Docosahexaenoic Acid Production Research Articles

Photoautotrophic Production of Docosahexaenoic Acid- and Eicosapentaenoic Acid-Enriched Biomass by Co-Culturing Golden-Brown and Green Microalgae

Marine microalgae offer a sustainable alternative source for the human diet’s essential omega-3-fatty acids, including docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, none of them can produce DHA and EPA in a nutritionally balanced ratio of 1:1. As shown recently, the phototrophic co-cultivation of the golden-brown microalgae Tisochrysis lutea (DHA producer) with the green microalgae Microchloropsis salina (EPA producer) can provide microalgae biomass with a balanced DHA-to-EPA ratio with increased productivity compared to monocultures. This study evaluates whether other golden-brown (Isochrysis galbana) and green microalgae (Nannochloropsis oceanica, Microchloropsis gaditana) can enable the phototrophic batch production of omega-3 fatty acids in a nutritionally balanced ratio in co-culture. All co-cultivations applying a physically dynamic climate simulation of a repeated sunny summer day in Australia in LED-illuminated flat-plate gas lift photobioreactors resulted in increased biomass concentrations compared to their respective monocultures, achieving balanced DHA-to-EPA ratios of almost 1:1. Using urea instead of nitrate as a nitrogen source increased the EPA content by up to 80% in all co-cultures. Light spectra measurements on the light-adverted side of the photobioreactor showed that increased biomass concentrations in co-cultures could have been related to enhanced light use due to the utilization of different wavelengths of the two microalgae strains, especially with the use of green light (500–580 nm) primarily by golden-brown microalgae (I. galbana) and orange light (600–620 nm) predominantly used by green microalgae (N. oceanica). Phototrophic co-cultivation processes thus promise higher areal biomass yields if microalgae are combined with complimentary light-harvesting features.

Chitosan-based delivery of fish codon-optimised Caenorhabditis elegans FAT-1 and FAT-2 boosts EPA and DHA biosynthesis in Sparus aurata

AbstractOmega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are essential fatty acids required in healthy balanced diets for humans. To induce sustained production of n-3 LC-PUFA in gilthead seabream (Sparus aurata), chitosan-tripolyphosphate (TPP) nanoparticles encapsulating plasmids expressing fish codon-optimised Caenorhabditis elegans FAT-1 and FAT-2 were intraperitoneally administered every 4 weeks (3 doses in total, each of 10 μg plasmid per g of body weight). Growth performance and metabolic effects of chitosan-TPP complexed with pSG5 (empty plasmid), pSG5-FAT-1, pSG5-FAT-2 and pSG5-FAT-1 + pSG5-FAT-2 were assessed 70 days post-treatment. Tissue distribution analysis showed high expression levels of fish codon-optimised FAT-1 and FAT-2 in the liver (> 200-fold). Expression of fat-1 and fat-1 + fat-2 increased weight gain. Fatty acid methyl esters assay revealed that co-expression of fat-1 and fat-2 increased liver production and muscle accumulation of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 LC-PUFA, while decreased the n-6/n-3 ratio. Co-expression of fat-1 and fat-2 downregulated srebf1 and genes encoding rate-limiting enzymes for de novo lipogenesis in the liver, leading to decreased circulating triglycerides and cholesterol. In contrast, FAT-2 and FAT-1 + FAT-2 upregulated hepatic hnf4a, nr1h3 and key enzymes in glycolysis and the pentose phosphate pathway. Our findings demonstrate for the first time efficient and sustained production of EPA and DHA in animals after long-term treatment with chitosan-TPP-DNA nanoparticles expressing FAT-1 and FAT-2, which enabled the production of functional fish rich in n-3 LC-PUFA for human consumption.

Maternal circadian rhythm disruption affects neonatal inflammation via metabolic reprograming of myeloid cells.

Disruption of circadian rhythm during pregnancy produces adverse health outcomes in offspring; however, the role of maternal circadian rhythms in the immune system of infants and their susceptibility to inflammation remains poorly understood. Here we show that disruption of circadian rhythms in pregnant mice profoundly aggravates the severity of neonatal inflammatory disorders in both male and female offspring, such as necrotizing enterocolitis and sepsis. The diminished maternal production of docosahexaenoic acid (DHA) and the impaired immunosuppressive function of neonatal myeloid-derived suppressor cells (MDSCs) contribute to this phenomenon. Mechanistically, DHA enhances the immunosuppressive function of MDSCs via PPARγ-mediated mitochondrial oxidative phosphorylation. Transfer of MDSCs or perinatal supplementation of DHA relieves neonatal inflammation induced by maternal rhythm disruption. These observations collectively demonstrate a previously unrecognized role of maternal circadian rhythms in the control of neonatal inflammation via metabolic reprograming of myeloid cells.

Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of DHA-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. DS is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

Concentration-dependent dual roles of proanthocyanidins on oxidative stress and docosahexaenoic acid production in Schizochytrium sp. ATCC 20888

Antioxidant addition is an effective strategy to achieve docosahexaenoic acid (DHA) overproduction in oleaginous microorganisms. Nevertheless, antioxidants like phenolic compounds sometimes exert pro-oxidant activity. In this work, effects of proanthocyanidins (PAs) on fermentation performance and oxidative stress in Schizochytrium sp. were investigated. Low PAs addition (5 mg/L) reduced reactive oxygen species and enhanced lipogenic enzymes activities and NADPH, resulting in significant increase in lipid (20.3 g/L) by 33.6 % and DHA yield (9.8 g/L) by 53.4 %. In contrast, high PAs addition (500 mg/L) exerted pro-oxidant effects, aggravated oxidative damage and lipid peroxidation, leading to sharp decrease in biomass (21.3 g/L) by 35.1 %, lipid (8.2 g/L) by 46.0 %, and DHA (2.9 g/L) by 54.8 %. Therefore, the antioxidant concentration is especially crucial in DHA production. This study is the first to report concentration-dependant dual roles of PAs in oxidative stress and DHA production in Schizochytrium sp., providing new insights into microbial DHA production.

Enhanced fatty acid storage combined with the multi-factor optimization of fermentation for high-level production of docosahexaenoic acid in Schizochytrium sp.

Schizochytrium sp. hasreceived much attention for itsability to synthesize and accumulate high-level docosahexaenoic acid (DHA), which can reach nearly 40 % of total fatty acids. In this study, the titer of DHA in Schizochytrium sp. was successfully improved by enhancing DHA storage through overexpressing the diacylglycerol acyltransferase (ScDGAT2C) gene, as well as optimizing the supply of precursors and cofactors required for DHA synthesis by response surface methodology. Notably, malic acid, citric acid, and biotin showed synergistic and time-dependent effects on DHA accumulation. The maximum lipid and DHA titers of the engineered Schizochytrium sp. strain reached 84.28 ± 1.02 g/L and 42.23 ± 0.69 g/L, respectively, with the optimal concentration combination (1.62 g/L malic acid + 0.37 g/L citric acid + 8.28 mg/L biotin) were added 48 h after inoculation. This study provides an effective strategy for improving lipid and DHA production in Schizochytrium sp.

Lipidomic and transcriptomic analysis of the increase in eicosapentaenoic acid under cobalamin deficiency of Schizochytrium sp.

Schizochytrium sp. is a heterotrophic microorganism capable of accumulating polyunsaturated fatty acids and has achieved industrial production of docosahexaenoic acid (DHA). It also has the potential for eicosapentaenoic acid (EPA) production. In this study, it was found that the cell growth, lipid synthesis and fatty acid composition of Schizochytrium sp. were significantly affected by the level of cobalamin in the medium, especially with regard to the content of EPA in the fatty acids. The content of EPA in the fatty acids increased 17.91 times, reaching 12.00%, but cell growth and lipid synthesis were significantly inhibited under cobalamin deficiency. The response mechanism for this phenomenon was revealed through combined lipidomic and transcriptomic analysis. Although cell growth was inhibited under cobalamin deficiency, the genes encoding key enzymes in central carbon metabolism were still up-regulated to provide precursors (Acetyl-CoA) and reducing power (NADPH) for the synthesis and accumulation of fatty acids. Moreover, the main lipid subclasses observed during cobalamin deficiency were glycerolipids (including glycerophospholipids), with EPA primarily distributed in them. The genes involved in the biosynthesis of these lipid subclasses were significantly up-regulated, such as the key enzymes in the Kennedy pathway for the synthesis of triglycerides. Thus, this study provided insights into the specific response of Schizochytrium sp. to cobalamin deficiency and identified a subset of new genes that can be engineered for modification.

The Biosynthesis of Astaxanthin Esters in Schizochytrium sp. is Mediated by a Bifunctional Diacylglycerol Acyltransferase.

Astaxanthin esters are a major form of astaxanthin found in nature. However, the exact mechanisms of the biosynthesis and storage of astaxanthin esters were previously unknown. We found that Schizochytrium sp. synthesized both astaxanthin and docosahexaenoic acid (DHA)-enriched lipids. The major type of astaxanthin produced was free astaxanthin along with astaxanthin-DHA monoester and other esterified forms. DHA accounted for 41.0% of the total fatty acids from astaxanthin monoesters. These compounds were deposited mainly in lipid droplets. The biosynthesis of the astaxanthin esters was mainly carried out by a novel diacylglycerol acyltransferase ScDGAT2-1, while ScDGAT2-2 was involved only in the production of triacylglycerol. We also identified astaxanthin ester synthases from the astaxanthin-producing algae Haematococcus pluvialis and Chromochloris zofingiensis, as well as a thraustochytrid Hondaea fermentalgiana with an unknown carotenoid profile. This investigation enlightens the application of thraustochytrids for the production of both DHA and astaxanthin and provides enzyme resources for the biosynthesis of astaxanthin esters in the engineered microbes.

Batch Fermentation of Salt-Acclimatizing Microalga for Omega-3 Docosahexaenoic Acid Production Using Biodiesel-Derived Crude Glycerol Waste as a Low-Cost Substrate

Biodiesel produced from waste cooking oil (WCO) is on the rise and inevitably leads to issues in managing glycerol waste. Due to the presence of colour, odour and other minor compounds, the refining costs for this type of glycerol are higher and uneconomical. The potential of biodiesel-derived glycerol waste (BDGW) obtained from WCO to produce the highly added product of docosahexaenoic acid (DHA), also known as omega-3 polyunsaturated fatty acid, via the marine microalga of Schizochytrium limacinum ATCC MYA-1381 under aerobic batch fermentation was investigated. Cell growth, as well as DHA production, were performed under various operating conditions, including aeration rates and BDGW concentrations. The effect of the substrate type on cell growth and DHA yield was evaluated. The optimum operating condition was obtained when the air flow of a 0.25 vvm and 50 g/L of the glycerol concentration was fed into the fermenter and maximum cell dry weight (11.40 g/L) and DHA yield (665.52 mg/g) were achieved. However, cell growth and DHA yield were not significantly different when S. limacinum was grown using various carbon sources. Successfully, it clearly demonstrates that the BDGW can be used as a cheap carbon source for DHA production via marine microalgae using aerobic batch fermentation.

Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile

BackgroundThe hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1 gene of Schizochytrium limacinum SR21 (S. limacinum SR21) on lipid metabolism was investigated.ResultsPLD1 knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and real-time quantitative PCR analysis revealed the knockout of PLD1 reduced the interexchange and increased de novo synthesis of PLs, which altered the composition of PLs, accompanied by a final decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs.ConclusionsThese results indicate that PLD1 mainly performs the transphosphatidylation activity in S. limacinum SR21, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGs via an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in S. limacinum SR21.

Development of an alternative low-cost culture medium for a new isolated high-production DHA strain using kitchen wastewater

Docosahexaenoic acid (DHA) is a high-value polyunsaturated fatty acid. The industrial development of microbial DHA was restricted due to high medium cost. Kitchen wastewater was always rich in nitrogen, phosphorous, carbohydrates, which can be used for microorganism growth. This study developed of a primitive low-cost culture medium for a new isolated high-production DHA strain using kitchen wastewater. The results showed that, a total of 66 strains were isolated from mangrove habitats. The highest DHA-producing strain N6 was identified as Aurantiochytrium sp. (GenBank accession number OL455742) with a DHA production of 6.56 g/L. Then the optimization of kitchen wastewater medium was designed by Response Surface Methodology, and 7.14 ∼ 7.23 g/L of DHA production was obtained. At the same time, the lipid content in Aurantiochytrium sp. N6 was as high as 75.68%, which contributed to the increase of DHA production in optimal kitchen wastewater medium. Finally, the substrate cost of kitchen wastewater medium was reduced by 43.95%. Meanwhile, the dissolved organic carbon (DOC), dissolved nitrogen (DN) and dissolved phosphorus (DP) in kitchen wastewater were utilized by Aurantiochytrium sp. N6 more than 90%. This work enriched the competitive candidate DHA-producing microbes, and provided a perceptive idea for both the medium substrate cost reducing and kitchen wastewater recycling.

Integrated biorefinery approach for bioconversion of fish manure to volatile fatty acids and its valorization by Schizochytrium sp for docosahexaenoic acid, squalene, and carotenoids production

To meet environmental sustainable goals, an integrated approach was used to generate volatile fatty acids (VFAs) from fish manure through acidogenic fermentation and their valorization by Schizochytrium limacinum SR21 for value-added biomass production. Acidogenic fermentations were conducted at different pH values (5.0–9.0) and total solid (TS) contents (5 %, 8 %, and 12 %). The highest VFA yield from thermally pretreated fish manure was obtained at pH 9.0 and TS 12 %. In batch culture, 84.0 % of the acidogenic fermentation VFAs were utilized by Schizochytrium limacinum SR21. The highest significant biomass, total fatty acids (TFA), and docosahexaenoic acid (DHA) production were 19, 10.1, and 3.0 g/L, respectively, at C/N ratio of 90 in batch culture with yeast extract as a nitrogen source. Analysis of the effects of three nitrogen feeding strategies with simulated VFA in pH-auxostat fed-batch culture shows that DHA concentration of 15.3 g/L and TFA yield of 53.56 g/L were achieved with low nitrogen (10 g/L) feeding. Squalene and carotenoid contents were 85 mg/g and 0.43 mg/g of dry biomass in fed-batch culture. These findings shows that the production of bio-based volatile fatty acids (VFA) is a cost-effective eco-friendly waste management method and provides high-value-added biomass for biofuel and aquaculture feed production.

Essential omega-3 fatty acids are depleted in sea ice and pelagic algae of the Central Arctic Ocean.

Microalgae are the main source of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), essential for the healthy development of most marine and terrestrial fauna including humans. Inverse correlations of algal EPA and DHA proportions (% of total fatty acids) with temperature have led to suggestions of a warming-induced decline in the global production of these biomolecules and an enhanced importance of high latitude organisms for their provision. The cold Arctic Ocean is a potential hotspot of EPA and DHA production, but consequences of global warming are unknown. Here, we combine a full-seasonal EPA and DHA dataset from the Central Arctic Ocean (CAO), with results from 13 previous field studies and 32 cultured algal strains to examine five potential climate change effects; ice algae loss, community shifts, increase in light, nutrients, and temperature. The algal EPA and DHA proportions were lower in the ice-covered CAO than in warmer peripheral shelf seas, which indicates that the paradigm of an inverse correlation of EPA and DHA proportions with temperature may not hold in the Arctic. We found no systematic differences in the summed EPA and DHA proportions of sea ice versus pelagic algae, and in diatoms versus non-diatoms. Overall, the algal EPA and DHA proportions varied up to four-fold seasonally and 10-fold regionally, pointing to strong light and nutrient limitations in the CAO. Where these limitations ease in a warming Arctic, EPA and DHA proportions are likely to increase alongside increasing primary production, with nutritional benefits for a non-ice-associated food web.

Nutritional effects on biomass and metabolic products by Aurantiochytrium sp.

Docosahexaenoic acid, or Docosahexaenoic acid (DHA), plays a pivotal role in biological functions and is beneficial for both humans and animals. Traditionally, DHA is sourced from fish and fish oil. Due to the disadvantages associated with these sources, such as marine pollution and variable composition, there is a pressing need to explore alternative, reliable sources for DHA. Aurantiochytrium sp. has been identified as a promising candidate for DHA production. The primary aim of this study was to cultivate three strains in previously published media and, subsequently, to develop defined or semi-defined media for Aurantiochytrium sp.. These strains were grown in the selected media, and the dry weight of the cells was measured. Total fatty acids were extracted and analysed using gas chromatography. All three strains demonstrated satisfactory growth in media that incorporated glucose as a carbon source and monosodium glutamate as a nitrogen source. Strain B072 achieved the highest DHA concentration, with a peak of 5.0 g/l, and could accumulate lipids up to 57.57% (w/w). The DHA content in the biomass was 21.43% (w/w), with a biomass yield of 23.35 g/l. Vitamins and trace elements positively influenced the growth and DHA production of Aurantiochytrium sp.. Furthermore, strain B072 efficiently utilised ammonium for growth and fatty acid production.

Enhancing docosahexaenoic acid production in Aurantiochytrium species using atmospheric and room temperature plasma mutagenesis and comprehensive multi-omics analysis

Aurantiochytrium sp. belongs to marine heterotrophic single-cell protist, which is an important decomposer in marine ecosystem. Aurantiochytrium sp. has gained notoriety because of its ability to accumulate high-value docosahexaenoic acid (DHA), but the key factors of DHA synthesis were unclear at present. In this study, Atmospheric and Room Temperature Plasma technology was applied to the mutagenic breeding of Aurantiochytrium sp., and transcriptomics and proteomics were adopted to analyze the DHA-biosynthesis mechanism. According to the growth and DHA accumulation profiles, the mutant strain Aurantiochytrium sp. R2A35 was selected. The DHA content in total lipids was greatly improved from 49.39 % of the wild strain R2 to 63.69 % of the mutant strain. Moreover, the DHA content in the biomass of Aurantiochytrium sp. R2A35 as 39.72 % was the highest DHA productivity reported so far. The differentially expressed genes distinguished from transcriptome and the TMT-identified differential proteins distinguished from proteome confirmed that the expression of acetyl-CoA carboxylase and ketoacyl reductase was up-regulated by 4.78-fold and 6.95-fold, respectively and the fatty acid synthase was concurrently down-regulated by 2.79-fold, so that more precursor was transported to the polyketide synthase pathway, thereby increasing the DHA yield in Aurantiochytrium sp. R2A35. This research would provide reference for the DHA metabolism process and contribute to the understanding of the decomposer - Aurantiochytrium sp. in marine ecosystems.

Metabolic engineering of Schizochytrium sp. for superior docosahexaenoic acid production

Schizochytrium sp. is an industrial producer of docosahexaenoic acid (DHA), which is an important omega-3 polyunsaturated fatty acid used in the nutraceutical and food industry. Here, we rewired the carbon metabolism of Schizochytrium sp. ATCC20888 for superior DHA production and constructed 18 engineered strains through combinatorial gene manipulation of 8 crucial genes (acc, acl, ampD, dga1, mae, mfe1, pex10, and zwf) related to lipogenesis. Reducing β-oxidations (∆pex10, ∆mfe1), reinforcing the biosynthetic precursors supply (OACC, OACL, OAMPD), reinforcing NADPH supply (OMAE, OZWF), or promoting TAG formation (ODGA1) efficiently promoted DHA and lipid production of Schizochytrium. MAE and DGA1 overexpression exhibited cooperativity towards increasing DHA production and lipogenesis in the WT strain and ACC-overexpression strain, but had no cooperative advantage in the ∆pex10 strain. The highest lipid content (82.2%) and DHA yield (7.04 g/L) were obtained in an engineered strain OACL-ACC/∆pex10, which is 72.1% higher than DHA yield of parental strain. Therefore, inhibition of β-oxidations and ample biosynthetic precursors supply are the determining factors for high DHA productivity and lipid accumulation of Schizochytrium.

BZIP transcription factor FabR: Redox-dependent mechanism controlling docosahexaenoic acid biosynthesis and H2O2 stress response in Schizochytrium sp.

Schizochytrium sp. is an important industrial strain for commercial production of docosahexaenoic acid (DHA), which plays essential physiological roles in infant development and human health. The regulatory network for DHA biosynthesis and lipid accumulation in Schizochytrium remains poorly understood. FabR (fatty acid biosynthesis repressor), a basic leucine zipper (bZIP) transcription factor, was transcriptionally downregulated under low-nitrogen condition. Deletion of fabR gene (mutant ΔfabR) increased production of total lipids and DHA by 30.1% and 46.5%, respectively. ΔfabR displayed H2O2 stress resistance higher than that of parental strain or complementation strain CfabR. FabR bound specifically to 7-bp pseudo-palindromic sequence 5′-ATTSAAT-3′ in upstream regions and repressed transcription of fatty acid biosynthesis genes (acl, fas, pfa) and antioxidant defense genes (cat, sod1, sod2, gpx). DNA binding activity of FabR was regulated in a redox-dependent manner. Under oxidative condition, FabR forms intermolecular disulfide bonds between two Cys46 residues of dimers; its DNA binding activity is thereby lost, and the transcription of its target genes is enhanced through derepression. Our findings clarify the redox-dependent mechanism that modulates FabR activity governing lipid and DHA biosynthesis and H2O2 stress response in Schizochytrium.

Effects of Aurantiochytrium acetophilum as a source of docosahexaenoic acid for juvenile Atlantic salmon (Salmo salar) on growth, nutrient apparent digestibility, tissue fatty acid composition and expression of lipid metabolism-related genes

Thraustochytrids are a group of unicellular marine protists that are noted for their ability to produce high amounts of lipids, especially docosahexaenoic acid (DHA), making them a valuable source for replacing fish oil in aquafeeds. Varying levels of Aurantiochrytrium acetophilum meal (TM) were evaluated as replacement of fish oil in extruded diets for juvenile Atlantic salmon on growth, nutrient apparent digestibility, fatty acid composition in muscle and liver and expression of genes associated with lipid metabolism. Four experimental diets were tested: a control diet without the inclusion of A. acetophilum meal and three experimental diets derived from the control diet, in which fish oil was progressively replaced by the addition of A. acetophilum meal at 2.5% (TM 2.5), 6.5% (TM 6.5) and 12% (TM 12) respectively. After 12 weeks of feeding, the growth, feed conversion, condition factor and survival of the fish were not affected by the inclusion of A. acetophilum in the diet. However, the 12% inclusion significantly decreased the viscerosomatic index of the fish and the digestibility of protein, lipids, ash and energy compared to the other experimental diets. The lipid content and fatty acid profile of the liver and muscle were significantly affected by diet composition. In both tissues, fish fed the TM 12 diet retained the highest levels of DHA. However, it was also observed that EPA concentrations lessened with increasing dietary content of A. acetophilum meal. Finally, the decrease in the expression of Δ5fad in the fish fed with the TM 6.5 and TM 12 diets showed that fish do not require a higher expression of this desaturase for DHA production, due to the high content of this fatty acid in A. acetophilum meal.

Refactoring the architecture of a polyketide gene cluster enhances docosahexaenoic acid production in Yarrowia lipolytica through improved expression and genetic stability

BackgroundLong-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), are essential for human health and have been widely used in the food and pharmaceutical industries. However, the limited availability of natural sources, such as oily fish, has led to the pursuit of microbial production as a promising alternative. Yarrowia lipolytica can produce various PUFAs via genetic modification. A recent study upgraded Y. lipolytica for DHA production by expressing a four-gene cluster encoding a myxobacterial PKS-like PUFA synthase, reducing the demand for redox power. However, the genetic architecture of gene expression in Y. lipolytica is complex and involves various control elements, offering space for additional improvement of DHA production. This study was designed to optimize the expression of the PUFA cluster using a modular cloning approach.ResultsExpression of the monocistronic cluster with each gene under the control of the constitutive TEF promoter led to low-level DHA production. By using the minLEU2 promoter instead and incorporating additional upstream activating UAS1B4 sequences, 5' promoter introns, and intergenic spacers, DHA production was increased by 16-fold. The producers remained stable over 185 h of cultivation. Beneficially, the different genetic control elements acted synergistically: UAS1B elements generally increased expression, while the intron caused gene-specific effects. Mutants with UAS1B16 sequences within 2–8 kb distance, however, were found to be genetically unstable, which limited production performance over time, suggesting the avoidance of long repetitive sequence blocks in synthetic multigene clusters and careful monitoring of genetic stability in producing strains.ConclusionsOverall, the results demonstrate the effectiveness of synthetic heterologous gene clusters to drive DHA production in Y. lipolytica. The combinatorial exploration of different genetic control elements allowed the optimization of DHA production. These findings have important implications for developing Y. lipolytica strains for the industrial-scale production of valuable polyunsaturated fatty acids.

A phospholipid:diacylglycerol acyltransferase is involved in the regulation of phospholipids homeostasis in oleaginous Aurantiochytrium sp.

BackgroundThraustochytrids have gained attention as a potential source for the production of docosahexaenoic acid (DHA), where DHA is predominantly stored in the form of triacylglycerol (TAG). The TAG biosynthesis pathways, including the acyl-CoA-dependent Kennedy pathway and the acyl-CoA-independent pathway, have been predicted in thraustochytrids, while the specific details regarding their roles are currently uncertain.ResultsPhospholipid:diacylglycerol acyltransferase (PDAT) plays a key role in the acyl-CoA-independent pathway by transferring acyl-group from phospholipids (PL) to diacylglycerol (DAG) to from TAG. In thraustochytrid Aurantiochytrium sp. SD116, an active AuPDAT was confirmed by heterologous expression in a TAG-deficient yeast strain H1246. Analysis of AuPDAT function in vivo revealed that deletion of AuPDAT led to slow growth and a significant decrease in cell number, but improved PL content in the single cell during the cell growth and lipid accumulation phases. Interestingly, deletion of AuPDAT did not affect total lipid and TAG content, but both were significantly increased within a single cell. Moreover, overexpression of AuPDAT also resulted in a decrease in cell number, while the total lipid and cell diameter of a single cell were markedly increased. Altogether, both up-regulation and down-regulation of AuPDAT expression affected the cell number, which further associated with the total lipid and TAG content in a single cell.ConclusionsOur study demonstrates that AuPDAT-mediated pathway play a minor role in TAG synthesis, and that the function of AuPDAT may be involved in regulating PL homeostasis by converting PL to TAG in a controlled manner. These findings expand our understanding of lipid biosynthesis in Aurantiochytrium sp. and open new avenues for developing “customized cell factory” for lipid production.