2016
DOI: 10.1002/bit.25922
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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 Sa… Show more

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Cited by 10 publications
(14 citation statements)
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References 39 publications
(43 reference statements)
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“…Overexpression of this particular desaturase led to the enrichment of intracellular monounsaturated fatty acids and lipid accumulation (Qiao et al, 2015). Fatty acid desaturases, which are membrane-bound proteins, catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated and polyunsaturated fatty acids (PUFA) (Bai et al, 2016;Díaz et al, 2018). The fatty acid synthetic pathway includes a variety of desaturases, which are specific to the location, number, and stereochemistry of double bonds present in the fatty acids (Fickers et al, 2003).…”
Section: Introductionmentioning
confidence: 99%
“…Overexpression of this particular desaturase led to the enrichment of intracellular monounsaturated fatty acids and lipid accumulation (Qiao et al, 2015). Fatty acid desaturases, which are membrane-bound proteins, catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated and polyunsaturated fatty acids (PUFA) (Bai et al, 2016;Díaz et al, 2018). The fatty acid synthetic pathway includes a variety of desaturases, which are specific to the location, number, and stereochemistry of double bonds present in the fatty acids (Fickers et al, 2003).…”
Section: Introductionmentioning
confidence: 99%
“…Our directed evolution of the DESG 16:0-glycerolipid desaturase from Synechococcus elongatus PCC6301 targeted to the ER of yeast provided several variants with greatly improved activity (Bai et al, 2016). One of these, DES9*15, provides for considerable conversion of 16:0 to 16:1c both in yeast and when expressed in Arabidopsis seeds (Bai et al, 2016). We used this variant, herein called DES9*, in constructs similar to those described above for FAT-5 to target the protein to the ER or to the chloroplast.…”
Section: Supplementalmentioning
confidence: 99%
“…DNA sequences encoding the FAT-5 16:0-CoA desaturase from C. elegans (Watts and Browse, 2000) and the DES9*15 16:0-glycerolipid desaturase produced by directed evolution (Bai et al, 2016) were each recloned into a pENTR vector using the TOPO Cloning Kit (Invitrogen), following the manufacturer's instructions. After confirmation of the sequences, they were transferred by Gateway cloning into the binary plasmid PB2GW7, under control of the cauliflower mosaic virus 35S promoter (Karimi et al, 2002).…”
Section: Constructs Encoding the Desaturases And Plant Transformationmentioning
confidence: 99%
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