Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems. Issue 7 (3rd February 2016)

• Record Type: Journal Article
• Authors: Bai, Shuangyi; Wallis, James G.; Denolf, Peter; Browse, John
• Is Part Of: Biotechnology and bioengineering. Volume 113:Issue 7(2016)
• Page Start: 1522
• Page End: 1530
• Publication Date: 2016-02-03
• Subjects: Saccharomyces cerevisiae -- fatty acid -- directed evolution -- metabolic engineering -- Arabidopsis -- oilseed biotechnology -- cyanobacteria -- saturated fatty acids -- Synechococcus elongatus -- PCC6301 -- electron carrier -- Anacystis nidulans; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25922
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon‐optimized DSG open reading frame with an endoplasmic‐reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ . Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently‐detected single amino acid change, Q240R, yielded a nearly 25‐fold increase in total desaturation in S. cerevisiae . Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60‐fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine‐rich regions known to be critical for electron transfer mediated by these di‐iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522–1530. © 2016 Wiley Periodicals, Inc. Abstract : Directed evolution of the DSG cyanobacterial fatty acid desaturase expressed in Saccharomyces cerevisiae discovered amino acids important to increased desaturation. The evolved desaturases demonstrated up to 60‐fold more desaturation than the parent enzyme. Evolved enzymes also exhibited increased desaturation when expressed in Arabidopsis thaliana seed, where they increased monounsaturated fatty acids and reduced saturated fatty acids, in some cases by one‐half. This strategy may be useful in discovering modified versions of other prokaryotic desaturases to engineer lipid composition of eukaryotes.