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
Patent Application Publication Oct. 17, 2002 Sheet 3 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 4 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 5 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 6 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 7 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 8 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 9 0f 22 US 2002/0150968 A1
.» 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
Patent Application Publication Oct. 17, 2002 Sheet 10 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 11 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 12 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 13 0f 22 US 2002/0150968 A1
rbs ga/U spHAS
rbs m ugd p9
T7 pLGAP-HAS rbs terminator / 7» PR
Cl 857
FIG.13
Patent Application Publication Oct. 17, 2002 Sheet 14 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 15 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 16 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 17 0f 22 US 2002/0150968 A1
Gene cluster
rbs FucT
rbs pGF(10.6kb) PpK
. T7 7\. PR termmator Cl 857
FIG. 17
Patent Application Publication Oct. 17, 2002 Sheet 18 0f 22 US 2002/0150968 A1
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
Patent Application Publication Oct. 17, 2002 Sheet 19 0f 22 US 2002/0150968 A1
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