Plant Molecular Biology 34: 15–29, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Expression of de novo high-lysine
-helical coiled-coil proteins may
signiﬁcantly increase the accumulated levels of lysine in mature seeds of
transgenic tobacco plants
Sharon J. Keeler
, Cathy L. Maloney
, Patricia Y. Webber, Carole Patterson, Lee T. Hirata
S. Carl Falco and Janet A. Rice
E.I. Dupont de Nemours, Experimental Station, Wilmington, DE 19880-0402, USA; present addresses:
Department of Plant and Soil Sciences, University of Delaware, 149 Townsend Hall, Newark, DE 19717, USA
author for correspondence);
Waters Division, Millipore Inc., Miford, MA;
Zeneca, Inc., Wilmington, DE
Received 11 June 1996; accepted in revised form 5 February 1997
-helical coiled-coil, conglycinin, lysine, methionine, phaseolin, seed storage proteins, nutritional
We have designed protein molecules based on an
-helical coiled-coil structure. These proteins can be tailored to
complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of
the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides
and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein
containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed
speciﬁc bean phaseolin and soybean
-conglycinin promoters. Both promoters provided a level of expression in
the mature transgenic tobacco seeds which resulted in a signiﬁcant increase in the total lysine content of the seeds.
Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression.
Plants transformed with the soybean
-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine
phenotypethrough three generations. However, expression of the high-lysinephenotype in plants transformed with
the bean phaseolin/CP 3-5 was variable. This is the ﬁrst report of a signiﬁcant increase in seed lysine content due
to the seed-speciﬁc expression of a de novo protein sequence.
are deﬁcient in some of the ten essential amino acids
which are required in animal diets. In feed derived
from corn, lysine is the most limiting amino acid for
the dietary requirements of many animals followed by
tryptophan and the sulfur amino acids methionine and
cysteine. Soybean meal, which is rich in lysine and
tryptophan,is used to supplement corn in animal feed.
However, feed blends of corn and soybean typically
still include methionine as an additive. A mechanism
to increase the levels of particular amino acids within
the plant seed for a given crop and a speciﬁc end-
use would eliminate the need to supplement mixed or
single grain feeds with puriﬁed amino acids.
The amino acid content of seeds is primarily
determined by the storage proteins which can repres-
ent 50% or more of the total seed protein. Seed storage
proteins are synthesized during seed development and
serve as a major nutrient reserve after germination.
Expression of both dicot and monocot seed storage
protein genes has been reported in transgenic tobacco
and petunia [3, 4, 15, 21, 38, 47, 53]. Efforts have
been made to modify natural seed storage proteins by
addition or replacement of amino acids to increase the
lysine or methionine content of the protein (for review
see ). Expression in seeds of tobacco, Arabidopsis
or canola of these transgenic gene products has been
A number of sulfur-richplant seed storage proteins
have been identiﬁed and their corresponding genes
GR: 201001899, Pips nr. 135349 BIO2KAP
pla325us.tex; 11/04/1997; 11:45; v.7; p.1