Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Edgar B Cahoon, John Shanklin

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57 Citations (Scopus)

Abstract

We demonstrate that naturally occurring C14 and C16-specific acylacyl 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 Δ9-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 Δ9-18:0-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 Δ9-18:0-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.

Original languageEnglish (US)
Pages (from-to)12350-12355
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number22
DOIs
StatePublished - Oct 24 2000

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Fatty Acid Desaturases
Metabolic Engineering
Plant Oils
Seeds
Carrier Proteins
Unsaturated Fatty Acids
Escherichia coli
Oils
Acyl Carrier Protein
Monounsaturated Fatty Acids
Substrate Specificity
Arabidopsis
Growth

ASJC Scopus subject areas

  • General

Cite this

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title = "Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils",
abstract = "We demonstrate that naturally occurring C14 and C16-specific acylacyl 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 Δ9-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 Δ9-18:0-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 Δ9-18:0-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.",
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T1 - Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

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AU - Shanklin, John

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N2 - We demonstrate that naturally occurring C14 and C16-specific acylacyl 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 Δ9-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 Δ9-18:0-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 Δ9-18:0-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.

AB - We demonstrate that naturally occurring C14 and C16-specific acylacyl 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 Δ9-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 Δ9-18:0-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 Δ9-18:0-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.

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