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Biosynthesis of nervonic acid and perspectives for its production by microalgae and other microorganisms

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Nervonic acid (NA) is a major very long-chain monounsaturated fatty acid found in the white matter of mammalian brains, which plays a critical role in the treatment of psychotic disorders and neurological development. In the nature, NA has been synthesized by a handful plants, fungi, and microalgae. Although the metabolism of fatty acid has been studied for decades, the biosynthesis of NA has yet to be illustrated. Generally, the biosynthesis of NA is considered starting from oleic acid through fatty acid elongation, in which malonyl-CoA and long-chain acyl-CoA are firstly condensed by a rate-limiting enzyme 3-ketoacyl-CoA synthase (KCS). Heterologous expression of kcs gene from high NA producing species in plants and yeast has led to synthesis of NA. Nevertheless, it has also been reported that desaturases in a few plants can catalyze very long-chain saturated fatty acid into NA. This review highlights recent advances in the biosynthesis, the sources, and the biotechnological aspects of NA.
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Biosynthesis of nervonic acid and perspectives for its production
by microalgae and other microorganisms
Yong Fan
&Hui-Min Meng
&Guang-Rong Hu
&Fu-Li Li
Received: 2 December 2017 /Revised: 11 February 2018 /Accepted: 12 February 2018 /Published online: 24 February 2018
#Springer-Verlag GmbH Germany, part of Springer Nature 2018
Nervonic acid (NA) is a major verylong-chain monounsaturated fatty acid found in the white matter of mammalian brains, which
plays a critical role in the treatment of psychotic disorders and neurological development. In the nature, NA has been synthesized
by a handful plants, fungi, and microalgae. Although the metabolism of fatty acid has been studied for decades, the biosynthesis
of NA has yet to be illustrated. Generally, the biosynthesis of NA is considered starting from oleic acid through fatty acid
elongation, in which malonyl-CoA and long-chain acyl-CoA are firstly condensed by a rate-limiting enzyme 3-ketoacyl-CoA
synthase (KCS). Heterologous expression of kcs gene from high NA producing species in plants and yeast has led to synthesis of
NA. Nevertheless, it has also been reported that desaturases in a few plants can catalyze very long-chain saturated fatty acid into
NA. This review highlights recent advances in the biosynthesis, the sources, and the biotechnological aspects of NA.
Keywords Very long-chain monounsaturated fatty acids .Synthetic biology .Metabolic engineering .Cellular engineering .
3-Ketoacyl-CoA synthesis
Nervonic acid (C24:1 Δ15; cis-tetracos-15-enoic acid, ω-9;
NA) is a major very long-chain monounsaturated fatty acid
(VLCMFA), which is bound via an amide bond to a sphingo-
sine base (Jr et al. 1997; Poulos 1995). Nervonyl
sphingolipids are main components of the white matter of
brains and myelin sheath of nerve fibers (Martínez and
Mougan 2010). NA level has been found to be significantly
decreased in individuals with a psychotic disorder (Amminger
et al. 2012), whereas no significant differences have been
observed for other fatty acids, including docosahexaenoic acid
(DHA) or arachidonic acid (AA). As a major component of
the myelin sheath, reduced levels of NA may reflect the sub-
optimal myelin status in individuals at a high risk of develop-
ing psychotic disorders (Coupland and Raoul 2001).
The use of NA supplements has become an established
method for the treatment of symptoms of several neurological
disorders (Vozella et al. 2017). The NA content of
sphingomyelin in the tissues of developing rats can be elevat-
ed by feeding dietary NA (Strandvik et al. 2016). NA has also
been reported to act as an essential long-chain fatty acid in
infants, particularly during nerve cell development in prema-
ture infants (Farquharson et al. 1996).
NA is able to interact with DNA polymerase β(Mizushina
et al. 1999) by spatially binding with four amino acids (Leull,
Lys35, His51, and Thr79). This binding pocket is similar to
that of the human immunodeficiency virustype-1reverse tran-
scriptase (HIV-1 RT), which allowed the researchers to build a
structural model of HIV-1 RT. NA can function as a non-
competitive inhibitor of HIV-RT in a dose-dependent manner
(Kasai et al. 2002). More recently, monounsaturated fatty
acids were demonstrated to be able to extend lifespan in
Caenorhabditis elegans. Since lipid metabolism between
worms and mammal is conserved, suggesting the benefits of
long-chain monounsaturated fatty acids on health might also
be conserved (Han et al. 2017). This provides a new perspec-
tive to understand the physiological functions of VLCMFAs,
especially NA.
Yong Fan and Hui-Min Meng contributed equally to this work
*Fu-Li Li
Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of
Synthetic Biology, Qingdao Engineering Laboratory of Single Cell
Oil, Qingdao Institute of Bioenergy and Bioprocess Technology,
Chinese Academy of Sciences, 189 Songling Road,
Qingdao 266101, Peoples Republic of China
Applied Microbiology and Biotechnology (2018) 102:30273035
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Each elongation cycle involves four successive reactions. Malonyl-CoA and a long-chain acyl-CoA are condensed by KCS, which is then reduced by KCR to 3hydroxyacyl-CoA, and then 3-hydroxyacyl-CoA is dehydrated by HCD and subsequently reduced to form elongated acyl-CoA catalyzed by ECR (Harwood, 2005;Haslam and Kunst, 2013;Li-Beisson et al., 2013;Huai et al., 2015;Fan et al., 2018; Figure 2). ...
... After synthesis, EA is assembled and stored as TAGs, and the pathway consists of the sequential acylation and dephosphorylation of glycerol-3-phosphate (G3P). G3P is catalyzed by glycerol-3-phosphate acyltransferase (GPAT) to produce lysophospholipids (LPAs); LPA is catalyzed by lysophosphatidic acid acyltransferase (LPAT/LPAAT) to produce phosphatidic acid (PA); PA is catalyzed by phosphatidic acid phosphorylase (PAP) to produce diacylglycerol (DAG); and DAG is catalyzed by diacylglycerol acyltransferase (DGAT) to produce TAG (Stumpf and Pollard, 1983;Bates et al., 2009;Li-Beisson et al., 2013;Fan et al., 2018; Figure 2). GPAT, LPAAT, and DGAT are the three main enzymes of the Kennedy pathway and play very important regulatory roles in the biosynthesis of lipids and phospholipids. ...
... synthesis in plants. Phospholipid diacylglycerol acyltransferase (PDAT) allows the transfer of FAs from the sn-2 position of phosphatidylcholine (PC) to the sn-3 position of DAG, producing TAG and lysophosphatidylcholine (LPC) products (Bates et al., 2009;Lu et al., 2009;Taylor et al., 2011;Fan et al., 2018; Figure 2). PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and DAG by catalyzing the shuffling of acyl groups between them. ...
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Erucic acid (C22:1, ω-9, EA) is a very-long-chain monounsaturated fatty acid (FA) that is an important oleochemical product with a wide range of uses in metallurgy, machinery, rubber, the chemical industry, and other fields because of its hydrophobicity and water resistance. EA is not easily digested and absorbed in the human body, and high-EA rapeseed (HEAR) oil often contains glucosinolates. Both glucosinolates and EA are detrimental to health and can lead to disease, which has resulted in strict guidelines by regulatory bodies on maximum EA contents in oils. Increasingly, researchers have attempted to enhance the EA content in Brassicaceae oilseeds to serve industrial applications while conversely reducing the EA content to ensure food safety. For the production of both LEAR and HEAR, biotechnology is likely to play a fundamental role. Elucidating the metabolic pathways of EA can help inform the improvement of Brassicaceae oilseeds through transgenic technology. In this paper, we introduce the industrial applications of HEAR oil and health benefits of low-EA rapeseed (LEAR) oil first, following which we review the biosynthetic pathways of EA, introduce the EA resources from plants, and focus on research related to the genetic engineering of EA in Brassicaceae oilseeds. In addition, the effects of the environment on EA production are addressed, and the safe cultivation of HEAR and LEAR is discussed. This paper supports further research into improving FAs in Brassicaceae oilseeds through transgenic technologies and molecular breeding techniques, thereby advancing the commercialization of transgenic products for better application in various fields.
... Both of these lipid species are examples of very-long-chain fatty-acyl sphingolipids. While shorter fatty acids (≤C18) are prevalent in the human diet 44 , often serving as preferential substrates for ceramide synthesis by certain gut microbes, longer-chain fatty acids, such as nervonic acid (24:1), are often the product of elongation by host enzymes before incorporation into sphingolipids primarily found in brain tissue 45 . This result was corroborated in Bar et al., where only short-chain sphingolipids were observed to be associated with the microbiome 1 . ...
... Ceramides with a ≤C18 fatty-acyl group showed stronger correspondence with the gut microbiome, consistent with the high prevalence of these fatty acids in the diet 44 and their preferential incorporation into ceramides by certain gut taxa 53 . On the other hand, ceramides with very-long-chain fatty-acyl groups (22:1, 24:1), which are most abundant in brain tissue and often synthesized through elongation by host enzymes 45 , showed a stronger correspondence with host genetics. Importantly, ceramides with different fatty-acyl chain lengths have been implicated in a number of human diseases including Alzheimer's disease, depression and mood disorders 54 . ...
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Variation in the blood metabolome is intimately related to human health. However, few details are known about the interplay between genetics and the microbiome in explaining this variation on a metabolite-by-metabolite level. Here, we perform analyses of variance for each of 930 blood metabolites robustly detected across a cohort of 1,569 individuals with paired genomic and microbiome data while controlling for a number of relevant covariates. We find that 595 (64%) of these blood metabolites are significantly associated with either host genetics or the gut microbiome, with 69% of these associations driven solely by the microbiome, 15% driven solely by genetics and 16% under hybrid genome–microbiome control. Additionally, interaction effects, where a metabolite–microbe association is specific to a particular genetic background, are quite common, albeit with modest effect sizes. This knowledge will help to guide targeted interventions designed to alter the composition of the human blood metabolome.
... First discovered in the brain of sharks, nervonic acid, also known as selacholeic acid, is very abundant in nervous system and brain tissues of vertebrate animals, where it is bound to sphingosine via amide linkage to form nervonyl sphingolipids [370]. These sphingolipids are the principal components of white matter and the myelin sheath of nerve fibers [371][372][373]. ...
... Genetic engineering approaches have been implemented to obtain microorganisms, microalgae, and plants producing oils rich in nervonic acid suitable for nutraceutical and pharmacological applications [196,370,393]. Improvement of existing plant sources, especially in the Brassicaceae family, was considered to generate new elite cultivars. ...
Monounsaturated fatty acids are straight-chain aliphatic monocarboxylic acids comprising a unique carbon‑carbon double bond, also termed unsaturation. More than 50 distinct molecular structures have been described in the plant kingdom, and more remain to be discovered. The evolution of land plants has apparently resulted in the convergent evolution of non-homologous enzymes catalyzing the dehydrogenation of saturated acyl chain substrates in a chemo-, regio- and stereoselective manner. Contrasted enzymatic characteristics and different subcellular localizations of these desaturases account for the diversity of existing fatty acid structures. Interestingly, the location and geometrical configuration of the unsaturation confer specific characteristics to these molecules found in a variety of membrane, storage, and surface lipids. An ongoing research effort aimed at exploring the links existing between fatty acid structures and their biological functions has already unraveled the importance of several monounsaturated fatty acids in various physiological and developmental contexts. What is more, the monounsaturated acyl chains found in the oils of seeds and fruits are widely and increasingly used in the food and chemical industries due to the physicochemical properties inherent in their structures. Breeders and plant biotechnologists therefore develop new crops with high monounsaturated contents for various agro-industrial purposes.
... It binds to sphingosine to form sphingomyelin, which is an integral component of myelin (Martínez and Mougan, 2010). Some clinical studies and animal disease models suggest that, on the one hand, NA intake can promote brain development (Fan et al., 2018;. The NA level in sphingomyelin of red blood cells from premature infants may reflect NA levels in sphingomyelin of the brain and could thus reflect brain maturity (Babin et al., 1993); On the other hand, NA can relieve the symptoms of some neurodegenerative diseases (Zheng et al., 2017). ...
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Background Multiple sclerosis is a chronic demyelinating disease of uncertain etiology. Traditional treatment methods produce more adverse effects. Epidemiological and clinical treatment findings showed that unknown environmental factors contribute to the etiology of MS and that diet is a commonly assumed factor. Despite the huge interest in diet expressed by people with MS and the potential role diet plays in MS, very little data is available on the role of diet in MS pathogenesis and MS course, in particular, studies on fats and MS. The oil of Acer truncatum is potential as a resource to be exploited in the treatment of some neurodegenerative diseases. Objective Here, we investigated the underlying influences of Acer truncatum oil on the stimulation of remyelination in a cuprizone mouse model of demyelination. Methods Cuprizone (0.2% in chow) was used to establish a mouse model of demyelination. Acer truncatum oil was administrated to mice during remyelination. Following techniques were used: behavioral test, histochemistry, fluorescent immunohistochemistry, transmission electron microscope. Results Mice exposed to cuprizone for 6 weeks showed schizophrenia-like behavioral changes, the increased exploration of the center in the open field test (OFT), increased entries into the open arms of the elevated plus-maze, as well as demyelination in the corpus callosum. After cuprizone withdrawal, the diet therapy was initiated with supplementation of Acer truncatum oil for 2 weeks. As expected, myelin repair was greatly enhanced in the demyelinated regions with increased mature oligodendrocytes (CC1) and myelin basic protein (MBP). More importantly, the supplementation with Acer truncatum oil in the diet reduced the schizophrenia-like behavior in the open field test (OFT) and the elevated plus-maze compared to the cuprizone recovery group. The results revealed that the diet supplementation with Acer truncatum oil improved behavioral abnormalities, oligodendrocyte maturation, and remyelination in the cuprizone model during recovery. Conclusion Diet supplementation with Acer truncatum oil attenuates demyelination induced by cuprizone, indicating that Acer truncatum oil is a novel therapeutic diet in demyelinating diseases.
... The traditional urea inclusion method and crystallization method have a low efficiency [4]. NA can be chemically synthesized with cis-13-docosenyl methyl ester as a precursor; however, the yield of NA by chemical synthesis is low, and there are many by-products [5,6]. ...
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This study explored the physicochemical properties, fatty acid compositions, and thermal characteristics of Malania oleifera seed oil (MOSO). Our data showed that the refractive index and specific gravity of MOSO at ambient temperature were 1.461 and 0.913, respectively. The oil content, acid, peroxide, saponification, iodine value, average molecular weight, and an average number of carbon–carbon double bond per triglyceride were 47.8%, 3.62 mg KOH/g oil, 4.61 meq O2/kg oil, 163.16 mg KOH/g oil, 104.28 g I2/100 g oil, 1031.51 g/mol, and 4.24, respectively. The fatty acid profiles of the methyl esters showed the presence of 96.09% unsaturated fatty acids and 2.16% saturated fatty acids. Nervonic acid (NA) was the major component (45.04%). These results suggested that MOSO is an excellent source of essential fatty acids and NA and may be used as a potential NA resource of health supplements and medicines. Graphical abstract
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The ketogenic diet has been the most popular diet in the world in recent years. Therefore, the keto diet, short for ketogenic, involves eating a lot of fat, a moderate amount of protein, and very little carbohydrate. People are more likely to take a ketogenic diet for weight loss, but it can also help control certain medical conditions, such as epilepsy, heart problems, certain brain conditions, and even acne. This research paper aims to find out the benefits of KD and shares its outcomes on adults. In this research, the descriptive methodology has been adopted which refers to secondary sources of data. In this project, the examiner used a variety of research that has also been undertaken by numerous researchers including dietitians who tend to support the positive benefits of using ketogenic diets to manage losing weight as well as other health complications that could result from overweight. Finally, it is essential to note that the ketogenic diet has created debate, partially although traditional dietary education has, for years, illustrated the adverse effects of high overall including trans-fat intake. Volume 6 | Issue 2 | 35
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The chemical composition of the insect cuticle varies remarkably between species and theirlife stages. It can affect host resistance and substrate utilization by invading entomopathogen fungi,such as the soil fungusConidiobolus coronatus. In this study,Sarcophaga argyrostomaflies were exposedto sporulatingC. coronatuscolonies for 24 h; the pupae were resistant, but the adults demonstrated60% mortality. Although the pupae demonstrated no sign of infection nor any abnormal development,our findings indicate that after 24 h of contact with the fungus, the pupae demonstrated a 25.2-foldincrease in total cuticular free fatty acids (FFAs) and a 1.9-fold decrease in total internal FFAs. Also,the cuticular FFA increased from 26 to 30, while the internal FFA class increased from 13 to 23.In exposed adults, the total mass of cuticular FFAs increased 1.7-fold, while the number of FFAsstayed the same (32 FFAs). Also, the internal FFA class increased from 26 to 35 and the total FFAmass increased 1.1-fold. These considerable differences between adults and pupae associated withC. coronatusexposure indicate developmental changes in the mechanisms governing lipid metabolismand spatial distribution in the organism, and suggest that cuticular lipids play a vital role in thedefence against pathogenic fungi.
X‐linked adrenoleukodystrophy (X‐ALD) is an inherited, neurodegenerative rare disease that can result in devastating symptoms of blindness, gait disturbances, and spastic quadriparesis due to progressive demyelination. Typically, the disease progresses rapidly, causing death within the first decade of life. With limited treatments available, efforts to determine an effective therapy that can alter disease progression or mitigate symptoms have been undertaken for many years, particularly through drug repurposing. Repurposing has generally been guided through clinical experience and small trials. At this time, none of the drug candidates have been approved for use, which may be due, in part, to the lack of pharmacokinetic/pharmacodynamic (PK/PD) information on the repurposed medications in the target patient population. Greater consideration for the disease pathophysiology, drug pharmacology, and potential drug‐target interactions, specifically at the site of action, would improve drug repurposing and facilitate drug development. Incorporating advanced translational and clinical pharmacological approaches in preclinical studies and early stages clinical trials will improve the success of repurposed drugs for X‐ALD as well as other rare diseases.
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Producing valuable coproducts from oleaginous microalgae is an option to reduce the total cost of biofuel production. Here, the influence of nitrogen sources on biomass yield and lipid accumulation of a newly identified oleaginous green microalgal strain, Mychonastes afer HSO-3-1, was evaluated. Carbon assimilation and the following lipid biosynthesis of M. afer were inhibited to some extent under weak acidic conditions (6 < pH < 7) and any of the tested nitrogen source. The highest lipid productivity of 50.7 mg L−1 day−1 was achieved with a 17.6 mM nitrogen supplement in the form of urea. The cell polar lipid content was significantly higher than triacylglycerol (TAG), and saturated palmitic acid (C16:0) occupied a dominant position in the fatty acid profiles while culturing M. afer in acidic medium with NH4+ as the nitrogen source. Under neutral conditions, the lipid productivities of M. afer cultivated in media containing 17.6 mM of NaNO3, NH4Cl, and NH4NO3 were 76.2, 77.5, and 79.0 mg L−1 day−1, respectively. The greatest TAG content (58.56%) of total lipids was obtained when NaNO3 was used as the nitrogen source. There was no significant difference in the fatty acid composition of M. afer cells when they were cultivated in neutral media supplemented with NaNO3, urea, NH4Cl, and NH4NO3. Therefore, NH4+ was not a suitable nitrogen source for M. afer cultivation due to the additional labor, working procedures, and alkali required to adjust the medium pH. Considering that using urea as nitrogen source could reduce the cost of nutrient salts substantially and urea can be taken up and utilized by most microalgae, it is a preferred nitrogen source. The major properties of biodiesel derived from M. afer HSO-3-1 met biodiesel quality, and nervonic acid concentrations remained at approximately 3.0% of total fatty acids.
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Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. Recently advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium, Trichosporon and Lipomyces. This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years.
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Erucic acid (cis-docosa-13-enoic acid, C22:1∆13) and nervonic acid (cis-tetracosa-15-enoic acid, C24:1 ∆15) are important renewable feedstocks in plastic, cosmetic, nylon, and lubricant industries. Furthermore, nervonic acid is also applied to the treatment of some neurological diseases. However, the production of these two very long-chain fatty acids (VLCFA) is very limited as both are not present in the main vegetable oils (e.g., soybean, rapeseed, sunflower, and palm). Ectopic integration and heterologous expression of fatty acid elongases (3-ketoacyl-CoA synthases, KCS) from different plants in Rhodosporidium toruloides resulted in the de novo synthesis of erucic acid and nervonic acid in this oleaginous yeast. Increasing KCS gene copy number or the use of a push/pull strategy based on the expression of elongases with complementary substrate preferences increased significantly the amount of these two fatty acids in the microbial oils. Oil titers in 7-L bioreactors were above 50 g/L, and these two VLCFA represented 20–30% of the total fatty acids. This is the first time that microbial production of these types of oils is reported.
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Large-scale production of biofuels and other high-value products from microalgae focuses on strain selection with desired characteristics such as high lipid productivity and robustness. However, microalgae often suffer severe biomass losses due to contamination that comes in the form of herbivores, especially rotifers. In this study, we found that the total lipid content of 54 microalgal strains from Eastern China ranged from 12.6 to 59.9% of dry cell weight (dw), with an average of 35.4% dw. Lipid productivity of these microalgal strains varied from 0.04 to 0.14 g L⁻¹ day⁻¹, and 15 showed lipid production rates over 0.10 g L⁻¹ day⁻¹. The fatty acid profiles of 34 microalgal strains with lipid content over 40% dw were analyzed by gas chromatography. We found that the amount of C18:1 increased with increasing total lipid content, whereas C14:0 and C16:0 showed a negative correlation to the total amount of lipids. An experimental rotifer-microalgae predator-prey model was constructed, and the anti-rotifer robustness of these microalgal strains was evaluated. We found that Didymogenes sp. HN-4 can depress the growth of rotifers significantly. This study demonstrates the biodiversity of the native Chinese microalgae in lipid productivity and robustness, which lays the groundwork for mass algal culture.
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Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5mgl 1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.
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Chromatin and metabolic states both influence lifespan, but how they interact in lifespan regulation is largely unknown. The COMPASS chromatin complex, which trimethylates lysine 4 on histone H3 (H3K4me3), regulates lifespan in Caenorhabditis elegans. However, the mechanism by which H3K4me3 modifiers affect longevity, and whether this mechanism involves metabolic changes, remain unclear. Here we show that a deficiency in H3K4me3 methyltransferase, which extends lifespan, promotes fat accumulation in worms with a specific enrichment of mono-unsaturated fatty acids (MUFAs). This fat metabolism switch in H3K4me3 methyltransferase-deficient worms is mediated at least in part by the downregulation of germline targets, including S6 kinase, and by the activation of an intestinal transcriptional network that upregulates delta-9 fatty acid desaturases. Notably, the accumulation of MUFAs is necessary for the lifespan extension of H3K4me3 methyltransferase-deficient worms, and dietary MUFAs are sufficient to extend lifespan. Given the conservation of lipid metabolism, dietary or endogenous MUFAs could extend lifespan and healthspan in other species, including mammals.
Nervonic acid (C24:1, Δ15), a monounsaturated long chain fatty acid, is a major component of mammalian neural tissue. Lack of nervonic acid in human usually leads to neurological disorder. Recently, production of nervonic acid from plant for pharmaceutical, nutraceutical, and functional food received increasing attention. We have previously isolated a microalgal strain Mychonastes afer HSO-3-1, which can accumulate approximately 6% of nervonic acid and <0.5% of erucic acid in triacylglycerol. In this study, the full length of 3-ketoacyl-coA synthase gene MaKCS, encoding the first component of fatty acid elongation complex, was cloned and expressed in Saccharomyces cerevisiae BY4741 under the control of GAL1 promoter. S. cerevisiae harboring pYC230-Gal-MaKCS resulted in the production of 1.5% nervonic acid in total lipid. The expression of KCS gene in M. afer was found to be induced under stress conditions such as nitrogen deficiency and high light, which was revealed by real-time PCR analysis. MaKCS was demonstrated to be able to extend very long chain monounsaturated fatty acids including nervonic acid, which provides insights into how nervonic acid is synthesized in microalgae.
Sphingolipids have been implicated in age-related neurodegeneration. Previous studies have reported elevated ceramide levels in the brain of old rodents, but a systematic investigation of the impact of age on brain sphingolipid metabolism is still lacking. Here we quantified 17 key sphingolipid species in the hippocampus of young (3months), middle-aged (12months) and old (21months) male and female mice. Lipids were extracted and quantified by liquid chromatography/mass spectrometry; transcription of enzymes involved in sphingolipid biosynthesis was evaluated by qPCR. Age-dependent changes of multiple sphingolipid species - including ceramide (d18:1/18:0), sphingomyelin (d34:1), hexosylceramide (d18:1/16:0), ceramide (d18:1/24:0) - were found in mice of both sexes. Moreover, sex-dependent changes were seen with hexosylceramide (d18:1/18:0), ceramide (d18:1/22:0), sphingomyelin (d36:1) and sphingomyelin (d42:1). Importantly, an age-dependent accumulation of sphingolipids containing nervonic acid (24:1) was observed in 21month-old male (p=0.04) and female mice (p<0.0001). Consistent with this increase, transcription of the nervonic acid-synthesizing enzyme, stearoyl-CoA desaturase (Scd1 and Scd2), was upregulated in 21month-old female mice (Scd1 p=0.006; Scd2 p=0.009); a similar trend was observed in males (Scd1 p=0.07). In conclusion, the results suggest that aging is associated with profound sex-dependent and -independent changes in hippocampal sphingolipid profile. The results also highlight the need to examine the contribution of sphingolipids, and particularly of those containing nervonic acid, in normal and pathological brain aging.