Molecular Biology Reports 31: 177–189, 2004.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
177
The expression of three desaturase genes of Spirulina platensis in
Escherichia coli DH5α
Heterologous expression of Spirulina-desaturase genes
Hongsthong Apiradee
1
, Paithoonrangsarid Kalyanee
1
, Prapugrangkul Pongsathon
2
, Desh-
nium Patcharaporn
1
, Sirijuntarut Matura
2
, Subhudhi Sanjukta
2
, Cheevadhanarak Supapon
2
&
Tanticharoen Morakot
1,2
1
BEC Unit, National Center for Genetic Engineering and Biotechnology, 83 Moo8, Thakham, Bangkhuntien,
Bangkok 10150, Thailand (Phone: 662-452-3452 Ext 4071; Fax: 662-452-3455; E-mail: apiradee@biotec.or.th
and apiradee@pdti.kmutt.ac.th);
2
School of Bioresources and Technology, King Mongkut’s University of Techno-
logy Thonburi, Tungkru, Bangkok 10140, Thailand
Accepted 5 May 2004
Key words: desaturase, fatty acid desaturation, gene regulation, heterologous expression, Spirulina platensis,
temperature stress
Abstract
The genes from a cyanobacterium - Spirulina platensis strain C1 - that encode the acyl-lipid desaturases (desC,
desA and desD) involved in γ -linolenic (GLA) synthesis have been successfully expressed for the first time in
Escherichia coli by employing a pTrcHisA expression system. In this report, the authors describe the expression of
the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins.
The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively.
Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed
proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina
platensis. The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where
the enzyme substrates were provided in appropriate concentration. This indicates that the
9
and
12
desaturases
were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an
E. coli host cell using its cofactors system. In contrast,
6
desaturase activity can be detected only in vitro where
electron carriers are provided. This suggests that while this enzyme is expressed in the heterologous host in its
active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the
system of Spirulina platensis.
Introduction
Spirulina platensis is a cyanobacterium that is widely
used in human health food and animal feed due to
its high level of proteins, vitamins and unsaturated
fatty acids. It contains a high level of γ -linolenic acid
(GLA), at 1.5% of the dry weight, or 20–30% of the
total fatty acids. GLA is a precursor in prostaglandin
biosynthesis, which plays a role in a variety of import-
ant processes in human health and diseases. GLA has
thus been used commercially in pharmaceuticals and
food supplements [3, 11].
Unsaturated fatty acid biosynthesis, including
GLA production, is achieved by a process called desat-
uration. The desaturation of fatty acids is an enzymatic
reaction in which a double bond is introduced into a
fatty acid and two molecules of oxygen are completely
reduced to water [7, 16]. In the case of this cyanobac-
terium, three desaturase genes - desC, desA, and desD
- are responsible for the introduction of three double
bonds into fatty acids that have been esterified to gly-
cerolipids. The three double bonds are introduced at
the
9
,
12
and
6
positions of stearic acid (18:0),
oleic acid (18:1
9
), and linoleic acid (18:2
9,12
) re-