Abstract
When the gene desD encoding Spirulina Δ6-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 (γ-linolenic acid), the product of the reaction catalyzed by Δ6-desaturase. The results revealed that in E. coli cells, where cytochrome b 5 is absent and ferredoxin, a natural electron donor of Δ6-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 Δ6-desaturase in E. coli. In comparison to the co-expression of cytochrome b 5 with the Δ6-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Δ6-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 FADH2 (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.
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This research was funded by a grant from the Office of the National Research Council of Thailand.
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Kurdrid, P., Subudhi, S., Cheevadhanarak, S. et al. Effect of two intermediate electron donors, NADPH and FADH2, on Spirulina Δ6-desaturase co-expressed with two different immediate electron donors, cytochrome b 5 and ferredoxin, in Escherichia coli . Mol Biol Rep 34, 261–266 (2007). https://doi.org/10.1007/s11033-006-9040-8
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DOI: https://doi.org/10.1007/s11033-006-9040-8