title: Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems.
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.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington.
authors.forename: Shuangyi
authors.initials: S
authors.lastname: Bai
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington.
authors.forename: James G
authors.initials: JG
authors.lastname: Wallis
authors.affiliation: Bayer CropScience N.V., Technologiepark 38, Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Denolf
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington. jab@wsu.edu.
authors.forename: John
authors.initials: J
authors.lastname: Browse
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D010938:Plant Oils / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D010938:Plant Oils; D044943:Fatty Acid Desaturases
keywords: Anacystis nidulans; Arabidopsis; PCC6301; Saccharomyces cerevisiae; Synechococcus elongatus; cyanobacteria; directed evolution; electron carrier; fatty acid; metabolic engineering; oilseed biotechnology; saturated fatty acids