Revealing the complementation of ferredoxin by cytochrome b (5) in the Spirulina- (6)-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b (5) domain and Spirulina- (6)-acyl-lipid desaturase.
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-(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-(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-(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-(6) desaturase. Furthermore, the free form of cytochrome b (5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.
Hongsthong
Apiradee
A
National Center for Genetic Engineering and Biotechnology, 83 Moo8, Thakham, Bangkhuntien, Bangkok, 10150, Thailand. apiradee@biotec.or.th
Subudhi
Sanjukta
S
Sirijuntarut
Matura
M
Kurdrid
Pavinee
P
Cheevadhanarak
Supapon
S
Tanticharoen
Morakot
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D015786:Cytochromes b5 / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005288:Ferredoxins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D050602:Linoleoyl-CoA Desaturase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D053545:Spirulina / Q000201:enzymology* / Q000235:genetics; D017965:gamma-Linolenic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D005288:Ferredoxins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D017965:gamma-Linolenic Acid; D015786:Cytochromes b5; D050602:Linoleoyl-CoA Desaturase