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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.

SUBMITTER: Ruiz-Lopez N

PROVIDER: S-EPMC5466440 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

Ruiz-Lopez Noemi N Broughton Richard R Usher Sarah S Salas Joaquin J JJ Haslam Richard P RP Napier Johnathan A JA Beaudoin Frédéric F

Plant biotechnology journal 20170202 7

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 additi ...[more]

PMID: 27990737

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Project description:BackgroundSimmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.ResultsDuring 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.ConclusionsIn in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Project description:BACKGROUND:It is important to explore renewable alternatives (e.g. biofuels) that can produce energy sources to help reduce reliance on fossil oils, and reduce greenhouse gases and waste solids resulted from fossil oils consumption. Camelina sativa is an oilseed crop which has received increasing attention due to its short life cycle, broader adaptation regions, high oil content, high level of omega-3 unsaturated fatty acids, and low-input requirements in agriculture practices. To expand its Camelina production areas into arid regions, there is a need to breed for new drought-tolerant cultivars. Leaf cuticular wax is known to facilitate plant development and growth under water-limited conditions. Dissecting the genetic loci underlying leaf cuticular waxes is important to breed for cultivars with improved drought tolerance. RESULTS:Here we combined phenotypic data and single nucleotide polymorphism (SNP) data from a spring C. sativa diversity panel using genotyping-by-sequencing (GBS) technology, to perform a large-scale genome-wide association study (GWAS) on leaf wax compositions. A total of 42 SNP markers were significantly associated with 15 leaf wax traits including major wax components such as total primary alcohols, total alkanes, and total wax esters as well as their constituents. The vast majority of significant SNPs were associated with long-chain carbon monomers (carbon chain length longer than C28), indicating the important effects of long-chain carbon monomers on leaf total wax biosynthesis. These SNP markers are located on genes directly or indirectly related to wax biosynthesis such as maintaining endoplasmic reticulum (ER) morphology and enabling normal wax secretion from ER to plasma membrane or Golgi network-mediated transport. CONCLUSIONS:These loci could potentially serve as candidates for the genetic control involved in intracellular wax transport that might directly or indirectly facilitate leaf wax accumulation in C. sativa and can be used in future marker-assisted selection (MAS) to breed for the cultivars with high wax content to improve drought tolerance.

Dataset Information

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

Publications

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

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