Title: Methods for enzymatic conversion of GDP-mannose to GDP-fucose
Abstract: This invention provides methods for practical enzymatic conversion of GDP-mannose to GDP-fucose. These methods are useful for efficient synthesis of reactants used in the synthesis of fucosylated oligosaccharides.
Patent Number: 7,026,142 Issued on 04/11/2006 to Sjoberg
| Inventors:
|
Sjoberg; Eric R. (San Diego, CA)
|
| Assignee:
|
Neose Technologies, Inc. (Horsham, PA)
|
| Appl. No.:
|
206655 |
| Filed:
|
July 25, 2002 |
| Current U.S. Class: |
435/101; 435/94; 435/189; 435/233; 536/23.2 |
| Current Intern'l Class: |
C12N 9/02 (20060101); C12N 9/90 (20060101); C07H 21/04 (20060101); C12P 19/04 (20060101); C12P 19/24 (20060101) |
| Field of Search: |
435/233,183,72,94,97,101,189
536/232
|
References Cited [Referenced By]
U.S. Patent Documents
| 5352670 | Oct., 1994 | Venot et al.
| |
| 5374541 | Dec., 1994 | Wong et al.
| |
| 5374655 | Dec., 1994 | Kashem et al.
| |
| 5728568 | Mar., 1998 | Sullivan et al.
| |
| Foreign Patent Documents |
| 0870841 | Oct., 1998 | EP.
| |
| WO 99/0918/0 | Feb., 1999 | WO.
| |
Other References
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the MutT Family of Enzymes"; The Journal of Biological Chemistry; 1995; pp. 24086-24091;
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Broschal, et al., "Purification and characterization of GDP-D-mannose 4,6-dehydratase
from porcine thyroid," Eur. J. Biochem., 1985, pp. 397-401, vol. 153.
Cameron, et al., "Expression of human chromosome 19p α(1,3)-fucosyltransferase
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Genes Dev., 1990, pp. 1288-1303, vol. 4.
Kumar, et al., "Cloning a human α(1,3)-fucosyltransferase gene that encodes
ELFT but does not confer fucosyltransferase cDNA that can form the H blook group
antigen," J. Biol. Chem., 1991, pp. 21777-21783, vol. 266.
Larsen, et al., "Molecular cloning, sequence, and expression of a human GDP-L-fucose
:β-D-galactoside 2- α-L-fucosyltransferase cDNA that can form the H
blood group antigen," Proc. Nat'l Acad. Sci. USA, 1990, pp. 6675-6678, vol. 87.
Mollicone, et al., "Acceptor specificity and tissue distribution of three human
α-3-fucosyltransferases," Eur. J. Biochem., 1990, pp. 169-176, vol. 191.
Nunez, et al., "The synthesis and characterization of α- and β-L-fucopyranosyl
and GDP fucose," Can. J. Chem., 1981, pp. 2086-2095, vol. 59. phosphates.
Ohyama et al., "Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase,
a key enzyme for fucose metabolism defective in Lec13 cells," J. Biol. Chem., Jun.
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sialyl-lewis-a-determinant," Carbohydrate Res., 1989, pp. 1-11, vol. 190.
Prieels, et al., "Co-purification of the lewis blood group N-acetylglucosaminide
α1—4 fucosyltransferase and an N-acetylglucosaminide α1-3 fucosyltransferase
from human milk," J. Biol. Chem., 1981, pp. 10456-10463, vol. 256.
Reeves, P.R. et al., "Bacterial polysaccharide synthesis and gene nomenclature,"
Trends Microbiol., Dec. 1996, pp. 495-503, vol. 4(12).
Samesto, et al., "Purification of the β-N-acetylglucosaminide α1-3-fucosyltransferase
from human serum," J. Biol. Chem., 1992, pp. 2745-2752, vol. 267.
Shinoda, et al., "Enzymatic characterization of human α 1,3-fucosyltransferase
fuc-TVII synthesized in a B cell lymphoma cell line," J. Biol. Chem., 1997, pp.
31992-31997, vol. 272.
Staudacher, E., "α1,3-fucosyltransferases," TIGG, 1996, pp. 391-408, vol. 8.
Stevenson, G. et al., "Organization of the Escherichia coli K-12 gene
cluster responsible for production of the extracellular polysaccharide and colani
acid," J. of Bacteriol., 1996, pp. 4885-4893, vol. 178(16).
Sullivan, F.X. et al., "Molecular cloning of human GDP-mannose 4,6-dehydratase
and rconstruction of GDP-fucose biosynthesis in vitro," J. of Biol. Chem., 1998,
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Tonetti, M. et al., "Synthesis of GDP-L-fucose by the human FX protein," J. of
Biochem. Chem., 1996, pp. 27274-27279, vol. 271(44).
Weston, et al., "Isolation of a novel human α(1,3) fucosyltransferase gene
and molecular comparison to the human Lewis blood group α(1,3/1,4) fucosyltransferase
gene," J. Biol. Chem., 1992, pp. 4152-4160, vol. 267.
Weston, et al., "Molecular cloning of a fourth member of a human α(1,3)fucosyltransferase
gene family," J. Biol. Chem., 1992, pp. 24575-24584, vol. 267.
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Chandrasekaran, E.V. et al.; "Specificity Analysis of Three Clonal and Five Non-Clonal
α1,3-L-Fucosyltransferases with Sulfated, Sialylated, or Fucosylated Synthetic
Carbohydrates as Acceptors in Relation to the Assembly of 3′-Sialyl-6′-sulfo
Lewis x (the L-Selectin Ligand and Related Complex Structures"; 1996, Biochemistry,
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Methods Enzymol , vol. 28, pp. 454-461.
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polylactosamines containing either a sialyl Lewis x,a VIM-2, or a sialylated and
Internally difucosylated sequence"; 1998, Carbohydrate Research, vol. 305,
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That is Involved in Biosynthesis of the Sialyl Lewis x Carbohydrate Determinants
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|
Primary Examiner: Slobodyansky; Elizabeth
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional application of and claims the benefit of U.S. patent application
ser. No. 09/231,905, now U.S. Pat. No. 6,500,661 filed Jan. 14, 1999, which claims
the benefit of U.S. Provisional Application No. 60/071,076, filed Jan. 15, 1998,
which application is incorporated herein by reference for all purposes.
Claims
What is claimed is:
1. A method for enzymatic conversion of GDP-mannose to GDP-fucose, the method comprising:
a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase,
and NADP+;
b) incubating the reaction mixture for a sufficient time to convert at least
90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
c) adding to the reaction mixture a polypeptide comprising an amino acid sequence
with at least 95% identity to SEQ ID NO:1, wherein the polypeptide has GDP-4-keto-6-deoxymannose
3,5-epimerase and GDP-4-keto-6-galactose reductase activities; and
d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose
to GDP-fucose.
2. The method of claim 1, which method further comprises recycling NADP
+
or NAD
+ produced by the reductase activity to NADPH or NADH, respectively,
by including in the reaction mixture of step c) an enzyme that can reduce the NADP
+
or NAD
+, and a substrate for the enzyme.
3. The method of claim 2, wherein the enzyme is selected from the group consisting
of alcohol dehydrogenase, glucose dehydrogenase, formate dehydrogenase, hydrogenase,
and glucose-6-phosphate dehydrogenase.
4. The method of claim 3, wherein the enzyme is glucose dehydrogenase and the
substrate is glucose.
5. The method of claim 1, wherein the method further comprises an enzymatic system
for generating the GDP-mannose from mannose.
6. The method of claim 5, wherein the enzymatic system for generating the GDP-mannose
from mannose comprises:
hexokinase, which converts mannose to mannose-6-phosphate;
phosphomannomutase, which converts the mannose-6-phosphate to mannose-1-phosphate; and
GDP-mannose pyrophosphorylase, which converts the mannose-1-phosphate to GDP-mannose.
7. The method of claim 1, wherein the amino acid sequence is SEQ ID NO: 1.
8. The method of claim 1, wherein the method further comprises adding sufficient
divalent metal cation to said reaction mixture to restore a portion of said divalent
cation lost during the course of the reaction to thereby achieve or maintain a
concentration of said divalent metal cation in said reaction mixture between about
1 mM and about 75 mM, and wherein the addition of divalent metal cation occurs
without interruption of said enzymatic conversion.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the synthesis of oligosaccharides. In particular,
it relates to improved enzymatic synthesis of GDP-fucose, which can be used in
fucosylation reactions. The methods make possible the synthesis of complex fucosylated
oligosaccharides in a single vessel using readily available starting materials.
2. Background
Increased understanding of the role of carbohydrates as recognition elements
on the surface of cells has led to increased interest in the production of carbohydrate
molecules of defined structure. For instance, compounds comprising the sialyl Lewis
ligands, sialyl Lewis
x and sialyl Lewis
a are present in leukocyte
and non-leukocyte cell lines that bind to receptors such as the ELAM-1 and GMP
140 receptors. Polley et al.,
Proc. Natl. Acad Sci., USA, 88: 6224 (1991)
and Phillips et al.,
Science, 250: 1130 (1990), see, also, U.S. Ser. No. 08/063,181.
Because of interest in making desired carbohydrate structures, glycosyltransferases
and their role in enzyme-catalyzed synthesis of carbohydrates are presently being
extensively studied. These enzymes exhibit high specificity and are useful in forming
carbohydrate structures of defined sequence. Consequently, glycosyltransferases
are increasingly used as enzymatic catalysts in synthesis of a number of carbohydrates
used for therapeutic and other purposes.
In the application of enzymes to the field of synthetic carbohydrate chemistry,
the use of glycosyltransferases for enzymatic synthesis of carbohydrate offers
advantages over chemical methods due to the virtually complete stereoselectivity
and linkage specificity offered by the enzymes (Ito et al.,
Pure Appl. Chem.,
65:753 (1993); and U.S. Pat. Nos. 5,352,670, and 5,374,541). However, the commercial-scale
production of carbohydrate compounds is often complicated by the cost and difficulty
in obtaining reactants that are used in the enzymatic and chemical synthesis of
the carbohydrates.
Improved methods for enzymatic synthesis of carbohydrate compounds, and
precursors used in these syntheses, would advance the production of a number of
beneficial compounds. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods, expression vectors, and reaction mixtures
that are useful for the efficient production of fucosylated oligosaccharides. The
invention provides ways by which nucleotide sugars such as GDP-fucose can be formed
relatively inexpensively.
In a first embodiment, the invention provides expression vectors that include
a promoter operably linked to a nucleic acid that encodes a prokaryotic enzyme
that has both an epimerase and a reductase activity. These two activities catalyze
the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose.
In another embodiment, the invention provides a reaction mixture for synthesizing
GDP-fucose. The reaction mixture includes GDP-4-keto-6-deoxymannose, NADPH, and
a prokaryotic enzyme that has both an epimerase and a reductase activity. The prokaryotic
enzyme can catalyze the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose.
In a presently preferred embodiment, the GDP-4-keto-6-deoxymannose is formed by:
a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose-4,6-dehydratase,
and NADP
+; and b) incubating the reaction mixture for a sufficient time
to convert at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose.
Another embodiment of the invention provides methods for the enzymatic conversion
of GDP-mannose to GDP-fucose. These methods involve:
a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose 4,6-dehydratase,
and NADP
+;
b) incubating the reaction mixture for a sufficient time to convert at least
about
90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
c) adding to the reaction mixture one or more polypeptides having GDP-4-keto-6-deoxymannose
3,5-epimerase and GDP-4-keto-6-galactose reductase activities; and
d) incubating the reaction mixture for a sufficient time to convert the GDP-4-keto-6-deoxymannose
to GDP-fucose.
Also provided are methods for enzymatic synthesis of a fucosylated oligosaccharide.
These methods involve transferring a fucose from the GDP-fucose produced by the
methods of the invention to an acceptor saccharide. This can be accomplished by
the following additional steps: e) adding a fucosyltransferase and the acceptor
saccharide to the GDP-4-keto-6-deoxymannose produced in step b) or to the GDP-fucose
produced in step d); and f) incubating a reaction mixture for a sufficient time
to transfer the fucose from the GDP-fucose to the acceptor saccharide.
Additional embodiments provide methods by which one can generate GDP-fucose
starting from mannose. These methods involve the use of an enzymatic system for
converting mannose into GDP-mannose, which is then converted to GDP-fucose using
the above methods. The conversion of mannose to GDP-mannose involves the following
enzymes: hexokinase, which converts mannose to mannose-6-phosphate; phosphomannomutase,
which converts the mannose-6-phosphate to mannose-1-phosphate; and GDP-mannose
pyrophosphorylase, which converts the mannose-1-phosphate to GDP-mannose.
Also provided by the invention are methods for the synthesis of a fucosylated
oligosaccharide in which efficiency-enhancing steps are used. The methods involve
contacting an acceptor saccharide with a fucosylation reaction mixture that comprises
GDP-fucose and a fucosyltransferase which transfers fucose from the GDP-fucose
to provide said fucosylated oligosaccharide, wherein the efficiency of said fucosylation
is enhanced by one or more efficiency-enhancing steps selected from the group consisting of:
- 1) forming said GDP-fucose by enzymatic conversion of GDP-mannose to
GDP-fucose by:
- a) providing a reaction mixture that comprises GDP-mannose, GDP-mannose
4,6-dehydratase, and NADP+;
- b) incubating the reaction mixture for a sufficient time to convert
at least about 90% of the GDP-mannose to GDP-4-keto-6-deoxymannose;
- c) adding to the product of step b) one or more polypeptides having
GDP-4-keto-6-deoxymannose 3,5-epimerase and GDP-4-keto-6-galactose reductase activities; and
- d) incubating the reaction mixture for a sufficient time to convert
the GDP-4-keto-6-deoxymannose to GDP-fucose;
- 2) adding pyruvate kinase and a substrate for the pyruvate kinase to
the fucosylation reaction mixture, wherein GDP produced as a result of the transfer
of fucose from the GDP-fucose is converted to GTP; and
- 3) conducting the fucosylation in a reaction medium that comprises a
soluble divalent metal cation, wherein said medium is supplemented with said soluble
divalent metal cation to maintain the concentration of said divalent metal cation
between about 2 mM and about 75 mM.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic diagram of the enzymatic conversion of GDP-mannose
to GDP-fucose.
FIG. 2 shows an 8-15% SDS-polyacrylamide gel analysis of extracts from bacterial
cells that harbor expression plasmids for enzymes involved in synthesis of GDP-fucose.
Molecular weight standards are in the two outside lanes. Extracts are from cells
containing the expression vectors pTGK, pTGK:YEF B, pTGK:wcaH, and pTGK:GMD. SDS-PAGE
under reducing conditions. Lane 1, pTGK lysate; Lane 2, pTGK:YEF B1 lysate; Lane
3, pTGK:YEF B2 lysate; Lane 4, pTGK:GMD lysate.
FIG. 3 shows HPLC profiles of the reaction products obtained using the GDP-mannose
dehydratase expressed from the expression vector pTGK:GMD.
FIG. 4 shows the results of an assay designed to test whether the activity of
GMD is linear over time.
FIG. 5 shows that GDP-fucose is a potent inhibitor of GMD.
FIG. 6 shows that wcaH and human Fx are expressed by the corresponding expression
vectors in
E. coli. The analysis was performed using 8-15% SDS-PAGE under
reducing conditions.
FIG. 7A shows the results of an assay for GDP-mannose dehydratase activity as
the first step of a coupled assay for determining the activity of YEF B. FIG. 7
shows the effect of adding YEF B to the reaction mixture after completion of the
GDP-mannose dehydratase reaction.
FIG. 8 shows the results of a time course of the coupled GMD-YEF B assay.
FIG. 9 shows the results of a large-scale conversion of GDP-mannose to GDP-fucose
using GMD and YEF B.
FIG. 10 shows the formation of sialyl Lewis X (as indicated by increasing GDP)
over time. The decrease in pH of the reaction mixture that occurs as a result of
the reaction is also shown.
FIG. 11 shows the change in concentration of Mg
2+ over time during
the synthesis of sialyl Lewis X.
FIG. 12 shows a time course of a large-scale sialyl Lewis X synthesis reaction.
Samples were taken from the reaction mixture and analyzed by TLC. From left to
right, the lanes are as follows: Lane 1, reaction at 160 hours; Lane 2, 147 hours;
Lane 3, SLN; Lane 4, 43 hours; Lane 5, 1 hour.
FIG. 13 shows an NMR analysis of the final product obtained in the large-scale
fucosylation reaction that used Neu5Acα2,3Galβ1,4GlcNAcβ1,3Gal-OR
as a substrate.
FIG. 14 shows a schematic diagram of a GDP-mannose half-cycle fucosyltransferase reaction.
FIG. 15 shows a TLC analysis of the products of a GDP-mannose fucosyltransferase
half-cycle reaction. From left to right: Lane 1, SLN; Lane 2, reaction at 2 days;
Lane 3, sialyl Lewis X.
FIG. 16 shows a schematic diagram of a mannose fucosyltransferase full-cycle reaction.
DETAILED DESCRIPTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. Singleton et al (1994)
Dictionary of Microbiology
and Molecular Biology, second edition, John Wiley and Sons (New York) provides
one of skill with a general dictionary of many of the terms used in this invention.
Although any methods and materials similar or equivalent to those described herein
can be used in the practice or testing of the present invention, the preferred
methods and materials are described. For purposes of the present invention, the
following terms are defined below.
The following abbreviations for carbohydrate compounds are used herein:
| |
|
| |
Ara = |
arabinosyl; |
| |
Fru = |
fructosyl; |
| |
Fuc = |
fucosyl; |
| |
Gal = |
galactosyl; |
| |
GalNAc = |
N-acetylgalactosaminyl; |
| |
Glc = |
glucosyl; |
| |
GlcNAc = |
N-acetylglucosaminyl; |
| |
Man = |
mannosyl; and |
| |
Sia (NeuAc) = |
sialyl (typically N-acetylneuraminyl). |
| |
|
Oligosaccharides are considered to have a reducing end and a non-reducing
end, whether or not the saccharide at the reducing end is in fact a reducing sugar.
In accordance with accepted nomenclature, oligosaccharides are depicted herein
with the non-reducing end on the left and the reducing end on the right. All oligosaccharides
described herein are described with the name or abbreviation for the non-reducing
saccharide (e.g., Gal), followed by the configuration of the glycosidic bond (α
or β), the ring bond, the ring position of the reducing saccharide involved
in the bond, and then the name or abbreviation of the reducing saccharide (e.g.,
GlcNAc). The linkage between two sugars may be expressed, for example, as 2,3,
2→3, or (2,3). Each saccharide is a pyranose.
The term "sialic acid" refers to any member of a family of nine-carbon carboxylated
sugars. The most common member of the sialic acid family is N-acetyl-neuraminic
acid (2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid
(often abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is
N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc
is hydroxylated. A third sialic acid family member is 2-keto-3-deoxy-nonulosonic
acid (KDN) (Nadano et al. (1986)
J. Biol. Chem. 261: 11550-11557; Kanamori
et al. (1990)
J. Biol. Chem. 265: 21811-21819. Also included are 9-substituted
sialic acids such as a 9-O—C
1-C
6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac
or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review
of the sialic acid family, see, e.g., Varki (1992)
Glycobiology 2: 25-40;
Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag,
New York (1992)). The synthesis and use of sialic acid compounds in a sialylation
procedure is disclosed in international application WO 92/16640, published Oct.
1, 1992.
The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer
in either single- or double-stranded form, and unless otherwise limited, encompasses
known analogues of natural nucleotides that hybridize to nucleic acids in manner
similar to naturally occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence includes the complementary sequence thereof.
The term "operably linked" refers to functional linkage between a nucleic acid
expression control sequence (such as a promoter, signal sequence, or array of transcription
factor binding sites) and a second nucleic acid sequence, wherein the expression
control sequence affects transcription and/or translation of the nucleic acid corresponding
to the second sequence.
The term "recombinant" when used with reference to a cell indicates that the
cell replicates a heterologous nucleic acid, or expresses a peptide or protein
encoded by a heterologous nucleic acid. Recombinant cells can contain genes that
are not found within the native (non-recombinant) form of the cell. Recombinant
cells can also contain genes found in the native form of the cell wherein the genes
are modified and re-introduced into the cell by artificial means. The term also
encompasses cells that contain a nucleic acid endogenous to the cell that has been
modified without removing the nucleic acid from the cell; such modifications include
those obtained by gene replacement, site-specific mutation, and related techniques.
A "recombinant polypeptide" is one which has been produced by a recombinant cell.
A "recombinant expression cassette" or simply an "expression cassette" is a nucleic
acid construct, generated recombinantly or synthetically, with nucleic acid elements
that are capable of effecting expression of a structural gene in hosts compatible
with such control elements. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the recombinant expression
cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding
a desired polypeptide), and a promoter (e.g., a dual promoter that contains a tac
promoter component and a gal promoter component as described in PCT/US97/20528;
Int'l. Publ. No. WO 9820111) that is operably linked to the nucleic acid. Additional
factors necessary or helpful in effecting expression can also be used as described
herein. For example, an expression cassette can also include nucleotide sequences
that encode a signal sequence that directs secretion of an expressed protein from
the host cell. Transcription termination signals, enhancers, and other nucleic
acid sequences that influence gene expression, can also be included in an expression cassette.
A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is
one that originates from a source foreign to the particular host cell (e.g., from
a different species), or, if from the same source, is modified from its original
form. Thus, a heterologous nucleic acid operably linked to a promoter is from a
source different from that from which the promoter was derived, or, if from the
same source, is modified from its original form. For example, a UDPglucose 4-epimerase
gene promoter can be linked to a structural gene encoding a polypeptide other than
native UDPglucose 4-epimerase. A heterologous gene that encodes an enzyme involved
in conversion of GDP-mannose to GDP-fucose, for example, in a prokaryotic host
cell includes a gene that is endogenous to the particular host cell that has been
modified. Modification of the heterologous nucleic acid can occur, e.g., by treating
the DNA with a restriction enzyme to generate a DNA fragment that is capable of
being operably linked to the promoter. Techniques such as site-directed mutagenesis
are also useful for modifying a heterologous nucleic acid.
A "subsequence" refers to a sequence of nucleic acids or amino acids that comprise
a part of a longer sequence of nucleic acids or amino-acids (e.g., polypeptide) respectively.
The term "isolated" is meant to refer to material which is substantially or essentially
free from components which normally accompany the nucleic acid, polypeptide, or
other molecule as found in its native state. Typically, isolated molecules are
at least about 80% pure, usually at least about 90%, and preferably at least about
95% pure as measured by, e.g., band intensity on a silver stained gel or other
method for determining purity. Protein purity or homogeneity can be indicated by
a number of means well known in the art, such as polyacrylamide gel electrophoresis
of a protein sample, followed by visualization upon staining. For certain purposes
high resolution will be needed and HPLC or a similar means for purification utilized.
The terms "identical" or percent "identity," in the context of two or more nucleic
acids or polypeptide sequences, refer to two or more sequences or subsequences
that are the same or have a specified percentage of amino acid residues or nucleotides
that are the same, when compared and aligned for maximum correspondence, as measured
using one of the following sequence comparison algorithms or by visual inspection.
The phrase "substantially identical," in the context of two nucleic acids or
polypeptides, refers to two or more sequences or subsequences that have at least
60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity,
when compared and aligned for maximum correspondence, as measured using one of
the following sequence comparison algorithms or by visual inspection. Preferably,
the substantial identity exists over a region of the sequences that is at least
about 50 residues in length, more preferably over a region of at least about 100
residues, and most preferably the sequences are substantially identical over at
least about 150 residues. In a most preferred embodiment, the sequences are substantially
identical over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence,
to which test sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence coordinates
are designated, if necessary, and sequence algorithm program parameters are designated.
The sequence comparison algorithm then calculates the percent sequence identity
for the test sequence(s) relative to the reference sequence, based on the designated
program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman,
Adv. Appl. Math 2:482
(1981), by the homology alignment algorithm of Needleman & Wunsch,
J. Mol. Biol.
48:443 (1970), by the search for similarity method of Pearson & Lipman,
Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally,
Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence
identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which
are described in Altschul et al. (1990)
J. Mol. Biol. 215: 403-410 and Altschuel
et al. (1997)
Nucleic Acids Res. 25: 3389-3402, respectively. Software for
performing BLAST analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying short words
of length W in the query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a database sequence.
T is referred to as the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits act as seeds for initiating searches to find
longer HSPs containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences, the parameters
M (reward score for a pair of matching residues; always>0) and N (penalty
score for mismatching residues; always<0). For amino acid sequences, a scoring
matrix is used to calculate the cumulative score. Extension of the word hits in
each direction are halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score goes to zero or
below, due to the accumulation of one or more negative-scoring residue alignments;
or the end of either sequence is reached. The BLAST algorithm parameters W, T,
and X determine the sensitivity and speed of the alignment. The BLASTN program
(for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of
10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,
Proc. Natl. Acad.
Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also
performs a statistical analysis of the similarity between two sequences (see, e.g.,
Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a match between
two nucleotide or amino acid sequences would occur by chance. For example, a nucleic
acid is considered similar to a reference sequence if the smallest sum probability
in a comparison of the test nucleic acid to the reference nucleic acid is less
than about 0.1, more preferably less than about 0.01, and most preferably less
than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially
identical is that the polypeptide encoded by the first nucleic acid is immunologically
cross reactive with the polypeptide encoded by the second nucleic acid, as described
below. Thus, a polypeptide is typically substantially identical to a second polypeptide,
for example, where the two peptides differ only by conservative substitutions.
Another indication that two nucleic acid sequences are substantially identical
is that the two molecules hybridize to each other under stringent conditions, as
described below.
The phrase "hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence under stringent
conditions when that sequence is present in a complex mixture (e.g., total cellular)
DNA or RNA.
The term "stringent conditions" refers to conditions under which a probe will
hybridize to its target subsequence, but to no other sequences. Stringent conditions
are sequence-dependent and will be different in different circumstances. Longer
sequences hybridize specifically at higher temperatures. Generally, stringent conditions
are selected to be about 5° C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength and pH. The Tm is the temperature
(under defined ionic strength, pH, and nucleic acid concentration) at which 50%
of the probes complementary to the target sequence hybridize to the target sequence
at equilibrium. (As the target sequences are generally present in excess, at Tm,
50% of the probes are occupied at equilibrium). Typically, stringent conditions
will be those in which the salt concentration is less than about 1.0 M Na ion,
typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30° C. for short probes (e.g., 10
to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater
than 50 nucleotides). Stringent conditions may also be achieved with the addition
of destabilizing agents such as formamide.
The phrases "specifically binds to a protein" or "specifically immunoreactive
with", when referring to an antibody refers to a binding reaction which is determinative
of the presence of the protein in the presence of a heterogeneous population of
proteins and other biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind preferentially to a particular protein and do not bind
in a significant amount to other proteins present in the sample. Specific binding
to a protein under such conditions requires an antibody that is selected for its
specificity for a particular protein. A variety of immunoassay formats may be used
to select antibodies specifically immunoreactive with a particular protein. For
example, solid-phase ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York,
for a description of immunoassay formats and conditions that can be used to determine
specific immunoreactivity.
"Conservatively modified variations" of a particular polynucleotide
sequence refers to those polynucleotides that encode identical or essentially identical
amino acid sequences, or where the polynucleotide does not encode an amino acid
sequence, to essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic acids encode any
given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all
encode the amino acid arginine. Thus, at every position where an arginine is specified
by a codon, the codon can be altered to any of the corresponding codons described
without altering the encoded polypeptide. Such nucleic acid variations are "silent
variations," which are one species of "conservatively modified variations." Every
polynucleotide sequence described herein which encodes a polypeptide also describes
every possible silent variation, except where otherwise noted. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is ordinarily the
only codon for methionine) can be modified to yield a functionally identical molecule
by standard techniques. Accordingly, each "silent variation" of a nucleic acid
which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a small
percentage of amino acids (typically less than 5%, more typically less than 1%)
in an encoded sequence are "conservatively modified variations" where the alterations
result in the substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar amino acids are
well known in the art.
One of skill will appreciate that many conservative variations of the GDP-fucose-synthesizing
enzymes and nucleic acid that encode the enzymes yield essentially identical products.
For example, due to the degeneracy of the genetic code, "silent substitutions"
(i e., substitutions of a nucleic acid sequence which do not result in an alteration
in an encoded polypeptide) are an implied feature of every nucleic acid sequence
which encodes an amino acid. As described herein, sequences are preferably optimized
for expression in a particular host cell used to produce the enzyme (e.g., yeast,
human, and the like). Similarly, "conservative amino acid substitutions," in one
or a few amino acids in an amino acid sequence are substituted with different amino
acids with highly similar properties (see, the definitions section, supra), are
also readily identified as being highly similar to a particular amino acid sequence,
or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively
substituted variations of any particular sequence are a feature of the present
invention. See also, Creighton (1984)
Proteins, W.H. Freeman and Company.
In addition, individual substitutions, deletions or additions which alter, add
or delete a single amino acid or a small percentage of amino acids in an encoded
sequence are also "conservatively modified variations".
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides methods for efficient production of substrates
such as nucleotide sugars (e.g., GDP-fucose) that are useful in synthesis of fucosylated
carbohydrate compounds. Expression vectors for producing enzymes that are used
in these methods are also provided. Methods for using these enzymes and methods
for producing nucleotide sugars to produce fucosylated oligosaccharides are also
provided by the invention.
The methods of the invention provide significant advantages over previously available
methods for fucosylation. For example, the methods provide a relatively inexpensive
way to make GDP-fucose, an expensive compound, starting from the lower-cost GDP-mannose,
or even from mannose. The invention provides methods by which the efficiency of
the GDP-fucose synthesis, and also the efficiency of subsequent fucosyltransferase
reactions, can be improved. The methods of the invention provide a high yield of
the desired fucosylated compound. Accordingly, the methods of the invention are
well suited for commercial-scale production of fucosylated compounds, including
those that are useful for diagnostic and therapeutic uses, foodstuffs, and the like.
A. Cloning and Expression of Nucleic Acids Encoding Enzymes Useful for Conversion
of GDP-mannose to GDP-fucose
The invention provides, in a first embodiment, methods of producing enzymes that
are useful for converting GDP-mannose to GDP-fucose. This biosynthetic pathway,
which is diagrammed in FIG. 1, involves three enzymatic activities. The first enzyme,
GDP-mannose dehydratase catalyzes the conversion of GDP-mannose to GDP-4-keto-6-D-deoxymannose.
This product is then epimerized to GDP-4-keto-6-L-deoxygalactose, which is in turn
reduced to GDP-L-fucose by a 4′ reductase. The latter two enzymatic activities
(epimerase and reductase) are both found in the human Fx protein (Tonetti et al.
(1996)
J. Biol. Chem. 271: 27274-27279; GenBank Accession No. U58766).
For use in commercial-scale enzymatic synthetic reactions, however, it is preferred
to use enzymes that are readily produced in prokaryotes, which are much easier
and more efficient to grow at large scale than mammalian cells. Mammalian enzymes
are often not expressed in the proper form at high yields when genes for the mammalian
enzymes are inserted into prokaryotic host cells. Thus, it was of great interest
to obtain a prokaryotic enzyme or enzymes to catalyze the epimerization and reduction.
However, prior to the instant invention, it was not known whether bacterial systems
for GDP-fucose synthesis required one or two separate polypeptides to catalyze
the epimerization and reduction of GDP-4-keto-6-D-deoxymannose to GDP-fucose. The
present invention provides this missing information, demonstrating that one enzyme
catalyzes both of these activities. In
E. coli, this enzyme is designated
YEF B. The need to produce only one enzyme to catalyze two activities simplifies
the development and scale-up of GDP-fucose production.
Accordingly, the present invention provides methods and vectors for
recombinant production of enzymes that are useful for producing GDP-fucose. Recombinant
production of a polypeptide generally involves obtaining a DNA sequence that encodes
the particular enzyme, modified as desired, placing the DNA in an expression cassette
under the control of a particular promoter, expressing the protein in a host, isolating
the expressed protein and, if required, renaturing the protein. More than one of
the enzymes can be expressed in the same host cells, either on the same expression
vector or on more than one expression vector that is present in the cells.
In one embodiment, the invention provides expression vectors that are useful
in
methods for producing enzymes involved in GDP-fucose synthesis in a host cell.
For example, expression vectors are provided that are useful for expressing YEF
B of
E. coli, which catalyzes the both the epimerization and reduction of
GDP-4-keto-6-deoxymannose to obtain GDP-fucose. The expression vectors can also
express related enzymes, in particular those from other prokaryotes, that also
have the dual epimerase/reductase activity.
1. Epimerase/Reductase-Encoding Nucleic Acids
The expression vectors of the invention include a nucleic acid that encodes an
enzyme that has both GDP-4-keto-6-D-deoxymannose epimerase activity and GDP-4-keto-6-L-deoxygalactose
reductase activity. A prokaryotic enzyme is encoded by the nucleic acid in presently
preferred embodiments. For example, one can use a nucleic acid that encodes a prokaryotic
epimerase/reductase from any prokaryotic species, including
E. coli. The
enzyme can be, for example, substantially identical to an
E. coli YEF B
polypeptide. In some embodiments, the expression vectors include a nucleic acid
that encodes an epimerase/reductase enzyme that has an amino acid sequence as shown
in SEQ ID NO:1. The nucleic acids can also encode polypeptides that have conservative
amino acid substitutions compared to the amino acid sequence of a native epimerase/reductase
enzyme, such as the
E. coli YEF B enzyme. Typically, the nucleic acids used
in the expression vectors of the invention are at least about 75% identical to
the nucleic acid sequence of the
E. coli YEF B coding region as shown in
GenBank Accession No. U38473 (nucleotides 9783-10748). More preferably, the nucleic
acids used in the expression vectors are at least about 85% identical to the
E.
coli YEF B coding region, and still more preferably are at least about 95%
identical. Typically, a computerized algorithm such as BLAST is used for the comparison,
preferably using default parameters. These percentage identities can be an overall
value for the entire coding regions, or can refer to the percentage identity over
a particular region of the coding regions. For example, in a presently preferred
embodiment, the nucleic acids used in the expression vectors of the invention are
at least about 85% identical over a region of at least 40 nucleotides in length,
using a pairwise BLAST algorithm (BLASTN 2.0.6 as implemented by the National Center
for Biotechnology Information) with the following parameters: Match: 1; Mismatch:
-2, Gap open: 5; Gap extension: 2, x-dropoff: 50; expect: 10.00; wordsize: 11;
with no filtering.
The nucleic acids that encode the epimerase/reductase enzyme can be obtained
using methods that are known to those of skill in the art. Suitable nucleic acids
(e.g., cDNA, genomic, or subsequences (probes)) can be cloned, or amplified by
in vitro methods such as the polymerase chain reaction (PCR), the ligase chain
reaction (LCR), the transcription-based amplification system (TAS), the self-sustained
sequence replication system (SSR). A wide variety of cloning and in vitro amplification
methodologies are well-known to persons of skill. Examples of these techniques
and instructions sufficient to direct persons of skill through many cloning exercises
are found in Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods
in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook
et al. (1989)
Molecular Cloning—A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et
al);
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing Associates, Inc. and
John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion et al., U.S. Pat.
No. 5,017,478; and Carr, European Patent No. 0,246,864. Examples of techniques
sufficient to direct persons of skill through in vitro amplification methods are
found in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat.
No. 4,683,202;
PCR Protocols A Guide to Methods and Applications (Innis
et al., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson
(Oct. 1, 1990)
C&
EN 36-47;
The Journal Of NIH Research (1991)
3: 81-94; (Kwoh et al. (1989)
Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli
et al. (1990)
Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989)
J. Clin. Chem., 35: 1826; Landegren et al., (1988)
Science 241: 1077-1080;
Van Brunt (1990)
Biotechnology 8: 291-294; Wu and Wallace (1989)
Gene
4: 560; and Barringer et al. (1990)
Gene 89: 117. Improved methods of
cloning in vitro amplified nucleic acids are described in Wallace et al., U.S.
Pat. No. 5,426,039.
Nucleic acids that encode enzymes having both epimerase and reductase activities,
or subsequences of these nucleic acids, can be prepared by any suitable method
as described above, including, for example, cloning and restriction of appropriate
sequences. As an example, one can obtain a nucleic acid that encodes a polypeptide
that has both epimerase and reductase activities by routine cloning methods. A
nucleotide sequence of a gene that encodes an enzyme known to have both activities,
such as a YEF B enzyme, can be used to provide probes that specifically hybridize
to a gene that encodes a suitable enzyme in a genomic DNA sample, or to a mRNA
in a total RNA sample (e.g., in a Southern or Northern blot). Suitable sequences
are provided in, for example, GenBank or other sequence database.
One suitable nucleotide sequence for use as a probe or in an expression vector
of the invention is found in an
E. coli gene cluster that encodes GDP-fucose-synthesizing
enzymes as described by Stevenson et al. (1996)
J. Bacteriol. 178: 4885-4893
(GenBank Accession No. U38473). This gene cluster had been reported to include
an open reading frame for GDP-mannose dehydratase (nucleotides 8659-9780 in GenBank
Accession No. U38473). Applicants discovered that this gene cluster also contains
an open reading frame that encodes an enzyme that has both 3,5 epimerization and
4-reductase activities (FIG. 1), and thus is capable of converting the product
of the GDP-mannose dehydratase reaction (GDP-4-keto-6-deoxymannose) to GDP-fucose.
This ORF, which is designated YEF B, wcaG, and fcl, is found at nucleotides 9783-10748
of GenBank Accession No. U38473. Prior to Applicants' discovery that YEF B encodes
an enzyme having two activities, it was not known whether one or two enzymes were
required for conversion of GDP-4-keto-6-deoxymannose to GDP-fucose by prokaryotes.
The gene cluster from
E. coli includes an additional ORF, which is designated
wcaH (nucleotides 10748-11230 of GenBank Accession No. U38473). This small open
reading frame, which encodes a GDP-mannose mannosyl hydrolase of 15 kd, is located
just downstream of the YEF B coding region (designated wcaG). Each of these enzymes
was expressed in bacteria as assessed by SDS-PAGE and ability to form GDP-fucose
from GDP-mannose.
Once the target epimerase/reductase nucleic acid is identified, it can be isolated
according to standard methods known to those of skill in the art (see, e.g., Sambrook
et al. (1989)
Molecular Cloning: A Laboratory Manual, 2
nd Ed., Vols.
1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987)
Methods in
Enzymology, Vol. 152:
Guide to Molecular Cloning Techniques, San Diego:
Academic Press, Inc.; or Ausubel et al. (1987)
Current Protocols in Molecular
Biology, Greene Publishing and Wiley-Interscience, New York).
A nucleic acid that encodes a prokaryotic epimerase/reductase can also be cloned
by detecting its expressed product by means of assays based on the physical, chemical,
or immunological properties. For example, one can identify a cloned epimerase/reductase-encoding
nucleic acid by the ability of a polypeptide encoded by the nucleic acid to catalyze
the conversion of GDP-4-keto-6-deoxymannose to GDP-fucose. In a preferred method,
reverse phase HPLC is used to determine the amounts of GDP-mannose, GDP-fucose,
and optionally one or more intermediates (e.g., GDP-4-keto-6-deoxymannose and GDP-4-keto-6-deoxygalactose)
at various times of reaction. Suitable assay conditions are described in the Examples.
In one embodiment, epimerase/reductase-encoding nucleic acids can be cloned using
DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example,
the nucleic acid sequence or subsequence is PCR amplified, using a sense primer
containing one restriction site (e.g., XbaI) and an antisense primer containing
another restriction site (e.g., HindIII). This will produce a nucleic acid encoding
the desired epimerase/reductase amino acid sequence or subsequence and having terminal
restriction sites. This nucleic acid can then be easily ligated into a vector containing
a nucleic acid encoding the second molecule and having the appropriate corresponding
restriction sites. Suitable PCR primers can be determined by one of skill in the
art using the sequence information provided in GenBank or other sources. Appropriate
restriction sites can also be added to the nucleic acid encoding the epimerase/reductase
or amino acid subsequence by site-directed mutagenesis. The plasmid containing
the epimerase/reductase-encoding nucleotide sequence or subsequence is cleaved
with the appropriate restriction endonuclease and then ligated into an appropriate
vector for amplification and/or expression according to standard methods.
Examples of suitable primers suitable for amplification of GDP-fucose-synthesizing
enzymes are shown in Table 1; each primer pair is designed to provide a 5′
XbaI restriction site and a 3′ HindIII site on the amplified fragment. The
plasmid containing the enzyme-encoding sequence or subsequence is cleaved with
the appropriate restriction endonuclease and then ligated into an appropriate vector
for amplification and/or expression according to standard methods.
| TABLE 1 |
|
| Enzyme |
5′Primer |
3′Primer |
|
| GDP-mannose dehydratase |
5′-CGCTCTAGATACATGT |
5′-GCGAAGCTTTTATGA |
|
| (E. coli) |
CAAAAGTCGCT-3′ |
CTCCAGCGCGAT-3′ |
| |
| YEF B (E. coli) |
5′-CGTCCTAGAGCGAT |
5′-GCGAAGCTTTTACCCCC |
| |
GAGTAAACAACGAGTT-3′ |
GAAAGCGGTC-3′ |
| |
| Wca H (E. coli) |
5′-GCTCTAGAGTAATGA |
5′-CCCAAGCTTTCATAAT |
| |
TGTTTTTACGTCAGG-3′ |
CCGGGTACTCCGGT-3′ |
| |
| Fx (human) |
5′-GCTCTAGAGACATG |
5′-ACGAAGCTTCACTTCC |
| |
GGTGAACCCCAGGGAT-3′ |
GGGCCTGCTCGTAGTTG-3′ |
|
As an alternative to cloning an epimerase/reductase-encoding nucleic acid, a
suitable
nucleic acid can be chemically synthesized from a known sequence that encodes a
YEF B polypeptide or a related enzyme that has both epimerase and reductase activities.
Direct chemical synthesis methods include, for example, the phosphotriester method
of Narang et al (1979)
Meth. Enzymol. 68: 90-99; the phosphodiester method
of Brown et al. (1979)
Meth. Enzymol 68: 109-151; the diethylphosphoramidite
method of Beaucage et al. (1981)
Tetra. Lett., 22: 1859-1862; and the solid
support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single
stranded oligonucleotide. This can be converted into double stranded DNA by hybridization
with a complementary sequence, or by polymerization with a DNA polymerase using
the single strand as a template. One of skill would recognize that while chemical
synthesis of DNA is often limited to sequences of about 100 bases, longer sequences
may be obtained by the ligation of shorter sequences. Alternatively, subsequences
may be cloned and the appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired DNA sequence.
A nucleic acid encoding a GDP-fucose-synthesizing enzyme can be identified by
detecting
its expressed product by means of assays based on the physical, chemical, or immunological
properties. For example, one can identify a cloned GDP-fucose-synthesizing nucleic
acid by the ability of a polypeptide encoded by the nucleic acid to catalyze the
conversion of GDP-mannose to GDP-fucose. Other physical properties of a polypeptide
expressed from a particular nucleic acid can be compared to properties of known
YEF B-like polypeptides to provide another method of identifying epimerase/reductase-encoding
nucleic acids. Alternatively, a putative epimerase/reductase gene can be mutated,
and its role as an epimerase/reductase established by detecting a variation in
the ability to produce GDP-fucose.
In some embodiments, it may be desirable to modify the epimerase/reductase-encoding
nucleic acids. One of skill will recognize many ways of generating alterations
in a given nucleic acid construct. Such well-known methods include site-directed
mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells
containing the nucleic acid to mutagenic agents or radiation, chemical synthesis
of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning
to generate large nucleic acids) and other well-known techniques. See, e.g., Giliman
and Smith (1979)
Gene 8:81-97, Roberts et al. (1987)
Nature 328: 731-734.
In a preferred embodiment, the recombinant nucleic acids present in the cells
of the invention are modified to provide preferred codons which enhance translation
of the nucleic acid in a selected organism (e.g., yeast preferred codons are substituted
into a coding nucleic acid for expression in yeast).
2. Expression Vectors and Methods for Expressing Enzymes Involve
