Probing carotenoid biosynthesis in developing seed coats of Bixa
orellana (Bixaceae) through expressed sequence tag analysis
Colette Jako, Catherine Coutu, Ingeborg Roewer, Darwin W. Reed, Lawrence
E. Pelcher, Patrick S. Covello *
National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9
Received 24 January 2002; received in revised form 11 April 2002; accepted 11 April 2002
Bixin is a commercially important diapocarotenoid derived from the seed coats of the tropical shrub Bixa orellana . Based on
available chemical data and by analogy to abscisic acid biosynthesis, a biosynthetic pathway for bixin starting from lycopene is
proposed. This putative pathway was investigated through expressed sequence tag (EST) analysis using a subtracted cDNA library.
This analysis identified ESTs corresponding to most of the enzymes of the carotenoid biosynthesis pathway including the 1-deoxy-
xylulose 5-phosphate pathway of plastids. ESTs corresponding to a number of genes encoding dioxygenases, aldehyde
dehydrogenases and carboxyl methyltransferases were also identified. These represent candidate enzymes for the last stage of the
bixin biosynthetic pathway. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Expressed sequence tag; Bixa orellana ; Carotenoid; Bixin; Dioxygenase; Carboxyl methyltransferase
In the past, investigations into natural products
chemistry and biochemistry have been understandably
dominated by the methods of qualitative and quantita-
tive analytical chemistry. Recently, the maturation of
methods in molecular genetics, especially high through-
put operations, has had an impact on how problems in
natural products chemistry can be approached. Ex-
pressed sequence tag (EST) analysis has become a
powerful tool in this regard, particularly for elucidating
biosynthetic pathways and the enzymes involved in
those pathways. Since most natural products research
is typically conducted outside of the standard model
organisms, the direct use of whole genome sequence
data is not possible. However, with the realization that
many of the enzymes involved in natural product
biosynthesis represent variations within known classes
of enzymes, similarity searches provide a means of
identifying corresponding gene candidates. Examples
of this EST approach to understanding natural products
biochemistry include the identification of genes encod-
ing enzymes involved in the biosynthesis of fatty acids
[1,2], monoterpenes  and phenylpropenes .
In this study, an investigation into biosynthesis of the
commercially important carotenoid pigment, bixin, is
reported. In nature, a wide variety of carotenoids exist,
providing pigmentation and/or biochemical activity for
a variety of organisms. . A selection of carotenoids
are of commercial value, including bixin (Fig. 1) [6,7],
the active ingredient in annatto, a colorant in the food
industry. It appears to be rare in nature, but is produced
in the seed coats of the tropical shrub, Bixa orellana .
Little is known about the biosynthesis of bixin and
norbixin, the free acid form of bixin, which is also
present in annatto extract derived from B. orellana seed
coats. As reflected in Fig. 1, we hypothesize that there
are a small number of structural genes involved in bixin
biosynthesis starting from the main carotenoid pathway
and that these genes should show similarity to those
encoding known dioxygenases, aldehyde oxidases, alde-
Abbreviations: DOXP, 1-deoxy-
-xylulose 5-phosphate; EST,
expressed sequence tag.
National Research Council of Canada, publication no. 43818.
* Corresponding author. Tel.: '
1-306-975-5269; fax: '
E-mail address: firstname.lastname@example.org (P.S. Covello).
Plant Science 163 (2002) 141 Á
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
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