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

Appl Microbiol Biotechnol. 2006 Oct;72(6):1192-201. doi: 10.1007/s00253-006-0407-5. Epub 2006 Mar 31.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Artificial Gene Fusion*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / physiology
  • Blotting, Western
  • Cytochromes b5 / genetics*
  • Cytochromes b5 / physiology
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Ferredoxins / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / physiology
  • Gas Chromatography-Mass Spectrometry
  • Gene Expression
  • Linoleoyl-CoA Desaturase / genetics*
  • Linoleoyl-CoA Desaturase / metabolism*
  • Molecular Sequence Data
  • Mucor / enzymology
  • Mucor / genetics
  • Mutagenesis, Site-Directed
  • Mutation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Spirulina / enzymology*
  • Spirulina / genetics
  • gamma-Linolenic Acid / metabolism

Substances

  • Bacterial Proteins
  • Ferredoxins
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • gamma-Linolenic Acid
  • Cytochromes b5
  • Linoleoyl-CoA Desaturase