmo / mg protein / min
Has a UDPPG pyrophosphorylase activity of X1023
4 times higher than the activity in This result is
pTS13 encodes UDP-glucose pyrophosphorylase gene
It shows that it is carried. pTS13 is UDP-glucose pyrophosphorylase gene
NABP as an electron acceptor to confirm that
Additional experiments were performed with other mutant strains. The result
The results are summarized in Table 3. Multiple measurements at different extract concentrations
The results are shown. X1023, X1024 and X1025 strains have pyrophosphorylase activity
Little or no. X1025 and X1023
X1040 and X1041 strains containing a plasmid that complements the deletion
Had a high activity. Similarly, X1052 is the plasmid p
Extremely higher activity than wild-type parent strain X77 without TS13
Had. pTS13 is a high copy number plasmid
Since it is derived from Sumid RSF1010, this difference in activity is
It may reflect the effect of the dose. X77 and X1040 strains
Prepare the cytoplasmic extract again, this time
Pyrophosphorylase in a spectrophotometer equipped with
Activity was measured directly. Specific activity is for X77 and X1040
They were 149 and 53.4 nmo / mg protein / min, respectively. These results indicate that the mutation in X649 is UDP-glucose
UDP-glucose by removing pyrophosphorylase activity
This confirms that the synthesis of is prevented. This activity
Is a gene for UDP-glucose pyrophosphorylase
Recovered by plasmid pTS13, which must be carrying
Can be Further increase UDP-glucose pyrophosphorylase activity
In addition, cells from the Tn903 series of sugar nucleotide variants
Measured in the quality extract. The reaction mixture contains no NaF
Was. The results are shown in Table 4. All strains have measurable pyrophosphorylase activity
Was. The absence of phosphoglucomutase activity was due to X82
8, X866 and X872 strains can synthesize UDP-glucose
It is enough to explain what you cannot do. Example 9. This example has a defect in sugar nucleotide synthesis.
Phosphos in a transposon-induced mutant of Pestris
Homannomutase activity is described. Preliminarily identified sugar nucleotide mutants (Example 1)
Was prepared as described in Example 2.
Was. Mannose-6-phosphate is converted to GDP-mannose
Mannose-1-ho which is a substrate of pyrophosphorylase
Phosphomannommutase, an enzyme that converts to phosphate.
These extracts were measured for (PMM). Enzyme anti
The response is reversible. The measurements (Table 5) were determined by Pindar and Bucke, Biochem. 152 : 61
7-622 (1975), which is hereby incorporated by reference.
Modified from the method of
Phosphomannose isomerase (PMI), phosphog
Lucose isomerase (PGI) and glucose-6-pho
By the addition of sulfate dehydrogenase (G6PD)
Mannose-6-phosphate formation favors NADP reduction.
It is pulled up. The reaction rate becomes linear after a few minutes. This time is phosphorus
When the oxidized sugar intermediate is needed to reach a steady concentration
Between. These linear velocities are included in the reaction mixture.
In proportion to the amount of cytoplasmic extract obtained. For convenience, the actual reaction speed
Instead of determining the degree, mannose-1-phosphate
NADPH in the presence of _Two Generation and 30 in the absence of
PM by comparing after
M activity was determined. The results are summarized in Table 6. Initially, the X866 extract was converted to mannose-1-phosphate
It showed an increase in absorbance at 340 nm after addition. But
However, this increase can be attributed to phosphomannommutase activity.
And could not. The reaction rate is not linear,
It flattened well below the maximum absorbance to be achieved. X866
Extract is glucose-6-phosphate dehydro
It was still active when measured for genease activity (0.
77 μmo / m / min). X866 strain clearly lacks PMM activity
are doing. All other GDP-mannose variants are phosphomanno
It had mutase activity. GD in these mutants
The defective enzyme that prevents the synthesis of P-mannose is GDP-manno.
Must be a source pyrophosphorylase. GDP−
Prevents synthesis of mannose, in X652, X711 and X712
Since the deletion is corrected by plasmid pAS7,
Rasmid is the ancestor of GDP-mannose pyrophosphorylase.
Must carry a gene. Species changes may be made in the methods and products of this invention.
It will be apparent to those skilled in the art that Therefore,
As long as they fall within the scope of the appended claims and their equivalents,
Modifications of the present invention are intended to belong to the present invention.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁷ Identification code FI (C12P 19/30 C12R 1:64) (C12N 1/21 C12R 1:40) (C12N 1/21 C12R 1:39) (C12N 1/21 C12R 1:38) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:01) Accession number of microorganism ATCC 67048 Accession number of microorganism ATCC 67047 (72) Inventor Bander Slice, Rebecca, Double, United States, Colorado 80303, Voulder, Tantra Park Circle 1011 (56) References Journal of Bacterology, Vol. 169, no. 1 (1987), p. 351-358 Development Mental Biology, Vol. 110, (1985) p. 369-381 Meth. Enzymol. , Vol. 28 B, (1972), p. 430-435 J.P. Biol. Chem. Vol. 57, (1975), p. 115-126 Biochim, Biophys. Act, Vol. 33, (1959), p. 522 to 526 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12P 1/00-41/00 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG) )

Claims (1)

(57) [Claims] 1.1 A method for producing one or more sugar nucleotides, wherein (a) UD obtainable from plasmid pAS9 (ATCC 67050)
A DNA fragment encoding P-glucose dehydrogenase,
A DNA fragment encoding phosphoglucomutase obtainable from plasmid pAS7 (ATCC 67048), plasmid pAS7 (A
DNA fragment encoding phosphomannose mutase obtainable from TCC 67048), plasmid pAS7 (ATCC 67048)
DNA fragment encoding GDP-mannose pyrophosphorylase and plasmid pST13 (ATCC 67047) obtainable from
Obtaining a vector comprising at least one DAN fragment selected from the group consisting of DNA fragments encoding UDP-glucose pyrophosphorylase obtainable from (b) transferring said vector to a host microorganism; (c) Culturing the host microorganism under conditions suitable for the synthesis of at least one sugar nucleotide; and (d) recovering the sugar nucleotide. 2. The method according to claim 1, wherein the sugar nucleotide is UDP-glucuronic acid. 3. 2. The method of claim 1, wherein said sugar nucleotide is GDP-mannose. 4. 2. The method of claim 1, wherein the sugar nucleotide is UDP-glucose. 5. The cloned DNA fragment is Xanthomonas
The method according to any one of claims 1 to 4, obtained from campestris (Xanthomonas campestris). 6. 2. The host microorganism is Xanthomonas.
The method according to any one of claims 1 to 5. 7. The host microorganism is Pseudomonas putida (Pseu
domonas putida), Pseudomonas, Sephacia (Pseudo)
monas cepacia), Pseudomonas denitrificans, Pseudomonas denitrificans
Fluorescens (Pseudomonas fluorescens), Pseudomonas stutzeri, Escherichia coli and Enterobacter
locae). 6. The method according to claim 1, wherein the method is selected from the group consisting of: 8. The vector containing the cloned DNA fragment is a mutant that avoids the synthesis of UDP-glucose, GDP-manmose and UDP-glucuronic acid in mutant X652 and the synthesis of GDP-mannose in mutants X711 and X712. PAS7 complements the mutation to be avoided; and UDP in mutant X736
PAS9 complementing a mutation that circumvents the synthesis of gluconic acid; and pTS13 that complements a mutation that circumvents the synthesis of UDP-glucose and UDP-glucuronic acid in mutant strain X649. The described method.