Carbohydrate Research 337 (2002) 327 – 333
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Production of highly phosphorylated glycopolymers by expression
of R1 in Escherichia coli
Anders Viksø-Nielsen,
a
Patrick Hao-Jie Chen,
b
Ha ˚kan Larsson,
b
Andreas Blennow,
a
Birger Lindberg Møller
a,
*
a
Plant Biochemistry Laboratory, Department of Plant Biology, Centre for Molecular Plant Physiology (PlaCe),
Royal Veterinary and Agricultural Uniersity, Thoraldsensej 40, DK-1871 Frederiksberg C, Denmark
b
Department of Plant Biology, Swedish Uniersity of Agricultural Sciences, PO Box 7080, S -750 07 Uppsala, Sweden
Received 10 July 2001; accepted 4 December 2001
Abstract
The possible involvement of the starch bound R1 protein from potato (Solanum tuberosum L.) in the phosphorylation of starch
was investigated by functional expression and characterisation of R1 in Escherichia coli. By expression of R1 in E. coli it is shown
that it is possible to produce glycopolymers, e.g., glycogen, with an increased degree of phosphate substitution. The expression of
R1 in E. coli resulted in a sixfold increase in glycogen bound phosphate and in an increased accumulation of glycogen leading to
a glycogen excess (gex) phenotype. There was an overall shift in the unit-chain length of the isolated glycogen towards smaller
degrees of polymerisation. The pleiotropic effects on the glycogen biosynthetic and amylolytic enzyme activities was investigated
and showed an increase in ADPglucose pyrophosphorylase activity, as well as a decrease in exo-amylolytic activity. These results
are discussed in relation to starch phosphorylation and a possible role of R1 in this respect. © 2002 Published by Elsevier Science
Ltd.
Keywords: R1 starch protein; Glycogen; Phosphorylation; Bacterial recombinant proteins; Pyrophosphorylase; Potato
1. Introduction
Most bacteria store glucose in glycogen bodies, con-
sisting of water-soluble -(1 4)-linked polysaccharides
with up to 10% -(1 6)-branch points (reviewed in
Manners
1
), whereas plants store glucose in the form of
starch granules. In contrast to glycogen, starch consists
of amylose that is essentially linear, and of partially
crystalline amylopectin, that contains up to 5% -(1
6)-branch points (reviewed by Smith et al.
2
).
Amylopectin from potato tuber starch contains small
amounts of covalently bound phosphate, i.e., one glu-
cose unit out of 200–300 is phosphorylated.
3
The phos-
phate groups are bound as monoesters at C-6 (approx.
70%) and at C-3 (approx. 30%) positions of the glucose
residues. In addition, a small fraction (1%) may be
linked to C-2.
3,4
Phosphate is also present in glycogen
although to a much less extent, i.e., one glucose unit
out of 1600 is phosphorylated.
5
A high degree of phos-
phate substitution in starch results in starch gels with
high viscosity and stable starch pastes.
6,7
In contrast to starch, phosphate in glycogen is bound
as monoesters at the C-6 position or as glucosyl-1,6-
glucose phosphodiesters in glycogen in a ratio of 40:60,
respectively.
5,8,9
Due to the fact that both starch and
glycogen contain monoesterified phosphate, Escherichia
coli constitutes an ideal model organism when studying
the phosphorylation of starch, since the biosynthetic
pathways leading to starch or glycogen are comparable.
In both pathways ADPglucose pyrophosphorylase (EC
2.7.7.27) converts Glc-1P into ADPglucose, the sub-
strate for starch and glycogen synthases (EC
2.4.1.21).
2,10
An additional common step in starch and
Abbreiations: AGPase, ADPglucose pyrophosphorylase;
DP, degree of polymerisation; E. coli, Escherichia coli ; GBE,
Glycogen branching enzyme; Glc-1P, glucose-1-phosphate;
Glc-6P, glucose-6-phosphate; HPAEC, high-pressure anion-
exchange chromatography.
* Corresponding author. Tel.: +45-35-283352; fax: +45-
35-283333.
E -mail address: blm@kvl.dk (B. Lindberg Møller).
0008-6215/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII:S0008-6215(01)00326-3