Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.
When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.
Kurdrid
Pavinee
P
Pilot Plant Development and Training Institute, King Mongkut's University of Technology-Thonburi (Bangkhuntien), Bangkok 10150, Thailand.
Subudhi
Sanjukta
S
Cheevadhanarak
Supapon
S
Tanticharoen
Morakot
M
Hongsthong
Apiradee
A
D001483:Base Sequence; D015153:Blotting, Western; D002849:Chromatography, Gas; D045303:Cytochromes b / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D050602:Linoleoyl-CoA Desaturase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D053545:Spirulina / Q000201:enzymology / Q000235:genetics
D005227:Fatty Acids; D005288:Ferredoxins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; D009249:NADP; D045303:Cytochromes b; D050602:Linoleoyl-CoA Desaturase