Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils.
We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.
Cahoon
E B
EB
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Shanklin
J
J
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D002368:Castor Oil / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D016296:Mutagenesis; D012639:Seeds / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D002368:Castor Oil; D044943:Fatty Acid Desaturases