glycoconjugate and sugar nucleotide synthesis using solid supports

US 20020150968A1

(12) Patent Application Publication (10) Pub. No.: US 2002/0150968 A1 (19) United States

Wang et al. (43) Pub. Date: Oct. 17, 2002

(54) GLYCOCONJUGATE AND SUGAR (52) US. Cl. ........................... .. 435/53; 435/681; 435/96; NUCLEOTIDE SYNTHESIS USING SOLID 435/175 SUPPORTS

(76) Inventors: Peng G. Wang; Troy; MI (US); Xi Chen; NorristoWn; PA (US)

Correspondence Address: Brinks Hofer Gilson & Lione PO. Box 10395 Chicago, IL 60610 (US)

(21) Appl. No.: 09/757,846

(22) Filed: Jan. 10, 2001

Publication Classi?cation

(51) Int. Cl.7 ......................... .. C12P 33/20; C12P 21/02; C12P 19/20; C12N 11/18

(57) ABSTRACT

This invention relates to methods and compositions for the in vitro production of glycoconjugates. In particular, a preferred production system is provided that comprises a solid support, at least one sugar nucleotide producing enZyme; at least one glycosyltransferase; at least one bioen

ergetic; and at least one acceptor. The sugar nucleotide producing enZyme(s) is preferably immobilized on the solid support. The glycosyltransferase may be co-immobiliZed on the solid support With the sugar nucleotide producing enZyme(s), or may be provided to the solid support in solution.

Patent Application Publication

PEP ATP Galactose

Ki", MgzkykF XGalK P t yruva e ADP Gab LP

GaIT UDP-Gal

Oct. 17, 2002 Sheet 1 0f 22

UDP-Glc

£61014? Mn2+ PPi GalU

US 2002/0150968 A1

GalB 1,4Glc

UTPggUDP Galoc 1 ,3 Lac PykF

Pyruvate PEP

FIG. I

Patent Application Publication Oct. 17, 2002 Sheet 2 0f 22 US 2002/0150968 A1

Sal! rbs ' galK + galT

1U I Xba‘ ga {05 Sac H

rbéf kF- a1 ,3GalT

py

T7 terminator pLDR€gz£<TUF EcoR V Cla lg l PR

7» C1 -

repressor

pMB1

FIG. 2


ATP Galactose

XGalK ADP Gal-l-P

GalT UDP-Gal LacOR

UDP-Glc G1C_1_P OL1,3G&1T

PPI GaIUUTP UD; Gal0c1,3 LacOR \Ngly A

ADP ATP

Sal | rbs I ga/K + ga/T

Sac H Xba I

rbs a1,3Ga/T

ndK ,

T7 LDRZ - rbs terminator p __8 g :ETUN EcoR V Cla I ' 7\ PR

K cl -

repressor

FIG. 3


Pn ATP Galactosce PPK GalK

P 11-1 AD Gal-l-P

GalT U _ LacOR UDP-G% DP G1 1

K/Glc-l-P 0L1,3Ga1T PPi 6%

UIP UDP Gala 1,3LacOR \PBK/

ga/U Sac I l

a1,3Ga/T ppK Kpn I '

T7 _ pLDRZO-onKTUP fbs terminator ,_ 9 5 kb EcoR V

' 7L PR Cla I

7» cl -

repressor

pMB1

FIG. 4


Ac ATP Galactose

AcKX @alK PAP ADP Gill-l-P y PoxB

62 Pi UDP-GlcGaITUDPGal Lac \\ .fkGlobp yocl?GalT

Ppa PP1GalU\UTP kPi \Ngik/UDP Galocl,3Lac A

Py ADP ATP + O2OXBAP%< CK Ac

Gal + Lac a Galotl,3Lac + 2 H2O

ZATP + 21120 a ZADP + 2HOPO32'+ 2H+ ZADP + 2cH3c02P032‘+ 2H+ r: ZATP + 2CH3CO2H ZPyruvate + 202 + 2HOPO32' a 2 Ace + 2002 + 2H2O2

2H202 7-: + 02’

Balance:

Gal + Lac + 2CH3COCOOH + 02 1 I

Galal?Lac + H2O + 2CH3COOH + ZCOZ

FIG. 5


Glc - ~> GlC-6-P A‘

PQM 615-143

PPi ‘ “@uDP-Gm ROH GIcT

UTP UDP’ *GmOR Y

pnjq PpK Pn

GIucQse + ZPn + ROI-l --> 2Pn_1 + GICOR + PPi

KPH I rbs ' M

Xba I galU pg 5, Sal!

ppK glcT

T7 1 terminator pLDRZO-UDPGlc ELEECOR v

Cla If k PR

nMBl Opt

FIG. 6


Pn ADP PEP GICNAC X’pK XDykF (NagE PM ATP Py eicryAc-s-P

Agm1

GICRJAC-1-P GImU

PF’; UDP-GICN c ROH

GICNACT

UTWDP GICNACOR A

Pn-1 Pn * 2PM + GICNACOR + PPi GICNAC + 2Pn + ROH

8 nagE rb g/mu rbs

rbs agm 1 PykF

rbs rbs

lgtA Kg pp rbs

X PR T7 terminator 2 Cl -

repressor

Ampr pMB1ori

FIG. 7


Pn-1 ATP GaiNAc

XDDK P)Gdnase Pn AD

P

GaINAc-1-P pyrophophorylase

Pi UDP-GalNAc ROH

JGBaINAcT UTP UD >PPK< GaINAcOR

Pn-1 Pn

GalNAc + 2Pn + ROH ~2Pn4 + GaINAcOR + PPi

‘r0 hos hor Iase Ga/NAc kinase rbs py p p y

rbs ppK Ga/NAcT

T7 _

terminator Cla I

pLDRZO -UDPGalNAc

repressor Amp

pMB1 ori

FIG. 8


.» PEP Glucose (external)

Xglueose transporter* P m

PYR G1c-6-P g Glc-l-P ZNAD Ugd ZNADH

GaiU A UDP-Gle UDP-GlcA

PP 1 ROH

UGT2B7 -— from glycolysis UTP ppk UDP GlcAOR

'4” pLDRZO-GlcA ?x PR

Ppk T7 c1857

terminator

pMBl ori Ampr

FIG. 9


MaENACNaIEé N NA Pyruvate eu C NeuA PP i

P CTP CMP-NeuNAc “'1 \ ROH

Ppk 15m CMP

P n CDP

\cmk/ ADP/\ATP \PPI/

NeruNAcOR

SP6 I 5 EcoR I

SiaT neuA

Pvu H7?‘ Sal I r: promotor

nan

Bam HI‘\\ T7 terminator Cl 857

Cla I r pMB1ori

Amp

FIG.1O


PTS PMM Man ——- Man-6-P ———~ Man_1_p

ppi GIVIP GDP~Man ROH

(ManT GTP GDP ManOR \_/

Pn TR pk

a/g2 tTY . t pL-ManA1A2 ,bs ermma or

9600 bp k PR

Cl 857

Ampr pMB1

FIG.1I


GDP-4-keto-6-deoxy-Man Man BIS» Man-6-P Man-l-P GMQ/ ‘~ \GFS

PPi GMP GDP-Man GDP-F116 ROH

FucT

GTPVGDP ManOR 4m Pn—l Pn

gmd rbs marrC

gfs manB

rbs rbs pL-MFucot1,3FT T7 promotor

10.5 kb a1,3FucT U

c! 857 _ T7

termlnator

pMB1 ori Amp

FIG. 12


rbs ga/U spHAS

rbs m ugd p9

T7 pLGAP-HAS rbs terminator / 7» PR

Cl 857

FIG.13


H OH 2 HNaO2C OH OH OH

HO O O AcHN O O \ O

HO OH HO \ OH

Target I. (Human milk) OH Neu5Aoo¢2,3GalB1,4GIcNa

Target ll. (Human milk Neu5Aco¢2,6Ga1B1 ,4GIcNAcB1,3GaIB1,4G1c

OH OH OH

% m m w 0 OH HO OH AcHN OH HO OH

Target Ill. (Human milk) ’ Neu5Aco¢2,6Ga|[31,3 (NeuACa2,6)GicNAcB1,3GalB1,4Glc

H OH 2 H H020 OH OH OH

O O .. . .

ACHN OH HO O-Ceramnde/Sphmgosme HO OH

Target IV. (3M3 (Bovine milk) (Neu5Aca2,3GaIB1,4GIc-O-Ceramide/Sphingosine

FIG. 14


HO OH '

f HO \ HO 0 OR I

HOOUDP HO HO H\O OH

140% a1,3Ga|T H0 \

HOOUDP HO OH 0

SUSA UDP Ho??Ho OH OH HoOw/owmq \ HO Fructose HO HO

(energy source) Sucrose

T7 terminator Cla | 041, 3Ga/T

pLDRZO-aES ~ 7.8 kb

rbs' EcoR V

k CI -

repressor

Amp

pMB1

FIG. 15


GFS candidate PMI/GMP GMD 5

Y Y HPOO45 HPOO43 HP0044 r5 kb

L 1-41kb ? L 1.14% 3 I I

40651bp 44172bp

FIG. 16


Gene cluster

rbs FucT

rbs pGF(10.6kb) PpK

. T7 7\. PR termmator Cl 857

FIG. 17


D _ Fructose U P G10 HO-R

SS GluT

Sucrose GIC_O_R UDP

susA

T7 rbs r terminator Amp

glcT pLDRZO-GlcSS

rbs

7L PR

repressor

FIG.18


ZNAD ZNADH Ugd

UDP-Glc UDP-GlcA

ROH Fructose SS )UGT2B7 (energy source) UDP

Sucrose G16 AOR

rbs ugd susA

T7 terminator UGT2B7

rbs

7» PR Amp

7t Cl -

repressor

pMB1

FIG. 19

glycoconjugate and sugar nucleotide synthesis using solid supports

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