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.

Overexpression of △12, △15-Desaturases for Enhanced Lipids Synthesis in Yarrowia lipolytica

“…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).…”

Phosphatidylglycerol Composition Is Central to Chilling Damage in the Arabidopsis fab1 Mutant

“…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.…”

“…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).…”

“…These results indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids; photosynthesis is the one process that requires high levels of polyunsaturation. Mutants with smaller changes in unsaturation are often similar to wild type (WT) under typical growthchamber conditions and reveal their phenotypes only under more-extreme conditions (Wallis andBrowse, 2002, 2010). Several mutants grow more slowly and become chlorotic at temperatures in the range 2° to 10°C (Hugly and Somerville 1992;Routaboul et al, 2000), indicating a role for fatty acid composition in maintaining photosynthesis at these low temperatures.…”

“…Here, we have taken a new approach to investigating the role of HMP-PG in damage and death of fab1 plants at chilling temperatures by using a 16:0-CoA desaturase from Caenorhabditis elegans, FAT-5 (Watts and Browse, 2000), and a glycerolipid desaturase, DES9*15, derived from a cyanobacterial enzyme by directed evolution (Bai et al, 2016). When expressed in the fab1 mutant background, both the FAT-5 enzyme targeted to the ER and the DES9*15 enzyme targeted to the chloroplast reduced leaf 16:0 to levels close to WT levels.…”