Improving Fatty Acid Composition of Lipids Synthesized by Brachionus plicatilis in Large Scale Experiments

Nannochloropsis oculata and Chlorella sp. were cultivated in industrial scale bioreactors 300?L, under full sunlight and artificial light during night-time at various temperatures. The most abundant fatty acids were C18:3n-3 and C16:0 in Chlorella sp., whereas in N. oculata C16:0, eicosapentaenoic acid (EPA) and C16:1n-9 were predominant. Brachionus plicatilis cultivated in 2,500-L tanks on N. oculata, Chlorella sp. and Saccharomyces cerevisiae, was able to de novo synthesize as well as to elongate and desaturate pre-existing fatty acids in the feed. When fed with S. cerevisiae, B. plicatilis synthesized lipids containing EPA and docosahexaenoic acid (DHA), despite the fact that these fatty acids were absent in yeast lipids. Brachionus fed with Nannochloropsis synthesized DHA in non-negligible quantities. Brachionus enriched with various commercial preparations incorporated into its lipids substrate fatty acids but also synthesized new fatty acids such as C16:1n-9, C18:1n-9, C18:2n-6, EPA and DHA. Neutral lipid was the main lipid fraction while phospholipids predominated over glycolipids plus sphingolipids. The major amounts of EPA and DHA were observed in neutral lipids, noteworthy quantities of these fatty acids were also incorporated into body phospholipids. Interestingly, the high n-3 fatty acids content of rotifer lipids was retained even after the rotifers were grown in low n-3 fatty acid media.     

FAME Production and Fatty Acid Profiles from Moist Chlorella sp. and Nannochloropsis oculata Biomass

In the present study, we investigated the production of fatty acid methyl esters (FAME) from moist Chlorella sp. and Nannochloropsis oculata biomass using a hydrolysis–esterification process. Additionally, we evaluated for the first time the fatty acid profile before and after this process. Hydrolysis of the lipid fraction was performed on a moist biomass in the presence of differing amounts of an acid catalyst in both 50 and 100 % w/w water relative to the biomass. The esterification of the crude extracts of the free fatty acids (FFA) was then investigated. The experiments show that in the presence of 50 % w/w water relative to the biomass, the hydrolysis–esterification process results in higher FFA and FAME yields. The analysis of the fatty ester profiles did not reveal any degradation of the FFA from the microalgae biomass under the hydrolysis–esterification conditions. The results were compared with both extraction–transesterification and direct transesterification processes using dry biomass. The extraction–transesterification and hydrolysis–esterification processes resulted in similar FAME yields and similar profiles of the fatty esters from dry and moist biomass materials, respectively.     

SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza

Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA₃), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.     

Synergistic Effect of Methyl Jasmonate and Abscisic Acid Co-Treatment on Avenanthramide Production in Germinating Oats

The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant, anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls (232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable method for avenanthramide production in germinating oats.     

The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica)

The total lipid and fatty acid content of 3 algal species,Pyramimonas virginica, Pseudoisochrysis paradoxa andChlorella sp., which have been successful as food sources for rearing larvae of the American oyster,Crassostrea virginica, was determined. Of the fatty acids of ω6 and ω3 families which have been shown to be essential fatty acids for normal growth in many animals, only the ω6 fatty acids were found to be higher in these 3 species of algae than in the traditional oyster larvae diet which consists of the algaeMonochrysis lutheri andIsochrysis galbana. The major fatty acid constituents of the total lipids of the 3 species were the C12, C14, C16 and C18 saturated fatty acids and the C16 and C18 mono- and polyunsaturated acids. These components constituted 70–93% of the total lipid in cultures of all ages. There were modest amounts of C20 and C22 polyunsaturated acids; some of these existed only in trace amounts. InP. virginica andChlorella sp., hexadecanoic acid was dominant (23–39%). The presence of large quantities of tetradecanoic acid (22–26%) and oleic acid (17–21%) was characteristic ofP. paradoxa. Chlorella sp. had the highest proportion of octadecatrienoic acid (18∶3ω3) which accounted for up to 17% of the total lipids. γ-Linolenic acid (18∶3ω6) was found only inChlorella sp., but in the 5th-day culture only. The lowest proportion of total polyethylenic acid was inP. paradoxa; however, lipid analyses showed this alga had the most lipid/individual cell. Some variations were observed in the fatty acid composition with age of the culture. Contribution No. 883 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062.     

2-Hydroxyhexadecanoic and 8,9,13-trihydroxydocosanoic acid accumulation by yeasts treated with fumonisin B1

Fumonisin B₁ is a sphingolipid-like compound that enhances the accumulation of yeast sphingolipids and 2-hydroxy fatty acids. These lipids occur both as freely extractable and cell bound components in yeast fermentations. Both free and bound 2-hydroxy fatty acids produced byPichia sydowiorum NRRL Y-7130 were increased when fumonisin B₁ (50 mg/L) was added to the usual growth medium containing yeast extract/malt extract/peptone/glucose. Fumonisin-treated cultures contained 38 mg/L more 2-hydroxyhexadecanoic and 15 mg/L more 2-hydroxyoctadecanoic acids than did untreated cultures. By contrast, fumonisin inhibited the accumulation of free 8,9,13-trihydroxydocosanoic acid inRhodotorula sp. YB-2501 cultures, leading to 240 mg/L lower trihydroxy acid production than by untreated cultures.     

Expression of Genes Controlling Unsaturated Fatty Acids Biosynthesis and Oil Deposition in Developing Seeds of Sacha Inchi (Plukenetia volubilis L.)

Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed.     

The Synergism of Biochemical Components Controlling Lipid Oxidation in Lamb Muscle

Lipid oxidation of M. longissimus lumborum in fresh or vacuum packaged (aged) lamb meat stored at 3 °C for 0 or 4 weeks, respectively and displayed under refrigerated conditions for a further 4 days was assessed by measuring the concentration of malondialdehyde (MDA) in meat using the thiobarbituric acid reactive substances procedure. The effects of vitamin E, heme iron and polyunsaturated fatty acids (n-6 and n-3) on lipid oxidation were examined. Results showed a strong positive relationship between heme iron, n-6 and n-3 fatty acids and lipid oxidation when vitamin E was below 2.95 mg/kg muscle. When lipid oxidation was related to vitamin E concentration and the other three variables, respectively, any increase in heme iron or n-6 or n-3 fatty acids concentration did not influence lipid oxidation. Management of diet to elevate muscle vitamin E concentration above 3.45 mg/kg muscle is beneficial to maintain the level of lipid oxidation below 2.4 mg MDA/kg muscle in meat stored for up to 4 weeks. This demonstrates that vitamin E concentration in muscle has a greater influence on controlling lipid oxidation in muscle tissues than do heme iron or polyunsaturated fatty acids.     

Aryl Hydrocarbon Receptor (AhR) Activation by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Dose-Dependently Shifts the Gut Microbiome Consistent with the Progression of Non-Alcoholic Fatty Liver Disease

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids, including taurolithocholic acid and deoxycholic acid (microbial modified bile acids involved in host bile acid regulation signaling pathways). To investigate the effects of TCDD on the gut microbiota, the cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 µg/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and o-succinylbenzoate synthase, a menaquinone biosynthesis associated gene. Analysis of the gut microbiomes from cirrhosis patients identified an increased abundance of genes from the mevalonate-dependent IPP biosynthesis as well as several other menaquinone biosynthesis genes, including o-succinylbenzoate synthase. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.     

Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid’s (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.     

Sustainable Hydrogen Photoproduction by Phosphorus-Deprived Marine Green Microalgae Chlorella sp.

Previously it has been shown that green microalga Chlamydomonas reinhardtii is capable of prolonged H₂ photoproduction when deprived of sulfur. In addition to sulfur deprivation (-S), sustained H₂ photoproduction in C. reinhardtii cultures can be achieved under phosphorus-deprived (-P) conditions. Similar to sulfur deprivation, phosphorus deprivation limits O₂ evolving activity in algal cells and causes other metabolic changes that are favorable for H₂ photoproduction. Although significant advances in H₂ photoproduction have recently been realized in fresh water microalgae, relatively few studies have focused on H₂ production in marine green microalgae. In the present study phosphorus deprivation was applied for hydrogen production in marine green microalgae Chlorella sp., where sulfur deprivation is impossible due to a high concentration of sulfates in the sea water. Since resources of fresh water on earth are limited, the possibility of hydrogen production in seawater is more attractive. In order to achieve H₂ photoproduction in P-deprived marine green microalgae Chlorella sp., the dilution approach was applied. Cultures diluted to about 0.5–1.8 mg Chl·L⁻¹ in the beginning of P-deprivation were able to establish anaerobiosis, after the initial growth period, where cells utilize intracellular phosphorus, with subsequent transition to H₂ photoproduction stage. It appears that marine microalgae during P-deprivation passed the same stages of adaptation as fresh water microalgae. The presence of inorganic carbon was essential for starch accumulation and subsequent hydrogen production by microalgae. The H₂ accumulation was up to 40 mL H₂ gas per 1iter of the culture, which is comparable to that obtained in P-deprived C. reinhardtii culture.     

Rapid Detection and Quantification of Triacylglycerol by HPLC–ELSD in Chlamydomonas reinhardtii and Chlorella Strains

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3–6 % DW (Chlamydomonas strains) and 7–12 % DW (Chlorella strains) respectively by both HPLC–ELSD and TLC/GC methods. HPLC–ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC–ELSD, while it was undetectable by TLC/GC method.     

不同碳氮组合对小球藻异养培养油脂积累的影响

目的探讨不同碳氮组合对小球藻Chlorella pro tothecoides CS-41异养培养油脂积累的影响。方法以不同浓度葡萄糖为碳源,尿素为氮源,异养培养小球藻,采用干重法测定小球藻生物量,氯仿/甲醇法提取总脂,并采用GC-MS分析脂肪酸含量。结果小球藻最佳生长条件的碳氮组合配比为葡萄糖浓度40g/L,尿素浓度3g/L,此时小球藻生物量达最高,为9.1g/L,脂肪酸产率最大,为1.26g/L;总脂含量最高,为4.38g/L。结论获得了碳氮最佳配比,使小球藻脂肪酸含量提高到1.26g/L,为发酵扩大化培养小球藻制备生物柴油奠定了基础。     

Increased Lipid Synthesis and Decreased β-Oxidation in the Liver of SHR/NDmcr-cp (cp/cp) Rats, an Animal Model of Metabolic Syndrome

SHR/NDmcr-cp (cp/cp) rats (SHR/NDcp) are an animal model of metabolic syndrome. A previous study of ours revealed drastic increases in the mass of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), cis-vaccenic (18:1n-7) and 5,8,11-eicosatrienoic acids in the liver of SHR/NDcp. However, detailed information on the class of lipid accumulated and the mechanism responsible for the overproduction of the accumulated lipid in the liver was not obtained. This study aimed to characterize the class of lipid accumulated and to explore the mechanism underlying the lipid accumulation in the liver of SHR/NDcp, in comparison with SHR/NDmcr-cp (+/+) (lean hypertensive littermates of SHR/NDcp) and Wistar Kyoto rats. In the liver of SHR/NDcp, de novo synthesis of fatty acids (16:0, 18:1n-9 and 16:1n-7) and triacylglycerol (TAG) synthesis were up-regulated and fatty acid β-oxidation was down-regulated. These perturbations of lipid metabolism caused fat accumulation in hepatocytes and accumulation of TAG, which were enriched with 16:0, 18:1n-9 and 16:1n-7, in the liver of SHR/NDcp. On the other hand, no changes were found in hepatic contents of diacylglycerol and unesterified fatty acid (FFA); among FFA, there were no differences in the hepatic concentrations of unesterified 16:0 and stearic acid between SHR/NDcp and two other groups of rats. Moreover, little change was brought about in the expression of genes responsive to endoplasmic reticulum stress in the liver of SHR/NDcp. These results may reinforce the pathophysiological role of stearoyl-CoA desaturase 1 and fatty acid elongase 6 in the liver of SHR/NDcp.     

The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.     

Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185

Thraustochytrium sp. 26185 is a marine protist that can produce a large amount of docosahexaenoic acid (DHA, 22:6n‐3), an ω3 very long chain polyunsaturated fatty acid (VLCPUFA) of nutritional importance. However, the mechanism of how this fatty acid is synthesized and assembled into the storage lipid triacylglycerol is unclear. Here we report sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Genome sequencing produced a total of 2,418,734,139 bp clean sequences with about 62 fold genome coverage. Annotation of the genome sequences revealed 10,797 coding genes. Among them, 10,216 genes could be assigned into 25 KOG classes where 451 genes were specifically assigned to the group of lipid transport and metabolism. Detailed analysis of these genes revealed co‐existence of both aerobic pathway and anaerobic pathways for the biosynthesis of DHA in this species. However, in the aerobic pathway, a key gene encoding stearate Δ9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. Genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine:diacylglycerol cholinephosphotransferase, an important enzyme in bridging two types of glycerolipids, diacylglycerols (DAG) and phosphatidylcholines (PtdCho). These results shed new insight on the biosynthesis and assembly of VLCPUFA in the Thraustochytrium.