C20 Polyunsaturated Fatty Acids Research Articles

Insect immune resolution with EpOME/DiHOME and its dysregulation by their analogs leading to pathogen hypersensitivity

Upon immune challenge, recognition signals trigger insect immunity to remove the pathogens through cellular and humoral responses. Various immune mediators propagate the immune signals to nearby tissues, in which polyunsaturated fatty acid (PUFA) derivatives play crucial roles. However, little was known on how the insects terminate the activated immune responses after pathogen neutralization. Interestingly, C20 PUFA was detected at the early infection stage and later C18 PUFAs were induced in a lepidopteran insect, Spodoptera exigua. This study showed the role of epoxyoctadecamonoenoic acids (EpOMEs) in the immune resolution at the late infection stage to quench the excessive and unnecessary immune responses. In contrast, dihydroxy-octadecamonoenoates (DiHOMEs) were the hydrolyzed and inactive forms of EpOMEs. The hydrolysis is catalyzed by soluble epoxide hydrolase (sEH). Inhibitors specific to sEH mimicked the immunosuppression induced by EpOMEs. Furthermore, the inhibitor treatments significantly enhanced the bacterial virulence of Bacillus thuringiensis against S. exigua. This study proposes a negative control of the immune responses using EpOME/DiHOME in insects.

Identification of Conjugated Dienes of Fatty Acids in Vischeria sp. IPPAS C-70 under Oxidative Stress.

The microalgae Vischeria sp. IPPAS C-70 produces eicosapentaenoic acid. Several stresses cause the formation of fatty acid peaks that resemble hexadecadienoic acids. We used the integrated technique including TLC, HPLC, and GC-MS to search and determine these fatty acids. Double bond positioning in these fatty acids indicated that they were conjugated dienes and allenes. We identified and described natural nine isomers of C16 polyunsaturated fatty acids, including common methylene-interrupted dienes (Δ6,9-16:2, Δ7,10-16:2, Δ9,12-16:2), and unusual conjugated dienes (Δ6,8-, Δ7,9-, Δ8,10-, Δ9,11-, and Δ10,12-16:2), as well as allenic diene (Δ9,10-16:2). We hypothesize that the formation of conjugated dienes and allenes among fatty acids is the result of oxidative stress caused by H2O2. Hydrogen peroxide also caused an increase in saturated at the expense of unsaturated fatty acids, suggesting inhibition either fatty acid desaturases activities or the corresponding gene expression.

Biotransformation of C20- and C22-polyunsaturated fatty acids and fish oil hydrolyzates to R,R-dihydroxy fatty acids as lipid mediators using double-oxygenating 15R-lipoxygenase

C20- and C22-dihydroxy fatty acids (DiHFAs) are bioactive lipid mediators (LMs) in humans.

Sustainable utilization of unsaleable walnut kernel in substitution for corn grain in the diet of fattening lambs improves growth performance and meat quality.

As a cost-effective typical co-product of walnut processing; this study aimed to investigate the effects of feeding poor quality, unsaleable walnut kernel, also known as wasted walnut (WW) kernel, on growth performance, carcass characteristics, meat quality and fatty acid (FA) composition of fattening lambs. The experiment was conducted using 24 fattening lambs (27 ±1.07 kg initial BW) assigned to two total mixed concentrate-based diets, over a 80-day experimental period on a completely randomized design. Dietary treatments were: 1) WW (10% WW), and 2) CON (without WW). Average daily and total gain, and final weight increased with WW inclusion (P < 0.05). Also, a significant enhancement in feed conversion ratio (P < 0.05) and carcass cooler shrink loss (P = 0.03) were observed by feeding WW. Dry matter intake (DMI) was not influenced by feeding WW as did carcass prime cuts, meat, bone and fat-tail; however, the backfat thickness was increased (P = 0.01). WW inclusion reduced cholesterol and increased protein load in muscle tissue (P ≤ 0.05). Moreover, this inclusion led to higher concentration of vaccenic acid (VA), conjugated linoleic acid (CLA) and C20 polyunsaturated FAs (PUFA), total n3 and n6 PUFA in muscle and fat-tail (P ≤ 0.05). Due to enhanced animal performance, also chemical and FA composition of muscle and adipose tissue, WW can be included in fattening lamb diets as a rich source of protein and PUFA.

Sexual dimorphism in the gonad lipidome of blue mussels (Mytilus sp.): New insights from a global lipidomics approach

Blue mussels (Mytilus sp.) are an economically important species for European aquaculture. Their importance as a food source is expected to increase in the coming net-zero society due to their low environmental footprint; however, their production is affected by anthropogenic stressors and climate change. During reproduction, lipids are key molecules for mussels as they are the main source of energy on which newly hatched embryos depend in the first days of their development. In this work, blue mussels of different origins are analysed, focusing on the differences in lipid composition between the ovary (BMO) and the testis (BMT). The lipidome of blue mussel gonads (BMG) is studied here by combining traditional lipid profiling methods, such as fatty acid and lipid class analysis, with untargeted liquid chromatography-mass spectrometry (LC-MS) lipidomics. The approach used here enabled the identification of 770 lipid molecules from 23 different lipid classes in BMG. BMT, which consists of billions of spermatocytes, had greater amounts of cell membrane and membrane lipid components such as FA18:0, C20 polyunsaturated fatty acids (PUFA), free sterols (ST), ceramide phosphoethanolamines (CerPE), ceramide aminoethylphosphonates (CAEP), cardiolipins (CL), glycerophosphocholines (PC), glycerophosphoethanolamines (PE) and glycerophosphoserines (PS). In BMO, saturated fatty acids (FA14:0 and FA16:0), monounsaturated fatty acids (MUFA) and other storage components such as C18-PUFA accumulated in triradylglycerolipids (TG) and alkyldiacylglycerols (neutral plasmalogens, TG O-), which, together with terpenes, wax esters and cholesterol esters, make up most of oocytes yolk reserves. BMO also had higher levels of ceramides (Cer) and generally alkyl/alkenyl glycerophospholipids (mainly plasmanyl/plasmenyl PC), suggesting a role for these lipids in vitellogenesis. Non-methylene interrupted dienoic fatty acids (NMID FA), typically found in plasmalogens, were the only membrane-forming PUFA predominantly detected in BMO. The results of this study are of great importance for clarifying the lipid composition of BMG and provide an important basis for future studies on the reproductive physiology of these organisms.

Effect of Bovine Milk Peptides on Cell Inflammation, Proliferation and Differentiation: Milk Potential Benefits Are Preserved in an Unconventional Cow Feeding Strategy.

Animal feeding through the reuse of agro-industrial by-products in one of the ultimate goals of sustainable agriculture. Olive oil pomace (OOP) produced as a waste product during olive oil milling has been used as an ingredient in the diet for Holstein lactating cows. Recent findings have shown no decrease in animal performance, feed intake or detrimental effect on rumen microbiota. In contrast, an improvement in C18 polyunsaturated fatty acids has been observed. In this work, the milk protein content from cows fed a commercial diet (CON) or an experimental one supplemented with OOP was determined and compared, and the peptides derived from the simulated gastrointestinal digestion of raw milk were analyzed. After fractionation via RP-HPLC, peptides were characterized for their biological activity on different cell lines. The ability to reduce both the intracellular ROS content and the expression of inflammatory markers, such as Cyclooxygenase, isoenzyme 2 (COX-2) and inducible Nitric Oxide Synthase (iNOS), as well as the remarkable properties to induce cell differentiation and to slow down the proliferation of human intestinal cancer cells, enable us to define them as bioactive peptides. In spite of there being no observed significant difference between the healthy activity of CON and OOP peptides, the results allow us to broaden the knowledge about the biological activity of these bioactive peptides and to confirm that agro-industrial by-products may be successfully incorporated into the feeding strategy of dairy cows.

Composition of galactolipids, betaine lipids and triglyceride‐associated fatty acids of the symbiotic dinoflagellate Zooxanthella (Brandtodinium) nutricula: A glimpse into polyunsaturated fatty acids available to its polycystine radiolarian host

SUMMARYZooxanthella nutricula is a photosynthetic dinoflagellate symbiont of polycystine radiolarians. As such, it is hypothesized to provide fixed organic carbon, including in the form of acylglycerolipids and sterols, to its non‐photosynthetic host. We have previously characterized the sterols of Z. nutricula that may be transferred to its host and, in the present study, have turned our attention to three classes of fatty acid‐containing lipids, chloroplast‐associated galactolipids, betaine lipids, which are non‐phosphorylated phospholipid analogs present in many eukaryotes, and triglycerides. Zooxanthella nutricula was observed using positive‐ion electrospray/mass spectrometry (ESI/MS) and ESI/MS/MS to produce the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) enriched in octadecapentaenoic (18:5(n‐3)) and octadecatetraenoic (18:4(n‐3)) acid to place it within a group of peridinin‐containing dinoflagellates in a C18/C18 (sn‐1/sn‐2 fatty acid regiochemistry) cluster, as opposed to another cluster with C20/C18 MGDG and DGDG, where the C20 fatty acid is eicosapentaenoic acid (20:5(n‐3)) and the C18 fatty acid is either 18:5(n‐3) or 18:4(n‐3). Zooxanthella nutricula was also observed to produce 38:10 (total number of fatty acid carbons:total number of double bonds), 38:6, and 44:7 diacylglycerylcarboxyhydroxymethylcholine (DGCC) as the sole type of betaine lipid. Although it is more difficult to determine which fatty acids are present in the sn‐1 and sn‐2 positions on the glycerol backbone of DGCC using ESI/MS/MS, gas chromatography/mass spectrometry (GC/MS)‐based examination indicated the putatively DGCC‐associated polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:6(n‐3)). Coupled with the C18 PUFAs of MGDG and DGDG, and fatty acids associated with triglycerides (also examined via GC/MS), Z. nutricula could serve as a rich source of PUFAs for its radiolarian host. These data demonstrate that Z. nutricula produces a similar set of PUFA‐containing lipids as Symbiodinium microadriaticum, a photosynthetic dinoflagellate symbiont of cnidarians, indicating a metabolic commonality in these phylogenetically discrete dinoflagellate symbionts with unrelated host organisms.

Construction of Eicosatetraenoic Acid Producing Cell Factory by Genetic Engineering of Mucor circinelloides

Eicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, Mucor circinelloides, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, M. circinelloides lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). M. circinelloides could produce DGLA by overexpressing the D6E(GLELO) gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (PpD17 and PaD17) genes from Phytophthora parasitica and Pythium aphanidermatum, respectively, were introduced into M. circinelloides to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains’ biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of cme2, fas2, and D6E, thereby contributing to lipid biosynthesis in M. circinelloides. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in PpD17 overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the PpD17 gene in M. circinelloides, which established a new scope for further research in the production of ETA in oleaginous fungi.

Identification of four secretory phospholipase A2s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses.

Eicosanoids are a group of the oxygenated C20 polyunsaturated fatty acids and play crucial roles in mediating various insect physiological processes. Catalytic activity of phospholipase A2 (PLA2) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis. This study identified four different secretory PLA2 (As-PLA2A-As-PLA2D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA2A and As-PLA2D are clustered with Group III PLA2s while As-PLA2B and As-PLA2C are clustered with Group XII and Group X PLA2s, respectively. Expression levels of these PLA2 genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA2 genes, which resulted in significant increases of the PLA2 enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca2+ dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA2s. In addition, the PLA2 activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA2, but not to intracellular PLA2 inhibitors. An addition of BPB to the immune challenge significantly prevented hemocyte-spreading behavior of A. sapporensis. BPB treatment also suppressed a cellular immune response measured by hemocyte nodule formation. However, the immunosuppression was significantly rescued by the AA addition. To determine the PLA2(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA2s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA2s. In all four PLA2s, the RNAi treatments prevented the cellular immune response even after the immune challenge. This study reports four secretory PLA2s encoded in A. sapporensis and their function in mediating cellular immunity.

Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia.

The dietary supply of polyunsaturated fatty acids (PUFA) crucially affects animals' performance at different temperatures. However, the underlying physiological mechanisms are still insufficiently understood. Here, we analyzed lifespan and heat tolerance of four genotypes of Daphnia magna reared on either the green alga Scenedesmus obliquus that lacks long-chain (> C18) PUFA, or the heterokont alga Nannochloropsis limnetica that contains C20 PUFA, both either at saturating and near-starvation levels. A significant genotype-by-diet interaction in lifespan was observed at saturating diets. The C20 PUFA-rich diet eliminated differences in lifespan among genotypes on the PUFA-deficient diet. Corrected for body length, acute heat tolerance was higher at low than at high food concentration, at least in the older of the two age groups analyzed. Genotypes differed significantly in heat tolerance, but there were no genotype-by-diet interactions. As predicted, the C20 PUFA-rich diet resulted in higher lipid peroxidation (LPO) and a lower mitochondrial membrane potential (ΔΨm). LPO levels averaged across clones and rearing conditions were inversely related to acute heat tolerance. Yet, heat tolerance was higher on the PUFA-rich diet than on the PUFA-deficient diet, particularly in older Daphnia, indicating that the C20 PUFA-rich diet allowed Daphnia to compensate for higher LPO. In contrast, Daphnia with intermediate levels of ΔΨm showed the lowest heat tolerance. Neither LPO nor ΔΨm explained the diet effects on lifespan. We hypothesize that antioxidants present in the PUFA-rich diet may have enabled higher heat tolerance of Daphnia despite higher LPO, which may also explain the lifespan expansion of otherwise short-lived genotypes.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Hepatic transcriptome analysis reveals that elovl5 deletion promotes PUFA biosynthesis and deposition

The safe and low-cost acquisition of polyunsaturated fatty acids (PUFAs) has become a research hotspot. Fatty acyl elongase 5 (Elovl5), a rate-limiting enzyme for fatty acid elongation, is principally in charge of extending C18 and C20 PUFA substrates. However, the role of elovl5 in regulating pathways and genes involved in PUFA synthesis remain largely unknown. Here, hepatic transcriptome analysis of wild-type and elovl5 knockout (elovl5−/−) zebrafish was performed to identify the potential regulatory targets related to PUFA deposition and synthesis. There were 1579 differentially expressed genes (DEGs), of which 787 had their expression levels increased while 792 had the opposite effect. Peroxisome proliferators-activated receptors (PPAR) signaling pathway was considerably enriched in DEGs, according to the KEGG analysis, in which fatp2, fabp7, and pparδ were engaged in PUFA absorption and deposition. Additionally, transcriptome analysis also revealed that cyp46a1 and cyp2r1 were implicated in the synthesis of bile acids and the metabolism of vitamin D, thus indirectly participating in PUFA biosynthesis and deposition. Finally, the DEGs, which improve PUFA level following elovl5 deletion, were verified through feeding experiment with two prepared diets soybean oil diet and linolenic acid oil diet. This study revealed potential regulatory targets that improve PUFA level after elovl5 deletion in teleosts.

The Influence of Ultrasound on the Growth of Nannochloris sp. in Modified Growth Medium.

The influence of ultrasound irradiation on the algal biomass productivity as well as its oil content and fatty acids profile, grown in a modified Zarrouk medium, i.e., deproteinized whey waste solution, was investigated. The algal samples (Nannochloris sp. 424-1 microalgae) were grown for 7 days in a thermostated incubator at 28 °C, shaken under continuous light. During this period, the algal biomass was subjected to induced stress by ultrasonic irradiation at different powers and sonication time. The obtained results demonstrate that ultrasound stressing of algae biomass has a positive effect on both the quantity of biomass and the oil obtained, also causing a shift in fatty acid composition by increasing the proportion of C16 and C18 polyunsaturated fatty acids. A low dosage level of exposure to the ultrasound led to algal biomass increase as well as lipid accumulation. For both types of irradiation modes which were investigated, daily and only initial irradiation, the beneficial effect of the ultrasound decreases as the exposure time increases and the excessive sonication becomes detrimental to microalgae growth.

Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (kcat/Km) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25°C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2g/l cells, and 7mM ARA as substrate in a 500ml-Erlenmeyer baffled flask with 50ml reaction solution with agitation at 200rpm in the presence of 10mM cysteine as a reduction agent, respectively. Under these conditions, 6.4mM 9S-hydroxyeicosatetraenoic acid, 6.2mM 9S-hydroxyeicosapentaenoic acid, and 5.9mM 11S-hydroxydocosahexaenoic acid were produced in 30min, 40min, and 60min with specific productivities of 1067μmol/min/g, 775μmol/min/g, and 492μmol/min/g, volumetric productivities of 213μM/min, 155μM/min, and 98μM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.

Dominant Elongase Activity of Elovl5a but Higher Expression of Elovl5b in Common Carp (Cyprinus carpio)

Most diploid freshwater and marine fish encode one elovl5 elongase, having substrate specificity and activities towards C18, C20 and C22 polyunsaturated fatty acids (PUFAs). The allo-tetraploid common carp is hypothesized to encode two duplicated elovl5 genes. How these two elovl5 genes adapt to coordinate the PUFA biosynthesis through elongase function and expression divergence requires elucidation. In this study, we obtained the full-length cDNA sequences of two elovl5 genes in common carp, named as elovl5a and elovl5b. Functional characterization showed that both enzymes had elongase activity towards C18, C20 and C22 PUFAs. Especially, the activities of these two enzymes towards C22 PUFAs ranged from 3.87% to 8.24%, higher than those in most freshwater and marine fish. The Elovl5a had higher elongase activities than Elovl5b towards seven substrates. The spatial-temporal expression showed that both genes co-transcribed in all tissues and development stages. However, the expression levels of elovl5b were significantly higher than those of elovl5a in all examined conditions, suggesting that elovl5b would be the dominantly expressed gene. These two genes had different potential transcriptional binding sites. These results revealed the complicated roles of elovl5 on PUFA synthesis in common carp. The data also increased the knowledge of co-ordination between two homoeologs of the polyploid fish through function and expression divergence.

Effects of dietary substitution of peanut meal for soybean meal on egg production, egg quality, oxidative status, and yolk fatty acid profile in laying ducks

There is an urgent need to evaluate and introduce alternatives for the unsustainable and traditional feed stuffs in poultry. This study evaluated the effects of graded replacement of soybean meal (SBM) by peanut meal (PNM) on egg production, egg quality, oxidative status, and yolk fatty acid profile in laying ducks. In total, 360 Longyan ducks aged 21 weeks were allocated to five treatments, each containing six replicates of 12 birds. Birds were fed the diets containing PNM replacing 0 (control), 25, 50, 75 or 100% of SBM for 16 weeks. With the increase of PNM level, egg production was improved (quadratic, P < 0.05), egg weight and feed consumption were decreased, feed conversion ratio and egg mass were impaired at 100% PNM, and yolk colour was enhanced (quadratic, P < 0.05). Plasma malondialdehyde concentration was increased at 100% PNM, while plasma glutathione concentration was decreased (quadratic, P < 0.01) as PNM substitution increased. Total cholesterol content in yolk decreased (P < 0.01) in response to increased PNM substitution. The contents of saturated fatty acids C20:0 and C22:0 in yolk increased (linear or quadratic, P < 0.05) with increased PNM substitution, but the contents of saturated fatty acid C18:0, monounsaturated fatty acid C22:1, and polyunsaturated fatty acids C18:3n-3 and C22:6n-3 in yolk were decreased (linear, P < 0.05) as PNM replacement increased. The contents in yolk of polyunsaturated fatty acid C18:2n-6, C20:2n-6, C20:3n-6, C20:4n-6, and total amount of polyunsaturated fatty acids increased with the highest contents obtained at 75% PNM substitution. The ratio between n-6 and n-3 increased (linear, P < 0.05) as PNM substitution increased. The transcript abundance in liver of peroxisome proliferators-activated receptors γ and fatty acid synthase showed quadratic (P < 0.05) responses with PNM replacement, with the highest expression of both genes being obtained with 75% substitution with PNM. Replacement of dietary SBM with 100% PNM decreased egg production and antioxidant capacity and increased the ratio of omega fatty acid in yolk between n-6 and n-3 in laying ducks. The obtained results indicate that PNM can be used to replace up to 75% of SBM in the diet of laying ducks without negative effects on the egg-laying production or egg quality. The regression model indicated that the maximal egg mass was obtained at no more than 67.6% replacement of SBM with PNM in the diet of laying ducks.

Long-chain polyunsaturated fatty acid biosynthesis in a land-crab with advanced terrestrial adaptations: Molecular cloning and functional characterization of two fatty acyl elongases.

Depending on the presence and activities of the front-end fatty acyl desaturases and elongation of very long-chain fatty acid (Elovl) enzymes, animals have different capacities for long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis. Successful land colonisation in brachyuran crabs requires a shift towards terrestrial food chain with limited LC-PUFA availability. We cloned and functionally characterised two elovl genes from the purple land crab Gecarcoidea lalandii. The two Elovl contained all the necessary motifs of a typical polyunsaturated fatty acids (PUFA) Elovl and phylogenetically clustered in the Elovl1 and Elovl6 clades, respectively. The G. lalandii Elovl1 elongated saturated fatty acids, with low activities towards C20 and C22 PUFA substrates. Moreover, the G. lalandii Elovl6 was particularly active in the elongation of C18 PUFA, although it also recognised monounsaturated fatty acids as substrates for elongation. Collectively, the herein characterised G. lalandii elovl paralogues fulfil all the elongation steps involved in the LC-PUFA biosynthetic pathways. Tissue distribution of the G. lalandii elovl genes, along with the FA composition analyses, suggest the hepatopancreas and gill as key metabolic sites for fatty acid elongation. However, current data suggest that G. lalandii is unable to rely solely on biosynthesis to fulfil LC-PUFA requirements, since front-end desaturase appears to be absent in this species and other decapods.

Can Artemia franciscana produce essential fatty acids? Unveiling the capacity of brine shrimp to biosynthesise long-chain polyunsaturated fatty acids

Artemia nauplii are widely used as live preys for feeding early life-cycle stages of marine finfish and shrimp. However, a major drawback associated to using Artemia is their deficient nutritional value for marine larvae that is primarily linked to suboptimal levels of long-chain polyunsaturated fatty acids (LC-PUFA), essential nutrients that guarantee normal growth and development of animals. While common marine hatchery procedures involve enrichment processes to address such nutritional deficiencies, the specific drivers accounting for the naturally occurring variability in LC-PUFA of Artemia are largely unknown. Biosynthesis, along diet, is one of the main factors determining the LC-PUFA profiles in animals and it depends upon the repertoire and function of fatty acyl elongases and desaturases existing in a particular species. The aim of this study was the molecular and functional characterisation of all elongase and desaturase enzymes involved in the LC-PUFA biosynthesis from Artemia franciscana, arguably the most commonly used Artemia species. Seven out of eight elongases (termed “Elo1-8”) found in A. franciscana had distinctive features of LC-PUFA biosynthesising elongases. Consistently, functional assays showed these elongases were able to elongate multiple substrates and thus enabling A. franciscana to perform all elongation reactions of the LC-PUFA biosynthetic pathways. While all seven functionally characterised elongases from A. franciscana showed activity towards multiple substrates including C18 to C22 polyunsaturated fatty acids, particularly high activity was detected for the Elovl8 orthologue (termed herein as “Elo7”). Additionally, A. franciscana was found to have three genes of the so-called “first” desaturases (“Des1-3”), and lack other key LC-PUFA biosynthesising desaturases such as methyl-end and front-end desaturases. Two desaturases have the expected ∆9 desaturase activity, whereas a third one showed ∆12 activity. Neither methyl-end nor front-end desaturases were found in A. franciscana. In conclusion, this study demonstrated that A. franciscana has high elongation capacity but its overall LC-PUFA biosynthesis can be regarded as limited due to the lack of an adequate complement of fatty acyl desaturases in its genome.

Comparative transcriptomic and lipidomic analyses indicate that cold stress enhanced the production of the long C18-C22 polyunsaturated fatty acids in Aurantiochytrium sp.

Aurantiochytrium sp. belonging to Thraustochytrids are known for their capacity to produce long-chain polyunsaturated fatty acids (PUFAs). However, effects of cold stress accompanied with staged-temperature control on the fatty acid metabolism in Aurantiochytrium sp. were rarely studied. In this study, cold stress (15°C, 5°C) was applied for Aurantiochytrium sp., with the physiological responses (morphology, growth, fatty acid profiling) and gene expression related FA synthesis, lipid metabolism, and regulatory processes was observed. Results showed that there is a significant change for the lipid types under 5°C (251 species) and 15°C (97 species) treatment. The 5°C treatment was benefit for the C18–C22 PUFAs with the yield of docosahexaenoic acid (DHA) increased to 1.25 times. After incubation at 15°C, the accumulation of eicosadienoic acid (EA) (20:2) was increased to 2.00-fold. Based on transcriptomic and qPCR analysis, an increase in genes involved in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways was observed under low-temperature treatment. With upregulation of 3-ketoacyl-CoA synthase (2.44-fold), ketoreductase (2.50-fold), and dTDP-glucose 4,6-Dehydratase (rfbB) (2.31-fold) involved in PKS pathway, the accumulation of DHA was enhanced under 5°C. While, FAS and fatty elongase 3 (ELO) involved in the FAS pathway were upregulated (1.55-fold and 2.45-fold, respectively) to accumulate PUFAs at 15°C. Additionally, glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP), phosphatidylserine synthase (PSS), and phosphatidylserine decarboxylase (PSD) involved in glycerophospholipid biosynthesis were upregulated at 5°C increasing the accumulation of phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). However, glycolysis and the TCA cycle were inhibited under 5°C. This study provides a contribution to the application of two-staged temperature control in the Aurantiochytrium sp. fermentation for producing cold stress-enhancing PUFAs, in order to better understand the function of the key genes for future genetic engineering.

Characterization and Preliminary Application of a Novel Lipoxygenase from Enterovibrio norvegicus.

Lipoxygenases have proven to be a potential biocatalyst for various industrial applications. However, low catalytic activity, low thermostability, and narrow range of pH stability largely limit its application. Here, a lipoxygenase (LOX) gene from Enterovibrio norvegicus DSM 15893 (EnLOX) was cloned and expressed in Escherichia coli BL21 (DE3). EnLOX showed the catalytic activity of 40.34 U mg−1 at 50 °C, pH 8.0. Notably, the enzyme showed superior thermostability, and wide pH range stability. EnLOX remained above 50% of its initial activity after heat treatment below 50 °C for 6 h, and its melting point temperature reached 78.7 °C. More than 70% of its activity was maintained after incubation at pH 5.0–9.5 and 4 °C for 10 h. In addition, EnLOX exhibited high substrate specificity towards linoleic acid, and its kinetic parameters of Vmax, Km, and Kcat values were 12.42 mmol min−1 mg−1, 3.49 μmol L−1, and 16.86 s−1, respectively. LC-MS/MS analysis indicated that EnLOX can be classified as 13-LOX, due to its ability to catalyze C18 polyunsaturated fatty acid to form 13-hydroxy fatty acid. Additionally, EnLOX could improve the farinograph characteristics and rheological properties of wheat dough. These results reveal the potential applications of EnLOX in the food industry.