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

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

Desaturase specificity is controlled by the physicochemical properties of a single amino acid residue in the substrate binding tunnel

Membrane fatty acyl desaturases (mFAD) are ubiquitous enzymes in eukaryotes. They introduce double bonds into fatty acids (FAs), producing structurally diverse unsaturated FAs which serve as membrane lipid components or precursors of signaling molecules. The mechanisms controlling enzymatic specificity and selectivity of desaturation are, however, poorly understood. We found that the physicochemical properties, particularly side chain volume, of a single amino acid (aa) residue in insect mFADs (Lepidoptera: Bombyx mori and Manduca sexta) control the desaturation products. Molecular dynamics simulations of systems comprising wild-type or mutant mFADs with fatty acyl-CoA substrates revealed that the single aa substitution likely directs the outcome of the desaturation reaction by modulating the distance between substrate fatty acyl carbon atoms and active center metal ions. These findings, as well as our methodology combining mFAD mutational screening with molecular dynamics simulations, will facilitate prediction of desaturation products and facilitate engineering of mFADs for biotechnological applications

Uncovering tomato quantitative trait loci and candidate genes for fruit cuticular lipid composition using the Solanum pennellii introgression line population

[EN] The cuticle is a specialized cell wall layer that covers the outermost surface of the epidermal cells and has important implications for fruit permeability and pathogen susceptibility. In order to decipher the genetic control of tomato fruit cuticle composition, an introgression line (IL) population derived from a biparental cross between Solanum pennellii (LA0716) and the Solanum lycopersicum cultivar M82 was used to build a first map of associated quantitative trait loci (QTLs). A total of 24 cuticular waxes and 26 cutin monomers were determined. They showed changes associated with 18 genomic regions distributed in nine chromosomes affecting 19 ILs. Out of the five main fruit cuticular components described for the wild species S. pennellii, three of them were associated with IL3.4, IL12.1, and IL7.4.1, causing an increase in n-alkanes (>= C-30), a decrease in amyrin content, and a decrease in cuticle thickness of similar to 50%, respectively. Moreover, we also found a QTL associated with increased levels of amyrins in IL3.4. In addition, we propose some candidate genes on the basis of their differential gene expression and single nucleotide polymorphism variability between the introgressed and the recurrent alleles, which will be the subjects of further investigation.Research at the IBMCP was supported by the Spanish Ministry of Education and Culture (BIO2013-42193-R) and H2020 TRADITOM (634561). AA, AG, and J-PF-M thank COST FA1106 Quality Fruit for STSM and networking activities. This work was supported by the Israel Science Foundation (ISF) personal grant to AA (grant no. 646/11). We would like to thank the Adelis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research, and the Raymond Burton Plant Genome Research Fund for supporting AA's laboratory activity. AA is the incumbent of the Peter J. Cohn Professorial Chair. We are very grateful to Prof. Dani Zamir for providing us the S. pennellii IL collection and to Prof. Antonio Heredia for his valuable advice in preparing the manuscript for publication. We would like to acknowledge the help offered by the Electron Microscopy Unit at the WIS (Israel) for the TEM sample preparation and imaging, especially Elena Kartvelishvily, Eugenia Klein, and Eyal Shimoni. Finally, we would also like to thank Calanit Raanan and Tamara Berkutzki (Department of Veterinary Resources, WIS) for their help in tissue fixation and embedding, as well as Hanna Levanony (Department of Plant Sciences, WIS) for her help in tissue staining for the light microscopy studies.Fernández Moreno, JP.; Levy-Samoha, D.; Malitsky, S.; Monforte Gilabert, AJ.; Orzáez Calatayud, DV.; Aharoni, A.; Granell Richart, A. (2017). Uncovering tomato quantitative trait loci and candidate genes for fruit cuticular lipid composition using the Solanum pennellii introgression line population. Journal of Experimental Botany. 68(11):2703-2716. https://doi.org/10.1093/jxb/erx134S27032716681

Functional characterisation of a Fads2 fatty acyl desaturase with delta6/delta8 activity and an Elovl5 with C16, C18 and C20 elongase activity in the anadromous teleost meagre (Argyrosomus regius)

The meagre, Argyrosomus regius, is a carnivorous fish with great potential to diversify finfish aquaculture in the Mediterranean. However, currently nothing is known about their essential fatty acid requirements. Meagres are marine fish but also display anadromous behaviour migrating to river estuaries to spawn, and thus may provide an insight to the influence of diadromy on biosynthetic ability for long-chain polyunsaturated fatty acid (LC-PUFA). Our primary aim was to characterise key cDNAs (fatty acyl desaturases and elongases) of the biosynthetic pathway as a key step to establish the capacity of meagre for LC-PUFA biosynthesis from shorter chain PUFA. The cDNA sequences of a fatty acyl desaturase (Fads) and elongase of very long-chain fatty acids (Elovl) were obtained using PCR-based methodologies, and function of the proteins was investigated by expression of the coding sequences of the putative desaturase and elongase in the yeast Saccharomyces cerevisiae. The tissue distribution of both cDNAs was studied by reverse transcription PCR. Our results demonstrated that meagre possesses at least one fatty acyl desaturase and one elongase involved in the endogenous production of LC-PUFA. The meagre desaturase and elongase were identified as orthologues of Fads2 and Elovl5, respectively. Functionally, the desaturase had dual Δ6/Δ8 activity, whereas the elongase exhibited high elongation efficiency for C18 and C20 PUFA with low activity towards C22 PUFA. However, we also showed that the meagre Elovl5 elongated 16:3n - 3 to 18:3n - 3, the first time that C16 elongation activity had been demonstrated for a fish elongase. Similar to other marine teleosts, expression of fads2 and elovl5 transcripts was highest in brain. The functions and expression of the meagre Fads2 and Elovl5 proteins suggest that the meagre has a ‘marine type' LC-PUFA biosynthetic pathway, and that its anadromous behaviour has no major influence

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

<p>Abstract</p> <p>Background</p> <p><it>Siraitia grosvenorii </it>(Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in <it>S. grosvenorii</it>, especially the late steps of the pathway.</p> <p>Results</p> <p>In this study, a cDNA library generated from of equal amount of RNA taken from <it>S. grosvenorii </it>fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as the candidates most likely to be involved in mogrosides biosynthesis.</p> <p>Conclusion</p> <p>A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from <it>S. grosvenorii</it>.</p

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

<p>Abstract</p> <p>Background</p> <p><it>Bupleurum chinense </it>DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of <it>B. chinense</it>, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.</p> <p>Results</p> <p>One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A <it>de novo </it>assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the <it>Bupleurum </it>genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel <it>Bupleurum </it>genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (<it>P450</it>s) and 102 glycosyltransferases (<it>GT</it>s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 <it>P450</it>s and 7 uridine diphosphate <it>GT</it>s (<it>UGT</it>s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two <it>P450</it>s and three <it>UGT</it>s were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with <it>β-AS </it>in methyl jasmonate-treated adventitious roots and on their similar expression patterns with <it>β-AS </it>in various <it>B. chinense </it>tissues.</p> <p>Conclusions</p> <p>A collection of high-quality ESTs for <it>B. chinense </it>obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of <it>B. chinense </it>and other <it>Bupleurum </it>species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the <it>P450</it>s and <it>UGT</it>s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.</p

The front-end desaturase : structure, function, evolution and biotechnological use

Very long chain polyunsaturated fatty acids such as arachidonic acid (ARA, 20:4n\u20136), eicosapentaenoic acid (EPA, 20:5n\u20133), docosapentaenoic acid (DPA, 22:5n\u20133) and docosahexaenoic acid (DHA, 22:6n\u20133) are essential components of cell membranes, and are precursors for a group of hormone-like bioactive compounds (eicosanoids and docosanoids) involved in regulation of various physiological activities in animals and humans. The biosynthesis of these fatty acids involves an alternating process of fatty acid desaturation and elongation. The desaturation is catalyzed by a unique class of oxygenases called front-end desaturases that introduce double bonds between the pre-existing double bond and the carboxyl end of polyunsaturated fatty acids. The first gene encoding a front-end desaturase was cloned in 1993 from cyanobacteria. Since then, front-end desaturases have been identified and characterized from a wide range of eukaryotic species including algae, protozoa, fungi, plants and animals including humans. Unlike front-end desaturases from bacteria, those from eukaryotes are structurally characterized by the presence of an N-terminal cytochrome b5\u2013like domain fused to the main desaturation domain. Understanding the structure, function and evolution of front-end desaturases, as well as their roles in the biosynthesis of very long chain polyunsaturated fatty acids offers the opportunity to engineer production of these fatty acids in transgenic oilseed plants for nutraceutical markets.Peer reviewed: YesNRC publication: Ye

A Peroxygenase Pathway Involved in the Biosynthesis of Epoxy Fatty Acids in Oat[W][OA]

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation