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标题：Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils
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作者：Edgar B. Cahoon ; John Shanklin
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2000
卷号：97
期号：22
页码：12350-12355
DOI：10.1073/pnas.210276297
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We demonstrate that naturally occurring C14 and C16-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, C18-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 C14 and C16 acyl-ACPs but a relative lack of C18 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 C14 and C16 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.