The invention provides isolated nucleic acid compounds encoding the glycosyltransferase protein GtfA of Amycolatopsis orientalis. Also provided are vectors carrying the gtfA gene, transformed heterologous host cells for expressing the GtfA protein, and methods for producing glycopeptide compounds using the cloned gtfA gene.

This application claims the benefit under Title 35, U.S. Code, .sctn.119(e) 
of U.S. provisional patent No. 60/026,069, filed Sep. 13, 1996. 
BACKGROUND OF THE INVENTION 
This invention relates to recombinant DNA technology. In particular the 
invention pertains to the cloning of glycosyltransferase gene gtfA from 
Amycolatopsis orientalis, the use of the cloned gene to express and purify 
the encoded enzyme, and a method of using the cloned enzyme for in vitro 
production of glycopeptide compounds. 
The use of antibiotic compounds has had a profound impact on the practice 
of medicine in the U.S. and around the world. Two highly effective 
antibiotic compounds of the glycopeptide class, vancomycin and 
teichoplanin, have been approved for use in humans. 
##STR1## 
The glycopeptide antibiotics comprise natural and semi-synthetic compounds 
of highly functionalized linear heptapeptides having a core structure 
composed of either seven modified or unusual aromatic amino acids, or a 
mix of aromatic and aliphatic amino acids. Natural glycopeptide compounds 
have been found in a variety of bacterial genera including Streptomyces, 
Actinoplanes, Nocardia, Amycolatopsis, Kibdelosporangia, and 
Pseudonocardia. M. Zmijewski and J. Fayerman. "Glycopeptide Antibiotics," 
In Genetics and Biochemistry of Antibiotic Production, Chap. 18. Ed. L. C. 
Vining and C. Studtard. Publ. Butterworth Heinemann, Boston (1995). 
Generally, glycopeptide compounds are differentiated by the placement of 
sugar substituents one the peptide core. In some instances differentiation 
arises from the positioning of fatty acid moieties on the sugar 
substituents. Research has shown that the sugar moieties attached to the 
core have an effect on the biological activity of glycopeptide molecules. 
At present, investigations into glycosylation of glycopeptides and 
glycopeptide cores are limited to preliminary observations on crude 
cellular extracts of bacterial strains that produce glycopeptide 
compounds. These experiments have demonstrated that the glycosylation 
reaction appears to involve one or more enzymatic activities which attach 
sugar residues onto a glycopeptide core. One study, for example, 
demonstrated a glycosylating activity in a crude cellular extract of a 
vancomycin-producing strain of Amycolatopsis orientalis. M. Zmijewski & B. 
Briggs. "Biosynthesis of vancomycin: identification of 
TDP-glucose:aglycosylvancomycin glucosyltransferase from Amycolatopsis 
orientalis" FEMS Microbiol. Lett. 59, 129-134 (1989). 
The glycosylation of glycopeptide compounds, intrinsically interesting from 
a scientific point of view, presents a number of practical considerations 
that warrant continued study of this subject. Recently, a number of 
glycopeptide resistant strains of pathogenic organisms have been 
encountered within the clinical environment. This trend toward diminished 
efficacy of glycopeptide compounds is alarming because of a similar 
phenomenon in the case of .beta.-lactam antibiotics. It is clear that the 
rise in antibiotic resistance has occurred by a plurality of molecular 
mechanisms and that resistant organisms possess a diverse repertoire for 
counteracting the otherwise lethal effect of antibiotic compounds. 
In light of the trend toward greater resistance, and in view of the absence 
of effective alternative treatments, there exists a pressing need to 
develop new antibiotic compounds. A useful strategy toward this end 
involves derivitizing presently available glycopeptide compounds by 
engineering in defined ways the placement and configuration of sugar 
moieties on the glycopeptide core structure. Achieving molecular 
rearrangements and substitutions on glycopeptide compounds by chemical 
means is difficult if not impossible in most cases. By contrast to 
chemical procedures, enzymatic methods, if available, would provide an 
effective means to engineer specific modifications onto the glycopeptide 
core. 
The challenge to provide an enzymatic means for modifying glycopeptide core 
molecules has been met by the present invention. Described herein are gtfA 
genes isolated from Amycolatopsis orientalis that encode 
glycosyltransferase enzyme GtfA, which adds epivancosamine onto 
glycopeptides of the vancomycin class. 
BRIEF SUMMARY 
The present invention is designed to meet the aforementioned need and 
provides, inter alia, the isolated gtfA gene and other nucleic acid 
molecules that encode the GtfA gene product from Amycolatopsis orientalis 
A82846. The invention also provides the GtfA protein product of the 
Amycolatopsis orientalis gtfA gene, in substantially purified form. 
Having the cloned gtfA gene of Amycolatopsis orientalis enables the 
production of recombinant GtfA protein from which glycopeptide compounds 
can be made in vitro. 
In one embodiment the present invention relates to an isolated DNA molecule 
encoding GtfA protein, said DNA molecule comprising the nucleotide 
sequence identified as SEQ ID NO. 1: 
ATGCGCGTGT TGATTACGGG GTGTGGATCG CGCGGAGATA CCGAACCGTT GGTGGCATTG 60 
GCGGCACGGT TGCGGGAACT CGGTGCGGAC GCGCGGATGT GCCTGCCGCC GGACTACGTG 120 
GAGCGGTGCG CCGAGGTCGG TGTGCCGATG GTGCCGGTCG GTCGGGCGGT GCGCGCAGGG 180 
GCACGCGAGC CGGGAGAACT GCCGCCGGGG GCGGCCGAAG TCGTGACCGA GGTGGTCGCC 240 
GAATGGTTCG ACAAGGTCCC GGCGGCCATC GAGGGGTGTG ACGCGGTGGT GACGACCGGC 300 
TTGCTGCCCG CCGCGGTCGC TGTCCGGTCG ATGGCCGAGA AGCTGGGCAT CCCGTACCGC 360 
TACACCGTGC TGTCTCCGGA CCATCTGCCG TCGGAGCAAA GCCAGGCGGA GCGGGACATG 420 
TACAACCAGG GCGCCGACAG GCTTTTCGGT GACGCGGTCA ACAGCCACCG GGCCTCGATC 430 
GGCCTGCCAC CGGTGGAGCA CCTCTACGAC TACGGCTACA CCGATCAGCC CTGGCTGGCG 540 
GCGGACCCGG TGCTGTCCCC GCTGCGGCCG ACGGACCTCG GCACTGTGCA GACCGGTGCG 600 
TGGATCCTGC CCGACGAACG GCCGCTTTCC GCGGAGCTGG AGGCGTTTCT GGCTGCCGGG 660 
TCGACGCCGG TGTACGTGGG TTTCGGCAGC TCGTCCCGAC CGGCAACCGC TGACGCCGCG 720 
AAGATGGCCA TCAAGGCGGT CCGTGCCAGT GGCCGCCGGA TCGTTCTCTC CCGCGGCTGG 780 
GCCGATTTGG TCCTGCCGGA CGACGGGGCC GACTGCTTCG TGGTCGGCGA AGTGAACCTT 840 
CAGGAGCTGT TCGGCCGGGT GGCCGCCGCC ATCCACCACG ACAGCGCGGG CACGACGCTG 900 
CTGGCCATGC GGGCGGGCAT CCCCCAGATC GTGGTGCGCC GCGTAGTGGA CAACGTGGTG 960 
GAGCAGGCGT ACCACGCCGA CCGGGTGGCC GAGCTGGGTG TCGGTGTGGC GGTCGACGGT 1020 
CCGGTCCCGA CCATCGACTC CTTGTCGGCC GCGCTCGACA CGGCTCTGGC CCCGGAGATC 1080 
CGTGCGCGAG CGACGACCGT GGCAGACACG ATTCGCGCCG ATGGGACAAC GGTGGCCGCG 1140 
CAGCTGCTGT TCGACGCGGT CAGCCTGGAA AAGCCGACTG TTCCCGCC 1188 
In another embodiment the present invention relates to a 
glycosyltransferase protein molecule, encoded by SEQ ID NO:1 wherein said 
glycosyltransferase protein molecule comprises the sequence identified as 
SEQ ID NO. 2. 
In a further embodiment the present invention relates to a ribonucleic acid 
molecule encoding GtfA protein, said ribonucleic acid molecule comprising 
the sequence identified as SEQ ID NO. 3: 
In yet another embodiment, the present invention relates to a recombinant 
DNA vector which incorporates the Amycolatopsis orientalis gtfA gene in 
operable linkage to gene expression sequences enabling the gtfA gene to be 
transcribed and translated in a host cell. 
In still another embodiment the present invention relates to homologous or 
heterologous host cells which have been transformed or transfected with 
the cloned gtfA gene of Amycolatopsis orientalis such that the gtfA gene 
is expressed in the host cell. 
In still another embodiment, the present invention relates to a method for 
producing glycopeptide compounds wherein recombinantly produced GtfA 
protein is utilized to add one or more sugar moieties onto a vancomycin 
glycopeptide in vitro. 
In a further embodiment the present invention relates to a composition 
comprising compound A82846B, said composition produced by the action of 
recombinant GtfA protein. 
DEFINITIONS 
"A82846B" refers to a glycopeptide produced by A. orientalis A82846 having 
the structure: 
##STR2## 
"AGV" denotes aglycosylvancomycin which comprises a vancomycin core having 
a free hydroxyl group on the B ring in place of the disaccharide moiety. 
"DVV" denotes desvancosaminyl vancomycin in which a glucose residue is 
attached onto AGV at the free hydroxyl position of the B ring. 
The terms "cleavage" or "restriction" of DNA refers to the catalytic 
cleavage of the DNA with a restriction enzyme that acts only at certain 
sequences in the DNA (viz. sequence-specific endonucleases). The various 
restriction enzymes used herein are commercially available and their 
reaction conditions, cofactors, and other requirements are used in the 
manner well known to one of ordinary skill in the art. Appropriate buffers 
and substrate amounts for particular restriction enzymes are specified by 
the manufacturer or can readily be found in the literature. 
The term "plasmid" refers to an extrachromosomal genetic element. The 
starting plasmids herein are either commercially available, publicly 
available on an unrestricted basis, or can be constructed from available 
plasmids in accordance with published procedures. In addition, equivalent 
plasmids to those described are known in the art and will be apparent to 
the ordinarily skilled artisan. 
"Recombinant DNA cloning vector" as used herein refers to any autonomously 
replicating agent, including, but not limited to, plasmids and phages, 
comprising a DNA molecule to which one or more additional DNA segments can 
or have been added. 
The term "recombinant DNA expression vector" as used herein refers to any 
recombinant DNA cloning vector, for example a plasmid or phage, in which a 
promoter and other regulatory elements are present to enable transcription 
of the inserted DNA. 
The term "vector" as used herein refers to a nucleic acid compound used for 
introducing exogenous DNA into host cells. A vector comprises a nucleotide 
sequence which may encode one or more protein molecules. Plasmids, 
cosmids, viruses, and bacteriophages, in the natural state or which have 
undergone recombinant engineering, are examples of commonly used vectors. 
The terms "complementary" or "complementarity" as used herein refers to the 
capacity of purine and pyrimidine nucleotides to associate through 
hydrogen bonding in double stranded nucleic acid molecules. The following 
base pairs are complementary: guanine and cytosine; adenine and thymine; 
and adenine and uracil. 
The term "glycopeptide" refers to a functionalized linear heptapeptide 
compound of natural or semi-synthetic origin, said compound having a core 
structure. 
"Glycopeptide core" or "core" or "core compound" interchangeably denote the 
progenitor structure of all glycopeptide compounds, comprising either 7 
modified or unusual aromatic amino acids, or a mix of aromatic and 
aliphatic amino acids. 
"Vancomycin glycopeptide" refers to any or all of the following: AGV, DVV, 
vancomycin. 
"Glycosylating substrate" refers to a compound which functions as a donor 
of a sugar moiety in an enzymatic glycosylation reaction, for example, 
uridine diphosphate-D-glucose. 
"Isolated nucleic acid compound" refers to any RNA or DNA sequence, however 
constructed or synthesized, which is locationally distinct from its 
natural location. 
A "primer" is a nucleic acid fragment which functions as an initiating 
substrate for enzymatic or synthetic elongation of, for example, a nucleic 
acid molecule. 
The term "promoter" refers to a DNA sequence which directs transcription of 
DNA to RNA. 
A "probe" as used herein is a labeled nucleic acid compound which 
hybridizes with another nucleic acid compound. 
The term "hybridization" as used herein refers to a process in which two or 
more strands of nucleic acid join through base pairing with complementary 
strands. "Selective hybridization" refers to hybridization under 
conditions of high stringency. The degree of hybridization between nucleic 
acid molecules varies with the degree of complementarity, the stringency 
of the hybridization conditions, and the length of the strands. 
The term "stringency" refers to a set of hybridization conditions, for 
example temperature and salt concentration, which may be varied to achieve 
"high stringency" or "low stringency" conditions, thereby varying the 
degree of hybridization of one nucleic acid molecule with another nucleic 
acid molecule. High stringency conditions disfavor non-homologous 
basepairing.

DETAILED DESCRIPTION 
The gtfA gene of Amycolatopsis orientalis encodes a glycosylating enzyme, 
GtfA. The enzyme is involved in glycosylating A82846B and will add 
epivancosamine onto a vancomycin glycopeptide compound in vitro. The 
enzyme will use TDP-epivancosamine or UDP-epivancosamine as a 
glycosylating substrate. 
The gtfA gene of Amycolatopsis orientalis comprises a DNA sequence of 1188 
nucleotide base pairs (SEQ ID NO. 1). There are no intervening sequences. 
Those skilled in the art will recognize that owing to the degeneracy of 
the genetic code (i.e. 64 codons which encode 20 amino acids), numerous 
"silent" substitutions of nucleotide base pairs could be introduced into 
the sequence identified as SEQ ID NO. 1 without altering the identity of 
the encoded amino acid(s) or protein product identified as SEQ ID NO:2. 
All such substitutions are intended to be within the scope of the 
invention. 
Gene Isolation Procedures 
Those skilled in the art will recogize that the gtfA gene may be obtained 
by a plurality of applicable techniques including, for example, polymerase 
chain reaction (PCR) amplification, or de novo DNA synthesis.(See e.g., J. 
Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)). 
Methods for constructing gene libraries in a suitable vector such as a 
plasmid or phage for propagation in procaryotic or eucaryotic cells are 
well known to those skilled in the art. See e.g. J. Sambrook et al. 
Supra!. Suitable cloning vectors are widely available. 
Skilled artisans will recognize that the gtfA gene of Amycolatopsis 
orientalis or fragment thereof could also be isolated by PCR amplification 
of Amycolatopsis orientalis genomic DNA using oligonucleotide primers 
targeted to any suitable region of SEQ ID NO. 1. Methods for PCR 
amplification are widely known in the art. See e.g. PCR Protocols: A Guide 
to Method and Application, Ed. M. Innis et al., Academic Press (1990), 
which hereby is incorporated by reference. The PCR amplification, which 
comprises genomic DNA, suitable enzymes, primers, and buffers, is 
conveniently carried out in a DNA THERMAL CYCLER.TM. (Perkin Elmer Cetus, 
Norwalk, Conn.). A positive PCR amplification is determined by detecting 
an appropriately-sized DNA fragment following agarose gel electrophoresis. 
Protein Production Methods 
One embodiment of the present invention relates to the substantially 
purified protein GtfA identified as SEQ ID NO:2 and encoded by the gtfA 
gene or functionally related proteins of Amycolatopsis orientalis. 
Skilled artisans will recognize that the proteins of the present invention 
can be synthesized or purified by any number of suitable methods. For 
example, the amino acid compounds of the invention can be made by chemical 
methods well known in the art, including solid phase peptide synthesis or 
recombinant methods. Both methods are described in U.S. Pat. No. 
4,617,149, incorporated herein by reference. 
The principles of solid phase chemical synthesis of polypeptides are well 
known in the art and are described in a number of general texts on the 
subject. See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981) 
Springer-Verlag, New York, 54-92. For example, peptides may be synthesized 
by solid-phase methodology using an Applied Biosystems 430A peptide 
synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles 
supplied by Applied Biosystems. Protected amino acids, such as 
t-butoxycarbonyl-protected amino acids, and other reagents are 
commercially available from many chemical supply houses. 
Sequential t-butoxycarbonyl chemistry using double- couple protocols are 
applied to the starting p-methyl benzhydryl amine resins for the 
production of C-terminal carboxamides. For the production of C-terminal 
acids, the corresponding pyridine-2-aldoxime methiodide resin is used. 
Asparagine, glutamine, and arginine are coupled using preformed hydroxy 
benzotriazole esters. Following completion of the synthesis the peptides 
may be deprotected and cleaved from the resin with anhydrous hydrogen 
fluoride containing 10% meta-cresol. Cleavage of the side chain protecting 
group(s) and of the peptide from the resin is carried out at zero degrees 
Celcius or below, preferably -20.degree. C. for thirty minutes followed by 
thirty minutes at 0.degree. C. 
The proteins of the present invention can also be produced by recombinant 
DNA methods using the cloned gtfA gene of Amycolatopsis orientalis. 
Recombinant methods are preferred if a high yield is desired. Expression 
of the cloned gtfA gene can be carried out in a variety of suitable host 
cells well known to those skilled in the art. The gtfA gene is introduced 
into a host cell by any suitable transformation, transfection, or 
conjugation means, well known to those skilled in the art. While 
chromosomal integration of the cloned gtfA gene is within the scope of the 
present invention, it is preferred that the gene be cloned into a suitable 
extra-chromosomally maintained expression vector so that the coding region 
of the gtfA gene is operably linked to a constitutive or inducible 
promoter. 
The basic steps in the recombinant production of the GtfA protein are: 
a) constructing a natural, synthetic or semi-synthetic DNA encoding GtfA 
protein; 
b) integrating said DNA into an expression vector in a manner suitable for 
expressing the GtfA protein, either alone or as a fusion protein; 
c) transforming, transfecting, or otherwise introducting said expression 
vector into an appropriate eukaryotic or prokaryotic host cell to form a 
recombinant host cell, 
d) culturing said recombinant host cell under conditions that favor 
expression of the GtfA protein; and 
e) recovering and purifying the GtfA protein by any suitable means. 
Expressing Recombinant GtfA Protein in Procaryotic and Eucaryotic Host 
Cells 
In general, prokaryotes are used for cloning DNA and for constructing the 
vectors of the present invention. Prokaryotes are also employed in the 
production of the GtfA protein. For example, the Escherichia coli K12 
strain 294 (ATCC No. 31446) is particularly useful for the expression of 
foreign proteins. Other strains of E. coli, bacilli such as Bacillus 
subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia 
marcescans, various Pseudomonas species, and other bacteria, such as 
Streptomyces, may also be employed as host cells in the cloning and 
expression of the recombinant proteins of this invention. 
Promoters suitable for driving the expression of gene sequences in 
prokaryotes include .beta.-lactamase e.g. vector pGX2907, ATCC 39344, 
contains a replicon and .beta.-lactamase gene!, lactose systems Chang et 
al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 
281:544 (1979)!, alkaline phosphatase, and the tryptophan (trp) promoter 
system vector pATH1 (ATCC 37695) which is designed to facilitate 
expression of an open reading frame as a trpE fusion protein under the 
control of the trp promoter!. Hybrid promoters such as the tac promoter 
(isolatable from plasmid pDR540, ATCC-37282) are also suitable. Still 
other bacterial promoters, whose nucleotide sequences are generally known, 
enable one of skill in the art to ligate such promoter sequences to DNA 
encoding the proteins of the instant invention using linkers or adapters 
to supply any required restriction sites. Promoters for use in bacterial 
systems also will contain a Shine-Dalgarno sequence operably linked to the 
DNA encoding the desired polypeptides. These examples are illustrative 
rather than limiting. 
The protein of this invention may be synthesized as the amino acid sequence 
identified as SEQ ID NO:2, or as a fusion protein comprising the protein 
of interest and another protein or peptide which may be removable by 
enzymatic or chemical cleavage. Expression as a fusion protein may prolong 
the lifespan, increase the yield of the desired peptide, or provide a 
convenient means for purifying the protein. A variety of peptidases (e.g. 
enterokinase and thrombin) which cleave a polypeptide at specific sites 
are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will 
cleave a polypeptide chain at specific sites. The skilled artisan will 
appreciate the modifications necessary to the amino acid sequence (and 
synthetic or semi-synthetic coding sequence if recombinant means are 
employed) to incorporate site-specific internal cleavage sites. See e.g., 
P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13, in 
Protein Purification: From Molecular Mechanisms to Large Scale Processes, 
American Chemical Society, Washington, D.C. (1990). 
In addition to prokaryotes, mammalian host cells and eukaryotic microbes 
such as yeast may also be used to isolate and express the genes of the 
present invention. The simple eucaryote Saccharomyces cerevisiae, is the 
most commonly used eukaryotic microorganism, although a number of other 
yeasts such as Kluyveromyces lactis are also suitable. For expression in 
Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used. 
See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et 
al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980). Plasmid 
YRp7 contains the TRP1 gene which provides a selectable marker for use in 
a trp1 auxotrophic mutant. 
Purification of Recombinantly-Produced GtfA Protein 
An expression vector carrying the cloned gtfA gene of Amycolatopsis 
orientalis is transformed, transfected, or otherwise introduced into a 
suitable host cell using standard methods. Cells which contain the vector 
are propagated under conditions suitable for expression of the 
Glycosyltransferase protein. If the gtfA gene is under the control of an 
inducible promoter, growth media and other conditions should incorporate 
the appropriate inducer. 
The recombinantly produced protein may be purified from cellular extracts 
of transformed cells by any suitable means. In a preferred protein 
purification method, the gtfA gene is modified at the 5' end to 
incorporate several histidine residues at the amino terminus of the GtfA 
protein product. The "histidine tag" enables a single-step protein 
purification method referred to as "immobilized metal ion affinity 
chromatography" (IMAC), essentially as described in M. C. Smith et al. 
"Chelating Peptide-immobilized metal-ion affinity chromatography," Chapter 
12, in Protein Purification: From Molecular Mechanisms to Large Scale 
Processes, American Chemical Society, Washington, D.C. (1990), and in U.S. 
Pat. No. 4,569,794 both of which hereby are incorporated by reference. The 
IMAC method enables rapid isolation of substantially pure protein. 
The gtfa gene, which comprises nucleic acid encoding SEQ ID NO:2, may also 
be produced using synthetic methodology. The synthesis of nucleic acids is 
well known in the art. See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, 
and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979). The DNA 
segments corresponding to the gtfA gene could be generated using a 
conventional DNA synthesizing apparatus, such as the Applied Biosystems 
Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln 
Center Drive, Foster City, Calif. 94404) which employ phosphoramidite 
chemistry. Alternatively, phosphotriester chemistry may be employed to 
synthesize the nucleic acids of this invention. See, e.g., M. J. Gait, 
ed., Oligonucleotide Synthesis, A Practical Approach, (1984).! 
The ribonucleic acids of the present invention may be prepared using the 
polynucleotide synthetic methods discussed supra, or they may be prepared 
enzymatically using RNA polymerases to transcribe a DNA template. 
The most preferred systems for preparing the ribonucleic acids of the 
present invention employ the RNA polymerase from the bacteriophage T7 or 
the bacteriophage SP6. These RNA polymerases are highly specific and 
require the insertion of bacteriophage-specific sequences at the 5' end of 
the template to be transcribed. See, J. Sambrook, et al., supra, at 
18.82-18.84. 
This invention also provides nucleic acids, RNA or DNA, which are 
complementary to SEQ ID NO:1 or SEQ ID NO:3. 
The present invention also provides probes and primers useful for a variety 
of molecular biology techniques. For example, the nucleic acid compounds 
of the present invention may be used to hybridize to genomic DNA which has 
been digested with one or more restriction enzymes and separated on an 
electrophoretic gel. The hybridization of radiolabeled probes onto such 
restricted DNA, usually fixed to a membrane after electrophoresis, is well 
known in the art. See, e.g., J. Sambrook, supra. A compound which 
comprises SEQ ID NO:1, SEQ ID NO:3 or a complementary sequence of SEQ ID 
NO:1 or SEQ ID NO:3, or a fragment thereof, and which is at least 15 base 
pairs in length, and which will selectively hybridize to Amycolatopsis 
orientalis DNA or mRNA encoding gtfA, is provided. Preferably, the 15 or 
more base pair compound is DNA. The probes and primers of this invention 
can be prepared by techniques well known to those skilled in the art (See 
e.g. Sambrook et al. supra). In a most preferred embodiment these probes 
and primers are synthesized using chemical means as described above. 
Another aspect of the present invention relates to recombinant DNA cloning 
vectors and expression vectors comprising the nucleic acids of the present 
invention. Many of the vectors encompassed within this invention are 
described above. The preferred nucleic acid vectors are those which 
comprise DNA. The most preferred recombinant DNA vectors comprise the 
isolated DNA sequence, SEQ ID NO:1. 
Choosing the most appropriate cloning vector or expression vector depends 
upon a number of factors including the availability of appropriate 
restriction enzyme sites, the type of host cell into which the vector is 
to be transfected or transformed, the purpose of the transfection or 
transformation (e.g., stable transformation as an extrachromosomal 
element, or integration into the host chromosome), the presence or absence 
of readily assayable or selectable markers (e.g., antibiotic resistance 
markers and metabolic markers), and the desired number of copies of the 
gene to be present in the host cell. 
Vectors suitable to carry the nucleic acids of the present invention 
comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic 
bacteriophages, stable bacteriophages, plasmids, viroids, and the like. 
The most preferred vectors are plasmids. 
When preparing an expression vector the skilled artisan understands that 
there are many variables to be considered, for example, whether to use a 
constitutive or inducible promoter. Inducible promoters are preferred 
because they enable high level, regulatable expression of an operably 
linked gene. A number of inducible promoters responding to a variety of 
induction signals are available, for example, carbon source, metal ions, 
and heat. The practitioner also understands that the amount of nucleic 
acid or protein to be produced dictates, in part, the selection of the 
expression system. The addition of certain nucleotide sequences, such as a 
sequence encoding a signal peptide preceding the coding sequence, is 
useful to direct localization of the resulting polypeptide. 
Host cells harboring the nucleic acids disclosed herein are also provided 
by the present invention. A preferred host is E. coli which has been 
transfected or transformed with a vector which comprises a nucleic acid of 
the present invention. 
The present invention also provides a method for constructing a recombinant 
host cell capable of expressing SEQ ID NO:2, said method comprising 
transforming or otherwise introducing into a host cell a recombinant DNA 
vector that comprises an isolated DNA sequence which encodes SEQ ID NO:2. 
A preferred host cell is any strain of E. coli which can accomodate high 
level expression of a gene(s) introduced by transformation or 
transfection. Preferred vectors for expression are those which comprise 
SEQ ID NO:1. A preferred expression vector for use in E. coli is plasmid 
pCZA364, which comprises SEQ ID NO:1. (See Example 1). Transformed host 
cells may be cultured under conditions well known to skilled artisans such 
that SEQ ID NO:2 is expressed, thereby producing GtfA protein in the 
recombinant host cell. 
The cloned GtfA enzyme is useful for glycosylating vancomycin glycopeptide 
compounds. A method embodied herein comprises glycosylating a vancomycin 
glycopeptide compound, by contacting the glycopeptide with the cloned GtfA 
protein in the presence of a suitable substrate, and monitoring the 
glycopeptide compound that is produced. 
The instant invention provides an enzymatic method for glycosylating 
glycopeptides of the vancomycin class using the cloned A. orientalis gtfA 
gene, said method comprising the steps of: 
a) expressing the cloned gtfA gene in a host cell so that GtfA enzyme is 
produced; 
b) exposing said GtfA enzyme to a glycopeptide compound, in vitro; 
c) introducing a suitable glycosylating substrate; and 
d) characterizing and/or purifying the product glycopeptide by any suitable 
means. 
The instant method can be used to enzymatically attach epivancosamine to 
glycopeptide molecules of the vancomycin class. 
The method can be implemented using substantially purified recombinant GtfA 
protein, as described herein, or using a crude cellular extract isolated 
from a recombinant cell culture that expresses the GtfA protein by virtue 
of having been transformed or transfected with the gtfA gene. 
A suitable substrate for the in vitro glycosylation reaction comprises 
TDP-epivancosamine. This substrate can be obtained by acid-catalyzed 
hydrolysis of compound A82846B using any suitable method known to skilled 
artisans (See e.g. M. Sim et al. "Synthesis and use of glycosyl 
phosphites: an effective route to glycosyl phophates, sugar nucleotides, 
and glycosides" J. Am. Chem. Soc. 115, 2260-67 (1993)). In one method for 
preparation of this substrate, following acid hydrolysis of A82846B the 
hydrolytic products are condensed with dibenzyl N,N-diethylphosphoramidite 
as a phosphitylating reagent so as to generate the appropriate dibenzyl 
glycosyl phosphite derivative. Oxidation and deprotection, followed by 
reaction with thymidine 5'-monophospho-morpholidate provides the desired 
sugar substrate. 
The following examples more fully describe the present invention. Those 
skilled in the art will recognize that the particular reagents, equipment, 
and procedures described are merely illustrative and are not intended to 
limit the present invention in any manner. 
EXAMPLE 1 
Construction of a DNA Vector for Expressing Amycolatopsis orientalis Gene 
gtfA in Escherichia coli 
Plasmid pCZA364 is an approximately 7 kilobase/pair expression vector 
suitable for expressing the gtfA gene at high levels in a procaryotic 
host, for example E. coli. The backbone of plasmid pCZA364 is derived from 
parent plasmid PET-11a (obtained from Novagen, Madison, Wis.), which 
contains an origin of DNA replication (ori), an ampicillin resistance gene 
(Amp), the T7 promoter region, and the lacI gene for repressing the lac 
operon. 
The gtfA gene cassette inserted into pCZA364 is generated using the PCR 
carried out on A. orientalis A82846 genomic DNA using standard conditions. 
Primers used in the amplification reaction are complementary to the 5' and 
3' ends of the gtfA gene sequence specified in SEQ ID NO: 1 and are 
engineered to contain NdeI and BglII restriction sites. The PCR-amplified 
gtfA gene sequence is digested with NdeI and BglII and ligated into 
pET11a, which has been digested with NdeI and BamHI. 
EXAMPLE 2 
Transformation of Escherichia coli with an Expression Plasmid Carrying the 
gtfA gene of Amycolatopsis orientalis 
Plasmid pCZA364 is transformed into E. coli BL21(DE3) (hsdS gal 
.lambda.cIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (See 
e.g. Sambrook et al. Supra). 
EXAMPLE 3 
In Vitro Glycosylation of Aglycosylvancomycin Using Cloned gtfA Gene 
Approximately 25 ml of a culture of E. coli BL21(DE3) cells transformed 
with plasmid pCZA364 is grown to an OD.sub.600 of about 0.6. Induction of 
gtfA gene expression is effected by adding 1 mM 
isopropyl-.beta.-D-thiogalactoside (IPTG) with shaking at room temperature 
for 2 to 3 hours. Thereafter, cells from about 2 ml of the induced culture 
are pelleted by centrifugation and resuspended in 2 ml of 50 mM Tris pH 
9.0, 100 .mu.g/ml lysozyme with incubation on ice for 10 minutes to effect 
cell lysis. After cell lysis the suspension is passed through a 23-gauge 
syringe and centrifuged at 10,000.times.g for 15 minutes to pellet cell 
debris. The resulting cell extract is used to attach epivancosamine onto 
AGV. 
The 1 ml glycosylation reaction contains: 
1 mg AGV in 50 mM Tris HCL, pH 9.0 
5 mg TDP-epivancosamine 
1 mg bovine serum albumin (BSA) 
20 .mu.l 1M MgCl2 
20 .mu.l 1M CaCl2 
5 .mu.l 1M dithiothreitol (DTT) 
445 .mu.l cell extract 
Distilled water to 1 ml. 
A control reaction contains cell extract from non-transformed BL21(DE3). 
After incubation overnight at 37.degree. C. with slight shaking the 
reaction is filtered through a 0.45 micron filter and analyzed by HPLC. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1188 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..1188 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGCGCGTGTTGATTACGGGGTGTGGATCGCGCGGAGATACCGAACCG48 
MetArgValLeuIleThrGlyCysGlySerArgGlyAspThrGluPro 
151015 
TTGGTGGCATTGGCGGCACGGTTGCGGGAACTCGGTGCGGACGCGCGG96 
LeuValAlaLeuAlaAlaArgLeuArgGluLeuGlyAlaAspAlaArg 
202530 
ATGTGCCTGCCGCCGGACTACGTGGAGCGGTGCGCCGAGGTCGGTGTG144 
MetCysLeuProProAspTyrValGluArgCysAlaGluValGlyVal 
354045 
CCGATGGTGCCGGTCGGTCGGGCGGTGCGCGCAGGGGCACGCGAGCCG192 
ProMetValProValGlyArgAlaValArgAlaGlyAlaArgGluPro 
505560 
GGAGAACTGCCGCCGGGGGCGGCCGAAGTCGTGACCGAGGTGGTCGCC240 
GlyGluLeuProProGlyAlaAlaGluValValThrGluValValAla 
65707580 
GAATGGTTCGACAAGGTCCCGGCGGCCATCGAGGGGTGTGACGCGGTG288 
GluTrpPheAspLysValProAlaAlaIleGluGlyCysAspAlaVal 
859095 
GTGACGACCGGCTTGCTGCCCGCCGCGGTCGCTGTCCGGTCGATGGCC336 
ValThrThrGlyLeuLeuProAlaAlaValAlaValArgSerMetAla 
100105110 
GAGAAGCTGGGCATCCCGTACCGCTACACCGTGCTGTCTCCGGACCAT384 
GluLysLeuGlyIleProTyrArgTyrThrValLeuSerProAspHis 
115120125 
CTGCCGTCGGAGCAAAGCCAGGCGGAGCGGGACATGTACAACCAGGGC432 
LeuProSerGluGlnSerGlnAlaGluArgAspMetTyrAsnGlnGly 
130135140 
GCCGACAGGCTTTTCGGTGACGCGGTCAACAGCCACCGGGCCTCGATC480 
AlaAspArgLeuPheGlyAspAlaValAsnSerHisArgAlaSerIle 
145150155160 
GGCCTGCCACCGGTGGAGCACCTCTACGACTACGGCTACACCGATCAG528 
GlyLeuProProValGluHisLeuTyrAspTyrGlyTyrThrAspGln 
165170175 
CCCTGGCTGGCGGCGGACCCGGTGCTGTCCCCGCTGCGGCCGACGGAC576 
ProTrpLeuAlaAlaAspProValLeuSerProLeuArgProThrAsp 
180185190 
CTCGGCACTGTGCAGACCGGTGCGTGGATCCTGCCCGACGAACGGCCG624 
LeuGlyThrValGlnThrGlyAlaTrpIleLeuProAspGluArgPro 
195200205 
CTTTCCGCGGAGCTGGAGGCGTTTCTGGCTGCCGGGTCGACGCCGGTG672 
LeuSerAlaGluLeuGluAlaPheLeuAlaAlaGlySerThrProVal 
210215220 
TACGTGGGTTTCGGCAGCTCGTCCCGACCGGCAACCGCTGACGCCGCG720 
TyrValGlyPheGlySerSerSerArgProAlaThrAlaAspAlaAla 
225230235240 
AAGATGGCCATCAAGGCGGTCCGTGCCAGTGGCCGCCGGATCGTTCTC768 
LysMetAlaIleLysAlaValArgAlaSerGlyArgArgIleValLeu 
245250255 
TCCCGCGGCTGGGCCGATTTGGTCCTGCCGGACGACGGGGCCGACTGC816 
SerArgGlyTrpAlaAspLeuValLeuProAspAspGlyAlaAspCys 
260265270 
TTCGTGGTCGGCGAAGTGAACCTTCAGGAGCTGTTCGGCCGGGTGGCC864 
PheValValGlyGluValAsnLeuGlnGluLeuPheGlyArgValAla 
275280285 
GCCGCCATCCACCACGACAGCGCGGGCACGACGCTGCTGGCCATGCGG912 
AlaAlaIleHisHisAspSerAlaGlyThrThrLeuLeuAlaMetArg 
290295300 
GCGGGCATCCCCCAGATCGTGGTGCGCCGCGTAGTGGACAACGTGGTG960 
AlaGlyIleProGlnIleValValArgArgValValAspAsnValVal 
305310315320 
GAGCAGGCGTACCACGCCGACCGGGTGGCCGAGCTGGGTGTCGGTGTG1008 
GluGlnAlaTyrHisAlaAspArgValAlaGluLeuGlyValGlyVal 
325330335 
GCGGTCGACGGTCCGGTCCCGACCATCGACTCCTTGTCGGCCGCGCTC1056 
AlaValAspGlyProValProThrIleAspSerLeuSerAlaAlaLeu 
340345350 
GACACGGCTCTGGCCCCGGAGATCCGTGCGCGAGCGACGACCGTGGCA1104 
AspThrAlaLeuAlaProGluIleArgAlaArgAlaThrThrValAla 
355360365 
GACACGATTCGCGCCGATGGGACAACGGTGGCCGCGCAGCTGCTGTTC1152 
AspThrIleArgAlaAspGlyThrThrValAlaAlaGlnLeuLeuPhe 
370375380 
GACGCGGTCAGCCTGGAAAAGCCGACTGTTCCCGCC1188 
AspAlaValSerLeuGluLysProThrValProAla 
385390395 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 396 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetArgValLeuIleThrGlyCysGlySerArgGlyAspThrGluPro 
151015 
LeuValAlaLeuAlaAlaArgLeuArgGluLeuGlyAlaAspAlaArg 
202530 
MetCysLeuProProAspTyrValGluArgCysAlaGluValGlyVal 
354045 
ProMetValProValGlyArgAlaValArgAlaGlyAlaArgGluPro 
505560 
GlyGluLeuProProGlyAlaAlaGluValValThrGluValValAla 
65707580 
GluTrpPheAspLysValProAlaAlaIleGluGlyCysAspAlaVal 
859095 
ValThrThrGlyLeuLeuProAlaAlaValAlaValArgSerMetAla 
100105110 
GluLysLeuGlyIleProTyrArgTyrThrValLeuSerProAspHis 
115120125 
LeuProSerGluGlnSerGlnAlaGluArgAspMetTyrAsnGlnGly 
130135140 
AlaAspArgLeuPheGlyAspAlaValAsnSerHisArgAlaSerIle 
145150155160 
GlyLeuProProValGluHisLeuTyrAspTyrGlyTyrThrAspGln 
165170175 
ProTrpLeuAlaAlaAspProValLeuSerProLeuArgProThrAsp 
180185190 
LeuGlyThrValGlnThrGlyAlaTrpIleLeuProAspGluArgPro 
195200205 
LeuSerAlaGluLeuGluAlaPheLeuAlaAlaGlySerThrProVal 
210215220 
TyrValGlyPheGlySerSerSerArgProAlaThrAlaAspAlaAla 
225230235240 
LysMetAlaIleLysAlaValArgAlaSerGlyArgArgIleValLeu 
245250255 
SerArgGlyTrpAlaAspLeuValLeuProAspAspGlyAlaAspCys 
260265270 
PheValValGlyGluValAsnLeuGlnGluLeuPheGlyArgValAla 
275280285 
AlaAlaIleHisHisAspSerAlaGlyThrThrLeuLeuAlaMetArg 
290295300 
AlaGlyIleProGlnIleValValArgArgValValAspAsnValVal 
305310315320 
GluGlnAlaTyrHisAlaAspArgValAlaGluLeuGlyValGlyVal 
325330335 
AlaValAspGlyProValProThrIleAspSerLeuSerAlaAlaLeu 
340345350 
AspThrAlaLeuAlaProGluIleArgAlaArgAlaThrThrValAla 
355360365 
AspThrIleArgAlaAspGlyThrThrValAlaAlaGlnLeuLeuPhe 
370375380 
AspAlaValSerLeuGluLysProThrValProAla 
385390395 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1188 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: mRNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AUGCGCGUGUUGAUUACGGGGUGUGGAUCGCGCGGAGAUACCGAACCGUUGGUGGCAUUG60 
GCGGCACGGUUGCGGGAACUCGGUGCGGACGCGCGGAUGUGCCUGCCGCCGGACUACGUG120 
GAGCGGUGCGCCGAGGUCGGUGUGCCGAUGGUGCCGGUCGGUCGGGCGGUGCGCGCAGGG180 
GCACGCGAGCCGGGAGAACUGCCGCCGGGGGCGGCCGAAGUCGUGACCGAGGUGGUCGCC240 
GAAUGGUUCGACAAGGUCCCGGCGGCCAUCGAGGGGUGUGACGCGGUGGUGACGACCGGC300 
UUGCUGCCCGCCGCGGUCGCUGUCCGGUCGAUGGCCGAGAAGCUGGGCAUCCCGUACCGC360 
UACACCGUGCUGUCUCCGGACCAUCUGCCGUCGGAGCAAAGCCAGGCGGAGCGGGACAUG420 
UACAACCAGGGCGCCGACAGGCUUUUCGGUGACGCGGUCAACAGCCACCGGGCCUCGAUC480 
GGCCUGCCACCGGUGGAGCACCUCUACGACUACGGCUACACCGAUCAGCCCUGGCUGGCG540 
GCGGACCCGGUGCUGUCCCCGCUGCGGCCGACGGACCUCGGCACUGUGCAGACCGGUGCG600 
UGGAUCCUGCCCGACGAACGGCCGCUUUCCGCGGAGCUGGAGGCGUUUCUGGCUGCCGGG660 
UCGACGCCGGUGUACGUGGGUUUCGGCAGCUCGUCCCGACCGGCAACCGCUGACGCCGCG720 
AAGAUGGCCAUCAAGGCGGUCCGUGCCAGUGGCCGCCGGAUCGUUCUCUCCCGCGGCUGG780 
GCCGAUUUGGUCCUGCCGGACGACGGGGCCGACUGCUUCGUGGUCGGCGAAGUGAACCUU840 
CAGGAGCUGUUCGGCCGGGUGGCCGCCGCCAUCCACCACGACAGCGCGGGCACGACGCUG900 
CUGGCCAUGCGGGCGGGCAUCCCCCAGAUCGUGGUGCGCCGCGUAGUGGACAACGUGGUG960 
GAGCAGGCGUACCACGCCGACCGGGUGGCCGAGCUGGGUGUCGGUGUGGCGGUCGACGGU1020 
CCGGUCCCGACCAUCGACUCCUUGUCGGCCGCGCUCGACACGGCUCUGGCCCCGGAGAUC1080 
CGUGCGCGAGCGACGACCGUGGCAGACACGAUUCGCGCCGAUGGGACAACGGUGGCCGCG1140 
CAGCUGCUGUUCGACGCGGUCAGCCUGGAAAAGCCGACUGUUCCCGCC1188 
__________________________________________________________________________