Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds.
The polyenoic fatty acid isomerase from Propioniumbacterium acnes (PAI) was expressed in E. coli and biochemically characterized. PAI catalyzes the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating (10E,12Z)-conjugated linoleic acid (CLA). PAI accepted a wide range of free polyunsaturated fatty acids as substrates ranging from 18:2 fatty acids to 22:6, converting them to fatty acids with two or three conjugated double bonds. For expression of PAI in yeast the PAI-sequence encoding 20 N-terminal amino acid residues was altered for optimal codon usage, yielding codon optimized PAI (coPAI). The percentage of 10,12-CLA of total esterified fatty acids was 8 times higher in yeast transformed with coPAI than in cells transformed with PAI. CLA was detected in amounts up to 5.7% of total free fatty acids in yeast transformed with coPAI but none was detected in yeast transformed with PAI. PAI or coPAI under the control of the constitutive CaMV 35S promoter or the seed-specific USP promoter was transformed into tobacco plants. CLA was only detected in seeds in coPAI-transgenic plants. The amount of CLA detected in esterified fatty acids was up to 0.3%, in free fatty acids up to 15%.
Hornung
Ellen
E
Georg-August-University Göttingen, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Krueger
Claudia
C
Pernstich
Christian
C
Gipmans
Martijn
M
Porzel
Andrea
A
Feussner
Ivo
I
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D006863:Hydrogen-Ion Concentration; D044243:Linoleic Acids, Conjugated / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003062:Codon; D044243:Linoleic Acids, Conjugated; D011994:Recombinant Proteins; D019748:Carbon-Carbon Double Bond Isomerases; C105113:polyenoic fatty acid isomerase