Role In Fatty Acid Biosynthesis Research Articles

Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis.

ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

Comparative transcriptomic and evolutionary analysis of FAD-like genes of Brassica species revealed their role in fatty acid biosynthesis and stress tolerance

BackgroundFatty acid desaturases (FADs) are involved in regulating plant fatty acid composition by adding double bonds to growing hydrocarbon chain. Apart from regulating fatty acid composition FADs are of great importance, and are involved in stress responsiveness, plant development, and defense mechanisms. FADs have been extensively studied in crop plants, and are broadly classed into soluble and non-soluble fatty acids. However, FADs have not yet been characterized in Brassica carinata and its progenitors.ResultsHere we have performed comparative genome-wide identification of FADs and have identified 131 soluble and 28 non-soluble FADs in allotetraploid B. carinata and its diploid parents. Most soluble FAD proteins are predicted to be resided in endomembrane system, whereas FAB proteins were found to be localized in chloroplast. Phylogenetic analysis classed the soluble and non-soluble FAD proteins into seven and four clusters, respectively. Positive type of selection seemed to be dominant in both FADs suggesting the impact of evolution on these gene families. Upstream regions of both FADs were enriched in stress related cis-regulatory elements and among them ABRE type of elements were in abundance. Comparative transcriptomic data analysis output highlighted that FADs expression reduced gradually in mature seed and embryonic tissues. Moreover, under heat stress during seed and embryo development seven genes remained up-regulated regardless of external stress. Three FADs were only induced under elevated temperature whereas five genes were upregulated under Xanthomonas campestris stress suggesting their involvement in abiotic and biotic stress response.ConclusionsThe current study provides insights into the evolution of FADs and their role in B. carinata under stress conditions. Moreover, the functional characterization of stress-related genes would exploit their utilization in future breeding programs of B. carinata and its progenitors.

In-silico identification of potential inhibitors against FabI protein in Klebsiella pneumoniae

The development of new antimicrobial drugs is needed to combat multi-drug resistant and novel hypervirulent strains of Klebsiella pneumoniae (KPN) that are associated with increased morbidity and mortality globally. The FabI protein plays a crucial role in fatty acid biosynthesis and has been identified as an important target for in-silico, in-vitro, and in-vivo drug discovery. In this study we have used computer integrated-drug discovery approaches and binding-free energy calculations to identify three novel inhibitors (21272541, 67724550, and 67724551) of the FabI protein. All inhibitors showed strong affinity including van der Waals energy, electrostatic energy, polar and non-polar energies; however, the 21272541 compound was the most effective inhibitor and bound with the strongest affinity (ΔGbind −59.02 kcal/mol) to the FabI protein. Nevertheless, all three inhibitors are promising targets for new novel antimicrobial drugs that could contribute to the management of antimicrobial resistant KPN infections based on various computational analysis. Additional in-vitro and in-vivo clinical studies will be needed to confirm drug effectiveness for the treatment of KPN infections.

Evaluation of the Composition and Accumulation Pattern of Fatty Acids in Tartary Buckwheat Seed at the Germplasm Level

Tartary buckwheat seeds not only contain higher contents of bioactive flavonoids, but also are rich in fatty acids. However, the composition, accumulation patterns, and biosynthesis genes of fatty acids in Tartary buckwheat seeds remain largely unclear. Here, we investigated the total lipid content, total flavonoid content, and ten fatty acids in the seeds of 31 different Tartary buckwheat accessions, analyzed the accumulation patterns of ten fatty acids during seed development, and identified the biosynthesis genes of fatty acids. The results indicated that there were significant differences in the total lipid content, total flavonoid content, and ten fatty acids among different Tartary buckwheat accessions. Among these ten fatty acids, the palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid were the most abundant fatty acids in Tartary buckwheat seeds. A total of ten fatty acids displayed five kinds of different accumulation patterns during the development of seeds. A total of 14 genes involved in the biosynthesis of main fatty acid were identified and it was found that FAD5 may play a crucial role in fatty acid biosynthesis in Tartary buckwheat seed. These results not only indicate that Tartary buckwheat is an excellent food source, but also provide helpful information for new cultivar breeding with high health-promotion value.

3-Oxoacyl acyl carrier protein reductase overexpression reveals its unprecedented roles in biofuel production and high-temperature tolerance in diatom

Due to its high lipid accumulation capability, Phaeodactylum tricornutum has attracted considerable attention as a promising candidate for biofuel production. However, the derived lipid cannot satisfy the industrial biofuel manufacturing requirements given its poor fatty acid characteristics. Genetic engineering, as a prospective approach, has been employed to overcome this bottleneck. Relevant research indicates that the fabG gene plays a crucial role in fatty acid biosynthesis in bacteria, but its significance in microalgae remains unclear. In this study, FabG was successfully introduced, transcribed, and overexpressed in P. tricornutum. FabG overexpression significantly enhanced lipid (1.49-fold) and fatty acid (1.31-fold) biosynthesis without altering growth and photosynthesis, and this method thus exhibits great potential in high-quality biodiesel production. Moreover, increased saturation of fatty acids (e.g., C16:0) and improved antioxidative activities enhanced the tolerance of transgenic P. tricornutum to high temperature stress. To conclude, this study reports a feasible approach for high-quality algae-based biofuel production via the unprecedented roles of FabG retrieved from diatom.

Evolution and Functional Characteristics of the Novel elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis.

Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a−/−) and elovl8b (elovl8b−/−) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21

BackgroundAs a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear.ResultsIn this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain.ConclusionTherefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Enhanced lipid production by addition of malic acid in fermentation of recombinant Mucor circinelloides Mc-MT-2

In our previous work, we reported a novel approach for increasing lipid production in an oleaginous fungus Mucor circinelloides by overexpression of mitochondrial malate transporter protein. This transporter plays a vital role in fatty acid biosynthesis during malate and citrate transport systems in oleaginous fungi. In this study, the controlling metabolic supplementation strategy was used to improve the lipid production by overexpression of malate transporter protein in M. circinelloides strain coded as Mc-MT-2. The effects of different metabolic intermediates on lipid production in batch fermentation by Mc-MT-2 were investigated. The optimal lipid production was obtained at 0.8% malic acid after 24 h of fermentation. Furthermore, in fed-batch bioreactors containing glucose as a carbon source supplemented with malic acid, the highest cell growth, and lipid production were achieved. The resulting strain showed the fungal dry biomass of 16 g/L, a lipid content of 32%, lipid yield of 5.12 g/L in a controlled bench-top bioreactor, with 1.60-, 1.60- and 2.56-fold improvement, respectively, compared with the batch control without supplementation of malic acid. Our findings revealed that the addition of malic acid during fermentation might play an important role in lipid accumulation in the recombinant M. circinelloides Mc-MT-2. This study provides valuable insights for enhanced microbial lipid production through metabolic supplementation strategy in large scale and industrial applications.

Proteomic analysis of metabolic mechanisms associated with fatty acid biosynthesis during Styrax tonkinensis kernel development.

Styrax tonkinensis is a white-flowered tree with considerable potential as a feedstock source for biodiesel production from the oily seed contained within its nutlike drupes. Transcriptome changes during oil accumulation have been previously reported, but not concurrent changes in the proteome. Using proteomic analysis of samples collected at 50, 70, 100 and 130 days after flowering (DAF), we identified 1472 differentially expressed proteins (DEPs). Based on their expression patterns, we grouped the DEPs into nine clusters and analyzed the pathway enrichment. Proteins related to starch and sucrose metabolism were most abundant at 50 DAF. Proteins involved in fatty acid (FA) biosynthesis were mainly grouped into a cluster that peaked at 70 DAF. Proteins related to protein processing in endoplasmic reticulum had two major patterns, trending either upwards or downwards, while proteins involved in amino acid biosynthesis showed more complex relationships. We identified 42 key enzymes involved in lipid accumulation during kernel development, including the acetyl-CoA carboxylase complex (ACC) and the pyruvate dehydrogenase complex (PDC). One oil body membrane protein, oleosin, continuously increased during kernel development. A regulatory network of oil accumulation processes was built based on protein and available transcriptome expression data, which were in good temporal agreement. This analysis placed ACC and PDC in the center of the network, suggesting that the glycolytic provision of substrate plays a central regulatory role in FA biosynthesis and oil accumulation. © 2021 Society of Chemical Industry.

Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production.

ABSTRACTThe medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6–C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.

Genome-wide DNA methylation analysis using next-generation sequencing to reveal candidate genes responsible for boar taint in pigs.

Boar taint (BT) is an offensive flavor observed in non-castrated male pigs that reduces the carcass price. Surgical castration effectively avoids the taint but is associated with animal welfare concerns. The functional annotation of farm animal genomes for understanding the biology of complex traits can be used in the selection of breeding animals to achieve favorable phenotypic outcomes. The characterization of pig epigenomes/methylation changes between animals with high and low BT and genome-wide epigenetic markers that can predict BT are lacking. Reduced representation bisulfite sequencing of DNA methylation patterns based on next-generation sequencing is an efficient technology to identify candidate epigenetic biomarkers associated with BT. Three different BT levels were analyzed using reduced representation bisulfite sequencing data to calculate the methylation levels of cytosine and guanine dinucleotide (CpG) sites. The co-analysis of differentially methylated CpG sites identified by this study and differentially expressed genes identified by a previous study found 32 significant co-located genes. The joint analysis of GO terms and pathways revealed that methylation and gene expression of seven candidate genes were associated with BT; in particular, FASN plays a key role in fatty acid biosynthesis, and PEMT might be involved in estrogen regulation and the development of BT. This study is the first to report the genome-wide DNA methylation profiles of BT in pigs using next-generation sequencing and summarize candidate genes associated with epigenetic markers of BT, which could contribute to the understanding of the functional biology of BT traits and selective breeding of pigs against BT based on epigenetic biomarkers.

13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides

BackgroundMitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation.ResultsThe metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains.ConclusionIntracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.

Preparative isolation and purification of steroidal glycoalkaloid from the ripe berries of Solanum nigrum L. by preparative HPLC-MS and UHPLC-TOF-MS/MS and its anti-non-small cell lung tumors effects in vitro and in vivo.

Overcoming epidermal growth factor receptor resistance is a critical problem that needs to be solved in clinical practice. Drugs that downregulate the fatty acid synthase-epidermal growth factor receptor will become novel treatments for non-small cell lung cancer. Solanum nigrum, extracted with water at 4°C, shows strong cytotoxic activity and inhibits tumor growth in Lewis tumor bearing-mice in a dose-dependent manner. A novel active compound in S. nigrum, solaoiacid, was successfully separated and purified from S. nigrum by preparative high-performance liquid chromatography with mass spectrometry and ultra high performance liquid chromatography with time-of-flight tandem mass spectrometry. The IC50 of solaoiacid on lung cancer cells was 2.3 µmol/L, which was significantly lower than that of the known steroidal glycoalkaloid. Label-free proteomics and STRING Network analysis were used to identify significantly deregulated proteins in lung cancer cells that were treated with the fresh ripe fruit extracts of S. nigrum. S. nigrum regulates multiple signal pathways, including the epidermal growth factor receptor pathway. S. nigrum downregulated 24 main proteins with direct roles in fatty acid biosynthesis. Both S. nigrum and solaoiacid showed strong downregulation of the fatty acid synthase-epidermal growth factor receptor and anti-non-small cell lung cancer effects, and thus will become a novel drug for the treatment of non-small cell lung cancer.

Boosting C16 fatty acid biosynthesis of Escherichia coli, yeast and tobacco by tung tree (Vernicia fordii Hemsl.) beta-hydroxyacyl-acyl carrier protein dehydratase gene

Beta-hydroxyacyl-acyl carrier protein dehydratase (HAD) catalyzes the formation of enoyl-[ACP] from beta -hydroxylacyl-[ACP] and plays a key role in determining fatty acid composition during the process of oil biosynthesis. In this study, only one HAD gene (GenBank KU358903) was identified and isolated from tung tree (Vernicia fordii Hemsl.). The VfHAD contained an open reading frame (ORF) of 663 bp, encoding a polypeptide of 220 amino acid residues. It shared 86, 85, and 83% amino acid identities with the HADs of Hevea brasiliensis, Jatropha curcas, and Ricinus communis, respectively. The VfHAD exhibited all essential catalytic sites and the characteristic “hot-dog” structure of FabZ protein. Quantitative real-time PCR (qRT-PCR) analysis showed that VfHAD mRNA was detected in all tung tree organs, but its transcript was the most abundant in tung seeds at 20 weeks after flowering when oil rapidly accumulated. The VfHAD was localized in chloroplast. Heterologous expression of VfHAD significantly increased C16 fatty acids content in Escherichia coli, yeast, and tobacco. This study suggested that VfHAD may play a key role in fatty acid biosynthesis in tung seeds and could be a useful target for genetic improvement of oil quality.

Regulation of multiple abiotic stress tolerance by LexA in the cyanobacterium Anabaena sp. strain PCC7120

The paradigm of involvement of LexA in regulation of only SOS-response in bacteria through the down-regulation of DNA repair genes was challenged in the unicellular cyanobacterium, Synechocystis PCC6803, wherein it was originally shown not to be associated with DNA repair and later also involved in management of carbon-starvation through up-regulation of C-metabolism genes. In the filamentous cyanobacterium, Anabaena sp. strain PCC7120, global stress management role for LexA and a consensus LexA-binding box (AnLexA-box) has been established using a LexA-overexpressing recombinant strain, AnlexA+. High levels of LexA rendered Anabaena cells sensitive to different DNA damage and oxidative stress-inducing agents, through the transcriptional down-regulation of the genes involved in DNA repair and alleviation of oxidative stress. LexA overexpression enhanced the ability of Anabaena to tolerate C-depletion, induced by inhibiting photosynthesis, by up-regulating genes involved in C-fixation and down-regulating those involved in C-breakdown, while maintaining the overall photosynthetic efficiency. A consensus LexA-binding box, AnLexA-box [AGT-N4–11-ACT] was identified upstream of both up- and down-regulated genes using a subset of Anabaena genes identified on the basis of proteomic analysis of AnlexA+ strain along with a few DNA repair genes. A short genome search revealed the presence of AnLexA box in at least 40 more genes, with functional roles in fatty acid biosynthesis, toxin-antitoxin systems in addition to DNA repair, oxidative stress, metal tolerance and C-metabolism. Thus, Anabaena LexA modulates the tolerance to multitude of stresses through transcriptional up/down-regulation of their functional genes directly by binding to the AnLexA Box present in their promoter region.

Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

Silencing of BnTT1 family genes affects seed flavonoid biosynthesis and alters seed fatty acid composition in Brassica napus

TRANSPARENT TESTA1 (TT1) is a zinc finger protein that contains a WIP domain. It plays important roles in controlling differentiation and pigmentation of the seed coat endothelium, and can affect the expression of early biosynthetic genes and late biosynthetic genes of flavonoid biosynthesis in Arabidopsis thaliana. In Brassica napus (AACC, 2n=38), the functions of BnTT1 genes remain unknown and few studies have focused on their roles in fatty acid (FA) biosynthesis. In this study, BnTT1 family genes were silenced by RNA interference, which resulted in yellow rapeseed, abnormal testa development (a much thinner testa), decreased seed weight, and altered seed FA composition in B. napus. High-throughput sequencing of genes differentially expressed between developing transgenic B. napus and wild-type seeds revealed altered expression of numerous genes involved in flavonoid and FA biosynthesis. As a consequence of this altered expression, we detected a marked decrease of oleic acid (C18:1) and notable increases of linoleic acid (C18:2) and α-linolenic acid (C18:3) in mature transgenic B. napus seeds by gas chromatography and near-infrared reflectance spectroscopy. Meanwhile, liquid chromatography–mass spectrometry showed reduced accumulation of flavonoids in transgenic seeds. Therefore, we propose that BnTT1s are involved in the regulation of flavonoid biosynthesis, and may also play a role in FA biosynthesis in B. napus.

Identification of microRNAs Actively Involved in Fatty Acid Biosynthesis in Developing Brassica napus Seeds Using High-Throughput Sequencing.

Seed development has a critical role during the spermatophyte life cycle. In Brassica napus, a major oil crop, fatty acids are synthesized and stored in specific tissues during embryogenesis, and understanding the molecular mechanism underlying fatty acid biosynthesis during seed development is an important research goal. In this study, we constructed three small RNA libraries from early seeds at 14, 21, and 28 days after flowering (DAF) and used high-throughput sequencing to examine microRNA (miRNA) expression. A total of 85 known miRNAs from 30 families and 1160 novel miRNAs were identified, of which 24, including 5 known and 19 novel miRNAs, were found to be involved in fatty acid biosynthesis.bna-miR156b, bna-miR156c, bna-miR156g, novel_mir_1706, novel_mir_1407, novel_mir_173, and novel_mir_104 were significantly down-regulated at 21 DAF and 28 DAF, whereas bna-miR159, novel_mir_1081, novel_mir_19 and novel_mir_555 were significantly up-regulated. In addition, we found that some miRNAs regulate functional genes that are directly involved in fatty acid biosynthesis and that other miRNAs regulate the process of fatty acid biosynthesis by acting on a large number of transcription factors. The miRNAs and their corresponding predicted targets were partially validated by quantitative RT-PCR. Our data suggest that diverse and complex miRNAs are involved in the seed development process and that miRNAs play important roles in fatty acid biosynthesis during seed development.

Analysis of Enoyl-Acyl Carrier Protein Reductase Structure and Interactions Yields an Efficient Virtual Screening Approach and Suggests a Potential Allosteric Site.

Enoyl-acyl carrier protein reductases have an important role in fatty acid biosynthesis and are considered essential for bacterial and protozoal survival. Here, we perform a computational assessment of enoyl-acyl carrier protein reductase structures, providing insights for inhibitor design that we incorporate into a virtual screening approach. Firstly, we analyse 80 crystal structures of 16 different enoyl-acyl carrier protein reductases for their active site characteristics and druggability, finding these sites contain a readily druggable pocket, of varying size and shape. Interestingly, a high affinity, potentially allosteric site was identified for pfFabl. Analysis of the ligand-protein interactions of four enoyl-acyl carrier protein reductases from different micro-organisms (InhA, pfFabl, saFabl and ecFabl), involving 59 available crystal structures, found three commonly shared interactions; constraining these interactions in docking improved enrichment of enoyl-acyl carrier protein reductase virtual screens, by up to 60% in the top 3% of the ranked library. This docking protocol also improved pose prediction, decreasing the root-mean-square deviation to crystallographic pose by up to 75% on average. The binding site analysis and knowledge-based docking protocol presented here can potentially assist in the structure-based design of new enoyl-acyl carrier protein reductase inhibitors.

Characterization of the fatty acyl elongase (elovl) gene family, and hepatic elovl and delta-6 fatty acyl desaturase transcript expression and fatty acid responses to diets containing camelina oil in Atlantic cod (Gadus morhua)

For aquaculture to become sustainable, there is a need to substitute fish oil [FO, rich in ω3 long chain polyunsaturated fatty acids (LC-PUFA) such as 20:5ω3 (EPA) and 22:6ω3 (DHA)] in aquafeed with plant oils such as camelina oil [CO, rich in C18 PUFA such as 18:3ω3 (ALA) and 18:2ω6 (LNA)]. The LC-PUFA are essential components in fish diets for maintaining optimal health, physiology and growth. However, most marine fish including Atlantic cod are inefficient at producing LC-PUFA from shorter chain precursors. Since elovl genes encode enzymes that play key roles in fatty acid biosynthesis, we hypothesized that they may be involved in Atlantic cod responses to diets rich in 18:3ω3 and 18:2ω6. Ten members of the cod elovl gene family were characterized at the mRNA level. RT-PCR was used to study constitutive expression of elovl transcripts in fifteen tissues. Some transcripts (e.g. elovl5) were ubiquitously expressed, while others had tissue-specific expression (e.g. elovl4a in brain and eye). Cod fed a CO-containing diet (100% CO replacement of FO and including solvent-extracted fish meal) had significantly lower weight gain, with significant up-regulation of elovl5 and fadsd6 transcripts in the liver as shown by QPCR analysis, compared with cod on a FO control diet after a 13-week trial. Multivariate statistical analyses (SIMPER and PCA) indicated that high 18:3ω3 and/or low ω3 LC-PUFA levels in the liver were associated with the up-regulation of elovl5 and fadsd6, which are involved in LC-PUFA biosynthesis in cod.