Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity

Abstract

Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

“…These microbial systems are routinely used to characterize the enzymatic activities of various acyl-ACP thioesterases (Jha et al, 2010;Jing et al, 2011;Jones et al, 1995;Kalinger et al, 2018;Leonard et al, 1998;Pulsifer et al, 2012). Additional focus has also been placed on engineering these microbial systems for increased medium-chain fatty acid yield, and for improved utilization of abundant carbon sources (Cao et al, 2014;Fan et al, 2013;Goh et al, 2012;Goh et al, 2014;Lennen et al, 2011;Lin et al, 2018;Steen et al, 2010;Wu and Sun, 2014;Xu et al, 2019). Another major area of research interest surrounds the development of transgenic oilseed crops that incorporate medium-chain fatty acids into triacylglycerols by heterologously expressing medium-chain specific thioesterases from other plant species, such as the specialized FATB enzymes from Cuphea spp.…”

“…Overexpressing LACS genes from Arabidopsis and Brassica napus improves storage lipid accumulation in yeast (Pulsifer et al, 2012;Tan et al, 2014). However, preferential activation of undesirable acyl chain lengths, low activity of LACS enzymes in heterologous hosts, or the inability of such hosts to efficiently incorporate acyl-CoA thioesters of certain chain lengths into larger lipid molecules, are major bottlenecks in the sustainable biological production of valuable lipids (Xu et al, 2019). As with acyl-ACP thioesterases, recombinant plant LACS enzymes that are engineered for increased activity in heterologous hosts, or for preference toward particular fatty acid chain lengths, could therefore be used for more efficient lipid production in microbial systems, or to more effectively alter storage lipid composition in oilseed crops.…”

“…Engineering of a LACS was first accomplished in E. coli, where variants of the LACS enzyme FadD with preference for medium-chain fatty acids were generated via error-prone PCR (Ford and Way, 2015). The first report on an engineered plant LACS was recently published, in which two mutants of the plastidlocalized Arabidopsis LACS9 with greatly increased activity were described (Xu et al, 2019). The amino acid substitutions responsible for their enhanced activity occur at sites that are conserved in LACS9 homologues across eudicot species, and introducing these substitutions in a LACS9 homolog from another eudicot plant may therefore have similar effects (Xu et al, 2019).…”

“…The first report on an engineered plant LACS was recently published, in which two mutants of the plastidlocalized Arabidopsis LACS9 with greatly increased activity were described (Xu et al, 2019). The amino acid substitutions responsible for their enhanced activity occur at sites that are conserved in LACS9 homologues across eudicot species, and introducing these substitutions in a LACS9 homolog from another eudicot plant may therefore have similar effects (Xu et al, 2019). Expression of a LACS9 mutant with increased activity could be used to enhance lipid accumulation in microbial systems, or to boost triacylglycerol production in oilseeds when combined with overexpression of one or multiple glyceraldehyde-3-phosphate acyltransferases involved in triacylglycerol biosynthesis (Chapman and Ohlrogge, 2012;Shockey et al, 2016;Xu et al, 2019).…”

Metabolism of Storage Lipids and the Role of Lipid Droplets in the Yeast Schizosaccharomyces pombe

“…In addition to understanding FA channeling mechanisms present in plants that naturally produce punicic acid, such studies can lead to the development of recombinant S. pombe strains capable of producing oils highly enriched in punicic acid. For the biotechnological production, increased accumulation of punicic acid in TAG might also be achieved by a directed evolution of enzymes involved in FA activation and storage lipid biosynthesis (Chen et al, 2017;Greer et al, 2015;Siloto et al, 2009b;Xu et al, 2017Xu et al, , 2019. Another possibility to increase valueadded lipid production in S. pombe is the conversion to "oleaginous yeast" capable of accumulating high amounts of TAG, which was recently done with S. cerevisiae (Kamisaka et al, 2013).…”