Glycosyltransferase gene gtfE from Amycolatopsis orientalis
AssigneeEli Lilly and Company
InventorsRichard H. Baltz, Patricia J. Solenberg, Patti J. Treadway
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Abstract
The invention provides isolated nucleic acid compounds encoding a glycosyltransferase enzyme of Amycolatopsis orientalis. Also provided are vectors carrying genes that encode said enzyme, transformed heterologous host cells for expressing said enzyme, and methods for producing glycopeptide compounds using the cloned genes that encode said enzyme.
Claims34
A substantially pure glycosyltransferase protein from Amycolatopsis orientalis having the amino acid sequence:
- 3 Met Arg Val Leu Leu Ser Thr Cys Gly Ser Arg Gly Asp Val Glu Pro 1 5 10 15 Leu Val Ala Leu Ala Val Arg Leu Arg Glu Arg Gly Ala Glu Val Arg 20 25 30 Met Cys Ala Pro Pro Asp Cys Ala Asp Arg Leu Ala Glu Val Asp Val 35 40 45 Pro His Leu Pro Leu Gly Ala Ser Ala Arg Pro Ser Ala Gly Gln Ala 50 55 60 Lys Pro Leu Thr Ala Glu Asp Met Leu Arg Phe Thr Thr Glu Thr Ile 65 70 75 80 Ala Thr Gln Phe Glu Arg Ile Pro Ala Ala Ala Glu Gly Cys Ala Ala 85 90 95 Val Val Thr Thr Gly Leu Leu Ala Ala Ala Ile Gly Val Arg Ser Val 100 105 110 Ala Glu Lys Leu Gly Ile Pro Tyr Phe Tyr Gly Phe His Cys Pro Ser 115 120 125 Tyr Val Pro Ser Pro Tyr Tyr Ala Pro Pro Pro Pro Leu Gly Glu Pro 130 135 140 Pro Ala Pro Asp Gly Thr Asp Ile Gln Ala Leu Trp Glu Arg Asn Asn 145 150 155 160 Gln Ser Ala Tyr Arg Arg Tyr Gly Glu Pro Leu Asn Ser Arg Arg Ala 165 170 175 Ala Ile Gly Leu Pro Pro Val Glu Asp Ile Phe Gly His Gly Tyr Thr 180 185 190 Asp His Pro Trp Met Ala Ala Asp Pro Val Leu Ala Pro Leu Gln Pro 195 200 205 Thr Asp Leu Asp Ala Val Gln Thr Gly Ala Trp Ile Leu Pro Asp Glu 210 215 220 Arg Pro Ile Ser Ala Glu Leu Glu Ala Phe Leu Asp Ala Gly Ala Pro 225 230 235 240 Pro Val Tyr Leu Gly Phe Gly Ser Leu Arg Ala Pro Ala Asp Ala Ala 245 250 255 Lys Val Ala Ile Glu Ala Ile Arg Ala His Gly His Arg Val Ile Leu 260 265 270 Ser Arg Gly Trp Ala Asp Leu Val Leu Pro Asp Asp Arg Glu Asp Cys 275 280 285 Phe Ala Ile Gly Glu Val Asn Gln Gln Val Leu Phe Arg Arg Val Ala 290 295 300 Ala Val Ile His His Gly Gly Ala Gly Thr Thr His Val Ala Thr Arg 305 310 315 320 Ala Gly Val Pro Gln Ile Leu Val Pro Gln Ile Ala Asp Gln Pro Tyr 325 330 335 Tyr Ala Ala Arg Val Ala Glu Leu Gly Val Gly Val Ala His Asp Gly 340 345 350 Pro Thr Pro Thr Phe Asp Thr Leu Ser Ala Ala Leu Thr Lys Ala Leu 355 360 365 Ala Pro Glu Thr Arg Val Arg Ala Glu Ala Val Ala Glu Thr Val Gln 370 375 380 Thr Asp Gly Ala Ala Val Ala Ala Asp Leu Leu Phe Ala Ala Val Thr 385 390 395 400 Gly Asn Gln Pro Ala Val Pro Ala 405
- which is SEQ ID NO 2.
A method for glycosylating a glycopeptide compound or glycopeptide core using an Amycolatopsis orientalis enzyme produced in a recombinant host, said method comprising the steps of:
- a) mixing said glycopeptide compound or glycopeptide core and a suitable glycosylating substrate;
- and
- b) adding to the mixture of step (a) a suitable amount of said enzyme wherein said enzyme has glycosyltransferase activity.
A method for glycosylating glycopeptide compounds in vivo using a recombinantly produced enzyme having glycosyltransferase activity, comprising the steps of:
- a) introducing, by any suitable means, an expression vector that encodes said enzyme activity into a bacterial strain that produces a glycopeptide compound, forming a recombinant strain;
- and
- b) fermenting said recombinant strain on a suitable growth medium such that said enzyme activity leads to a glycosylation of said glycopeptide compound.
A composition of matter comprising a modified aglycosylvancomycin core, wherein D-xylose is covalently attached to the hydroxyl group of the B ring of aglycosylvancomycin, said composition having the structure:
- 2
A composition of matter having the formula:
- 3
- and wherein said composition is produced by a method comprising the steps of:
- a) admixing in a suitable buffer
- i) compound A47934, and
- ii) a suitable glucosylating substrate, forming a reaction mixture;
- and
- b) adding to said mixture in (a) a suitable amount of Amycolatopsis orientalis glucosyltransferase enzyme activity.
A composition of matter comprising a modified aglycosylvancomycin core, wherein D-glucose is covalently attached to the hydroxyl group of the B ring of aglycosylvancomycin and wherein said compound is made by a method comprising the steps of:
- a) admixing in a suitable buffer
- i) aglycosylvancomycin, and
- ii) a suitable glycosylating substrate, forming a reaction mixture;
- and
- b) adding to said mixture in (a) a suitable amount of Amycolatopsis orientalis glucosyltransferase enzyme activity.
A composition of matter having the formula:
- 4
- and wherein said composition is produced by a method comprising the steps of:
- a) admixing in a suitable buffer
- i) compound A41030A, and
- ii) a suitable glucosylating substrate, forming a reaction mixture;
- and
- b) adding to said mixture in (a) a suitable amount of Amycolatopsis orientalis glucosyltransferase enzyme activity.
Dependencies are inferred from the claim text.