A single-cell platform for reconstituting and characterizing fatty acid elongase component enzymes

Abstract

Fatty acids of more than 18-carbons, generally known as very long chain fatty acids (VLCFAs) are essential for eukaryotic cell viability, and uniquely in terrestrial plants they are the precursors of the cuticular lipids that form the organism’s outer barrier to the environment. VLCFAs are synthesized by fatty acid elongase (FAE), which is an integral membrane enzyme system with multiple components. The genetic complexity of the FAE system, and its membrane association has hampered the biochemical characterization of FAE. In this study we computationally identified Zea mays genetic sequences that encode the enzymatic components of FAE and developed a heterologous expression system to evaluate their functionality. The ability of the maize components to genetically complement Saccharomyces cerevisiae lethal mutants confirmed the functionality of ZmKCS4, ZmELO1, ZmKCR1, ZmKCR2, ZmHCD and ZmECR, and the VLCFA profiles of the resulting strains were used to infer the ability of each enzyme component to determine the product profile of FAE. These characterizations indicate that the product profile of the FAE system is an attribute shared among the KCS, ELO, and KCR components of FAE.

The FUSED LEAVES1‐ADHERENT1 regulatory module is required for maize cuticle development and organ separation

“…KCS genes often show distinct substrate specificity, although the biological role of most KCS genes remain largely unknown (Haslam & Kunst, 2013). Previous phylogenetic analysis revealed eight evolutionarily conserved subclasses of KCS genes, including 21 from Arabidopsis and 26 from maize (Joubes et al ., 2008; Campbell et al ., 2019), and we included two additional genes present in the maize B73v3 reference genome. AD1/ZmKCS19 lies within the θ subclass that contains three uncharacterized Arabidopsis KCS genes, and four additional maize homologs (Fig.…”

Dynamic product-precursor relationships underlie cuticular lipid accumulation on maize silks

“…These include variations in the substrate preferences of 1) the VLCFA elongation system; 2) the reductase that converts VLCFAs to aldehydes; and 3) the decarbonylase that converts aldehydes to hydrocarbons. Indeed, chain length preferences have been identified for several of the 21 ketoacyl-CoA synthetases (KCSs) responsible for the condensation reaction of the fatty acid elongase that generates VLCFAs of Arabidopsis (Millar and Kunst, 1997; Fiebig et al, 2000; Blacklock and Jaworski, 2006; Joubès et al, 2008; Kim et al, 2013; Hegebarth et al, 2016; Hegebarth et al, 2017) and similar preferences are likely to exist amongst the 26 putative KCSs identified in maize (Campbell et al, 2019). Moreover, the observed substrate specificities of the alkane-forming reductase-decarbonylase enzyme complexes may be associated with homologs of the Arabidopsis CER1 and CER1-LIKE1 proteins, which can each form a unique reductase-decarbonylase complex with CER3 homologs (Pascal et al, 2019).…”

“…Moreover, the observed substrate specificities of the alkane-forming reductase-decarbonylase enzyme complexes may be associated with homologs of the Arabidopsis CER1 and CER1-LIKE1 proteins, which can each form a unique reductase-decarbonylase complex with CER3 homologs (Pascal et al, 2019). These hypotheses could be tested via heterologous expression of candidate genes within the recently engineered yeast system expressing the maize fatty acid elongase complex (Campbell et al, 2019; Pascal et al, 2019).…”

Biosynthesis and Functions of Very-Long-Chain Fatty Acids in the Responses of Plants to Abiotic and Biotic Stresses

“…Phylogenetic analyses then emphasized the variable genetic diversity of the FAE components across different plant species. Whereas the ECR subunit is often encoded by a single-copy gene, the condensing enzyme KCS is usually encoded by a very large multi-copy gene family: 21 putative isoenzymes were identified in arabidopsis [ 30 ], 28 in maize ( Zea mays ) [ 38 ], 30 in peanut ( Arachis hypogea ) [ 39 ] and as much as 58 in rapeseed ( Brassica napus ) [ 40 ]. Multiple-copy genes can encode the KCR and HCD components, but with a much lower diversity.…”

“…Multiple-copy genes can encode the KCR and HCD components, but with a much lower diversity. In maize, ZmKCR1 and ZmKCR2 are both able to catalyze the reduction of 3-ketoacyl-CoA intermediates with different chain-length outcomes [ 38 , 41 ]. However, only one duplicated homolog from arabidopsis shows this catalytic potential [ 31 ].…”