title: Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.
abstract: The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl-CoA substrate pool, through the co-expression of acyl-ACP thioesterases, to direct the accumulation of medium-chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl-CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non-native WEs in the seeds of transgenic Camelina plants. A total of fourteen constructs were prepared containing selected FAR and WS genes in combination with an acyl-ACP thioesterase. All enzyme combinations led to the successful production of wax esters, of differing compositions. The impact of acyl-CoA thioesterase expression on wax ester accumulation varied depending on the substrate specificity of the WS. Hence, co-expression of acyl-ACP thioesterases with Marinobacter hydrocarbonoclasticus WS and Marinobacter aquaeolei FAR resulted in the production of WEs with reduced chain lengths, whereas the co-expression of the same acyl-ACP thioesterases in combination with Mus musculus WS and M. aquaeolei FAR had little impact on the overall final wax composition. This was despite substantial remodelling of the acyl-CoA pool, suggesting that these substrates were not efficiently incorporated into WEs. These results indicate that modification of the substrate pool requires careful selection of the WS and FAR activities for the successful high accumulation of these novel wax ester species in Camelina seeds.
authors.affiliation: IHSM-UMA-CSIC, Universidad de Málaga, Málaga, Spain.|Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Noemi
authors.initials: N
authors.lastname: Ruiz-Lopez
authors.affiliation: Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Broughton
authors.affiliation: Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Usher
authors.affiliation: Instituto de la Grasa, Universitario Pablo de Olavide, Seville, Spain.
authors.forename: Joaquin J
authors.initials: JJ
authors.lastname: Salas
authors.affiliation: Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Haslam
authors.affiliation: Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Johnathan A
authors.initials: JA
authors.lastname: Napier
authors.affiliation: Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts, UK.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Beaudoin
mesh_terms: D028244:Camellia / Q000235:genetics / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism; D014885:Waxes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004952:Esters; D014885:Waxes; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase
keywords: Camelina; metabolic engineering; seed lipids; wax esters