Revealing the complementation of ferredoxin by cytochrome b(5) in the Spirulina-Delta(6)-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b(5) domain and Spirulina-Delta(6)-acyl-lipid desaturase

摘要

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b(5). The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-Delta(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-Delta(6) desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b(5) domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-Delta(6) desaturase fused or co-expressed with cytochrome b(5) in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b(5). Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b(5) domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b(5) domain in the desaturation process of Spirulina-Delta(6) desaturase. Furthermore, the free form of cytochrome b(5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.