The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5alpha. Heterologous expression of Spirulina-desaturase genes.
The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-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 delta9 and delta12 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, delta6 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.
Apiradee
Hongsthong
H
BEC Unit, National Center for Genetic Engineering and Biotechnology, 83 Moo8, Thakham, Bangkhuntien, Bangkok 10150, Thailand. apiradee@biotec.or.th
Kalyanee
Paithoonrangsarid
P
Pongsathon
Prapugrangkul
P
Patcharaporn
Deshnium
D
Matura
Sirijuntarut
S
Sanjukta
Subhudhi
S
Supapon
Cheevadhanarak
C
Morakot
Tanticharoen
T
D001426:Bacterial Proteins / Q000235:genetics*; D001692:Biological Transport; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000096:biosynthesis* / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D050356:Lipid Metabolism; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D053545:Spirulina; D013997:Time Factors
D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases