Eicosapentaenoic Acid Production Research Articles

Optimization and mechanism analysis of photosynthetic EPA production in Nannochloropsis salina: evaluating the effect of temperature and nitrogen concentrations

Microalgae, recognized as sustainable and eco-friendly photosynthetic microorganisms, play a pivotal role in converting CO2 into value-added products. Among these, Nannochloropsis salina (Microchloropsis salina) stands out, particularly for its ability to produce eicosapentaenoic acid (EPA), a crucial omega-3 fatty acid with significant health benefits such as anti-inflammatory properties and cardiovascular health promotion. This study focused on optimizing the cultivation conditions of Nannochloropsis salina to maximize EPA production. We thoroughly investigated the effects of varying temperatures and nitrogen (NaNO3) concentrations on biomass, total lipid content, and EPA proportions. We successfully identified optimal conditions at an initial NaNO3 concentration of 1.28 g.L-1 and a temperature of 21°C. This condition was further validated by response surface methodology, which resulted in the highest EPA productivity reported in batch systems (14.4 mg.L-1.day-1). Quantitative real-time PCR and transcriptomic analysis also demonstrated a positive correlation between specific gene expressions and enhanced EPA production. Through a comprehensive lipid analysis and photosynthetic pigment analysis, we deduced that the production of EPA in Nannochloropsis salina seemed to be produced by the remodeling of chloroplast membrane lipids. These findings provide crucial insights into how temperature and nutrient availability influence fatty acid composition in N. salina, offering valuable guidance for developing strategies to improve EPA production in various microalgae species.

Dual stress factors adaptive evolution for high EPA production in Schizochytrium sp. and metabolomics mechanism analysis.

Eicosapentaenoic acid (EPA) is a vital ɷ-3 polyunsaturated fatty acid (PUFA) for human body with various physiological functions. In this study, we proposed an adaptive evolutionary strategy based on high-temperature and high-oxygen two-factor stress to increase the EPA production capacity of Schizochytrium. High-temperature stress was used to increase EPA yield, and high oxygen was implemented to continuously stimulate cell growth and lipid accumulation. The biomass and EPA production of ALE-D50 reached 35.33g/L and 1.54g/L, which were 43.85% and 71.11% higher than that of the original strain, respectively. Lower in vivo reactive oxygen species levels indicated that the evolved strain possessed stronger antioxidant activity. Liquid chromatography-mass spectrometry metabolomics showed that enhanced glucose consumption and glycolysis metabolism, as well as a weakened tricarboxylic acid cycle and reduced amino acid metabolic tributaries in the evolved strain, might be associated with increased growth and EPA synthesis. Finally, the lipid production and EPA production in a fed-batch fermentation were further increased to 48.93g/L and 3.55g/L, improving by 54.30% and 90.86%, respectively. This study provides a novel pathway for promoting EPA biosynthesis in Schizochytrium.

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.

Optimization of Conjugated Linoleic Acid and Eicosapentaenoic Acid Production from Sesame Waste by Response Surface Methodology

Conjugated linoleic acid (CLA) and eicosapentaenoic acid (EPA) have been recognized for their physiological functions and potential as dietary supplements. This study is aimed at investigating the production and enhancing the efficiency of these two acids from Lactiplantibacillus plantarum using sesame waste as a natural, cost-effective, and readily available culture medium. Response surface methodology (RSM) was employed to optimize the production of CLA and EPA in a solid bed system using sesame waste paste as the fermentation substrate. The main processing parameters, including three humidity levels (60%, 70%, and 80%), three inoculum percentages (2%, 4%, and 6% v/v), and three fermentation temperatures (30°C, 37°C, and 44°C), were optimized. An experiment was conducted to validate the determined conditions, and a strong correlation was observed between the experimental results and the predictions made by the software. The optimal conditions for CLA and EPA production were determined to be an inoculation level of 6% with 80% humidity and at 37°C. Gas chromatography analysis of the fermented sesame waste medium revealed that the highest yields of CLAc9t11 and CLAt10c12, as well as eicosapentaenoic acid, were obtained in the medium fermented with 80% humidity and 6% inoculation at 37°C. The respective percentages of these fatty acids in the total fatty acid composition were found to be 0.351% (w/v) and 0.1% (w/v) for CLA and 0.139% (w/v) for EPA under the optimized conditions. These findings contribute to the understanding of CLA and EPA production and highlight the potential of L. plantarum and sesame waste as a fermentation substrate for their efficient production.

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.

Pleiotropic beneficial cardiometabolic actions of a high-purity eicosapentaenoic acid product in high cardiovascular risk individuals.

The ideal approach to the secondary dyslipidemia goal of lowering triglycerides (TG) is not well established. The available ω-3 fatty acid products differ from each other in composition and content. The purpose of the present study was to investigate the effect of a highly purified eicosapentaenoic acid (EPA) formulation on cardiometabolic biomarkers in high cardiovascular (CV) risk patients. The study included 226 subjects with high TG and ≥1 of the following CV risk factors: arterial hypertension, diabetes mellitus, ultrasound-documented atheromatosis, peripheral artery disease, previous myocardial infarction, or ischemic stroke. Participants received 2 g EPA twice daily for 3 months, along with typical nutritional counseling. Cardiometabolic hematological parameters (TG, low-density lipoprotein [LDL], high-density lipoprotein [HDL], non-HDL, total cholesterol [TChol], apolipoprotein A1 [Apo A1 ], apolipoprotein B [Apo B], glucose, glycated hemoglobin [HbA1 c], and C-reactive protein [CRP]) were measured at baseline and at 3 months. The mean patients' age was 61.1 ± 1.4 years and the mean baseline TG was 2.97 ± 0.15 mmol/L. Apart from Apo A1 , all other biomarkers significantly (p < 0.05) improved at 3 months, regardless of sex (except Apo B) and age: TG 1.75 ± 0.09 versus 2.97 ± 0.15 mmol/L, LDL 2.46 ± 0.08 versus 3.05 ± 0.13 mmol/L, HDL 1.22 ± 0.03 versus 1.11 ± 0.03 mmol/L, non-HDL 3.29 ± 0.10 versus 4.14 ± 0.16 mmol/L, TChol 4.55 ± 0.10 versus 5.15 ± 0.13 mmol/L, Apo A1 26.8 ± 9.3 versus 22.5 ± 8.6 μmol/L, Apo B 1.25 ± 0.23 versus 1.29 ± 0.23 μmol/L, glucose 5.66 ± 0.11 versus 5.99 ± 0.17 mmol/L, HbA1 c 5.83 ± 0.1 versus 5.97 ± 0.1% and CRP 1.92 ± 0.2 versus 5.26 ± 2.8 mg/L. In conclusion, adding highly purified EPA product (4 g daily) on nutritional counseling leads to a significant TG reduction. In addition, this treatment appears to have pleiotropic beneficial cardiometabolic actions.

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.

Characterization of a new HUP+ Phaeodactylum tricornutum strain having improved production of eicosapentaenoic acid

AbstractBACKGROUNDEicosapentaenoic acid (EPA, 20:5) is an essential fatty acid of the omega‐3 family and its importance for prevention and treatment of several disorders has been documented. Phaeodactylum tricornutum is a prominent candidate as an EPA producer. In several microalgal species the mixotrophic growth allows improvement of cell growth and EPA production, although P. tricornutum does not have glucose transporter proteins, limiting glucose assimilation. In the present study, the growth and EPA production of P. tricornutum UTEX 646 and its transformant T‐HUP*(8), in which the glucose transporter hup1 gene was expressed, was evaluated under mixotrophic cultivation using glucose as carbon source.RESULTSInsertion of the gene hup1 in the T‐HUP*(8) strain allowed an increase in the glucose uptake of 4.4‐fold compared to the glucose consumption observed in the wild type strain. In T‐HUP*(8) the lipid content and productivity were 42% and 63% higher than in the wild type; these differences were reflected in the EPA concentration, which was enhanced up to twofold in cultivation of T‐HUP*(8) strain. The improvement observed in lipid and EPA production in the strain T‐HUP*(8) can be explained by increases of 2.97‐, 5.71‐, and 9.44‐fold in the relative expression of the genes acc1, me1, and ptd5, respectively.CONCLUSIONIncrease in glucose consumption in cultivation of the P. tricornutum T‐HUP*(8) strain caused metabolic changes that increased the lipid content and EPA specific concentration, making this strain a good candidate for improved EPA production. © 2023 Society of Chemical Industry (SCI)

Influence of specific light availability and solvent on process economics — The production of fucoxanthin and eicosapentaenoic acid from P. tricornutum using flat-panel airlift photobioreactors with artificial light

This study presents an economic assessment of an up- and downstream process (USP and DSP) using P. tricornutum in flat-panel airlift photobioreactors (FPA-PBRs) with artificial illumination. Depending on the solvent, it is possible to obtain separately a crystalline fucoxanthin and eicosapentaenoic acid-rich (EPA) oil or a fucoxanthin- and EPA-rich oleoresin. The objective was to investigate the potential of optimizing light supply during USP and changing the extraction solvent during DSP to reduce fixed capital investment (FCI) and production costs. Comprehensive experimental data on the cultivation of P. tricornutum and the extraction and separation of the products were used to perform techno-economic assessments (TEA) and profitability analyses of different scenarios. In the first part, six scenarios were developed to determine the economic performance with a biomass concentration (c0) of either 3.0 or 6.0 g l−1 and a specific light availability (Ispec) of 2.0, 5.0 or 8.0 μmolphotons g−1 s−1 during the annual production of 10.0 t fucoxanthin ((component of biomass) until harvest. In the second part, the scenario with the economically most favourable results served as the basis for TEA depending on the solvent. It was demonstrated that production costs of dry weight biomass (CX) could be reduced from 644.31 to 184.30 € kgDW−1 by increasing Ispec and volumetric biomass productivity (QX) with the lowest biomass yield on light energy (YX,ph). A sensitivity analysis was performed to evaluate the possibilities of reducing costs and increasing profitability. Increasing QX proved to be the most effective, resulting in a decrease in CX of up to 20.1 %. Subsidising the FCI was most effective in reducing FCI, resulting in a decrease of up to 49.6 %. By using ethanol instead of a nonpolar solvent, the specific production costs of fucoxanthin (CFx) could be decreased by 60.0 % from around 39,825 € kgFx−1 to 16,111 € kgFx−1.

Temperature, phosphorus and species composition will all influence phytoplankton production and content of polyunsaturated fatty acids.

Temperature increases driven by climate change are expected to decrease the availability of polyunsaturated fatty acids in lakes worldwide. Nevertheless, a comprehensive understanding of the joint effects of lake trophic status, nutrient dynamics and warming on the availability of these biomolecules is lacking. Here, we conducted a laboratory experiment to study how warming (18-23°C) interacts with phosphorus (0.65-2.58μM) to affect phytoplankton growth and their production of polyunsaturated fatty acids. We included 10 species belonging to the groups diatoms, golden algae, cyanobacteria, green algae, cryptophytes and dinoflagellates. Our results show that both temperature and phosphorus will boost phytoplankton growth, especially stimulating certain cyanobacteria species (Microcystis sp.). Temperature and phosphorus had opposing effects on polyunsaturated fatty acid proportion, but responses are largely dependent on species. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) synthesizing species did not clearly support the idea that warming decreases the production or content of these essential polyunsaturated fatty acids. Our results suggest that warming may have different effects on the polyunsaturated fatty acid availability in lakes with different nutrient levels, and that different species within the same phytoplankton group can have contrasting responses to warming. Therefore, we conclude that future production of EPA and DHA is mainly determined by species composition.

High productivity of fucoxanthin and eicosapentaenoic acid in a marine diatom Chaetoceros gracilis by perfusion culture under high irradiance

For a high production rate of microalgae, a high-cell-density culture is an effective strategy for reducing the light intensity per dry weight in the culture to avoid photoinhibition under high light conditions. However, under high cell density microalgae are exposed to nutrient-deficient conditions, resulting in a reduced biomass production rate. To enhance the production rate of high-value bioactives such as fucoxanthin and eicosatetraenoic acid (EPA) by the diatom Chaetoceros gracilis, a perfusion culture was operated at harvesting rates of 10–40 % under light intensities of 300–2000 μmol m−2 s−1. At a harvesting rate of 20 % under a high light intensity of 2000 μmol m−2 s−1, the dry weight increased, and the areal productivity reached 15.6 ± 1.3 g-dry weight (dw) m−2 d−1, which is comparable to the maximum value previously reported in diatoms. The high biomass production, even at a low harvesting rate of 20 %, could result from sufficient nutrient supply by perfusion culture. At a harvesting rate of 20 %, the EPA areal productivity achieved 183 mg-dw m−2 d−1 even under 2000 μmol m−2 s−1 due to the increase in the dry weight using the perfusion culture. However, the fucoxanthin content decreased under 2000 μmol m−2 s−1. A strong relationship was observed between the specific light intensity per dry weight and fucoxanthin content in C. gracilis. This relationship indicates that the fucoxanthin content does not increase unless the specific light intensity per dry weight is adjusted below 20 μmol g-dw−1 s−1. Thus, perfusion culture proved to be useful to control the biomass-specific light intensity for the effective production of EPA and fucoxanthin in C. gracilis under high light conditions expected for outdoor culture.

Transcriptome Analysis Reveals an Eicosapentaenoic Acid Accumulation Mechanism in a Schizochytrium sp. Mutant.

Eicosapentaenoic acid (EPA) is an omega-3 long-chain polyunsaturated fatty acid (PUFA) essential for human health. Schizochytrium is a marine eukaryote that has been widely utilized for the synthesis of PUFAs. The current low potency and performance of EPA production by fermentation of Schizochytrium spp. limits its prospect in commercial production of EPA. Since the synthesis pathway of EPA in Schizochytrium spp. is still unclear, mutagenesis combined with efficient screening methods are still desirable. In this study, a novel screening strategy was developed based on a two-step progressive mutagenesis method based on atmospheric and room temperature plasma (ARTP) and diethyl sulfate (DES) after multiple stresses (sethoxydim, triclosan and 2,2'-bipyridine) compound screening. Finally, the mutant strain DBT-64 with increased lipid (1.57-fold, 31.71 g/L) and EPA (5.64-fold, 1.86 g/L) production was screened from wild-type (W) strains; the docosahexaenoic acid (DHA) content of mutant DBT-64 (M) was 11.41% lower than that of wild-type strains. Comparative transcriptomic analysis showed that the expression of genes related to the polyketide synthase, fatty acid prolongation, and triglyceride synthesis pathways was significantly upregulated in the mutant strain, while the expression of genes involved in the β-oxidation pathway and fatty acid degradation pathway was downregulated in favor of EPA biosynthesis in Schizochytrium. This study provides an effective strain improvement method to enhance EPA accumulation in Schizochytrium spp. IMPORTANCE Schizochytrium, a marine eukaryotic microorganism, has emerged as a candidate for the commercial production of PUFAs. EPA is an omega-3 PUFA with preventive and therapeutic effects against cardiovascular diseases, schizophrenia, and other disorders. Currently, the low potency and performance of EPA production by Schizochytrium spp. limits its commercialization. In this study, we performed two-step progressive mutagenesis based on ARTP and DES and screened multiple stresses (sethoxydim, triclosan, and 2,2'-bipyridine) to obtain the EPA-high-yielding Schizochytrium mutant. In addition, high expression of the polyketide synthase pathway, fatty acid elongation pathway, and triglyceride synthesis pathway in the mutants was confirmed by transcriptomic analysis. Therefore, the multistress screening platform established in this study is important for breeding EPA-producing Schizochytrium spp. and provides valuable information for regulating the proportion of EPA in microalgal lipids by means of genetic engineering.

Metabolic analysis of Schizochytrium sp. mutants with high EPA content achieved with ARTP mutagenesis screening.

Eicosapentaenoic acid (EPA) belonged to the ω-3 series of polyunsaturated fatty acids and had physiological functions lipid as regulating blood lipid and preventing cardiovascular diseases. Schizochytrium sp. was considered to be a potential industrial fermentation strain of EPA because of its fast growth, high oil content, and simple fatty acid composition. However, Schizochytrium sp. produced EPA with low production efficiency and a long synthesis path. This research aims to improve the yield of EPA in Schizochytrium sp. by ARTP mutagenesis and to reveal the mechanism of high-yield EPA through transcriptome analysis. ARTP mutagenesis screening yielded the mutant M12 that whereas the productivity of EPA increased 108% reaching 0.48g/L, the total fatty acid concentration was 13.82g/L with an increase of 13.7%. The transcriptomics revealed 2995 differentially expressed genes were identified between M12 and the wild-type strain and transcripts involved in carbohydrate, amino acid, energy, and lipid metabolism were up-regulated. Among them, the hexokinase (HK) and the phosphofructokinase genes (PFK), which can catalyze pyruvate to acetyl-CoA, were increased 2.23-fold and 1.78-fold. Glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GLDH), which can both generate NADPH, were increased by 1.67-fold and 3.11-fold. Furthermore, in the EPA synthesis module, the expression of 3-oxoacyl-[acyl-carrier protein] reductase(fabG) and carbonyl reductase 4 / 3-oxoacyl-[acyl-carrier protein] reductase beta subunit(CBR4), also up-regulated 1.11-fold and 2.67-fold. These may lead to increases in cell growth. The results provide an important reference for further research on promoting fatty acid and EPA accumulation in Schizochytrium sp.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.

Characterization of the Positive Transcription Regulator PfaR for Improving Eicosapentaenoic Acid Production in Shewanella putrefaciens W3-18-1.

The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster (pfaEABCD) was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of pfaABCD. The transcription start site of pfaR was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of pfaR. Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. IMPORTANCE We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.

Highly efficient co-production of fucoxanthin and eicosapentaenoic acid by heterotrophic cultivation of a newly isolated microalga Nitzschia sp. FZU62

Microalgae can accumulate various compounds simultaneously, thus co-production of high-value metabolites to improve the economic value of microalgae production is feasible. In this study, a microalga strain Nitzschia sp. FZU62 capable of co-producing fucoxanthin and eicosapentaenoic acid (EPA) was isolated and identified. Further, heterotrophic conditions were optimized to improve the co-production efficiency in flasks. Results showed that the optimal conditions for flask culture were cultivation for 84 h with an inoculum size of 0.47 g/L and a temperature of 19 °C. Besides, three fed-batch culture strategies with distinct nutrient compositions were explored to improve the heterotrophic performance of strain FZU62. Results indicated that the highest biomass, fucoxanthin, and EPA productivities were obtained by Fed-batch III, in which concentrated medium was pulse-fed to supply 60 % initial nutrients once glucose concentration dropped to about 2 g/L. Moreover, the heterotrophic performance was verified in a 5 L fermenter, resulting in excellent fucoxanthin and EPA productions of 63.03 and 398.60 mg/L, and productivities of 3.41 and 22.52 mg/L/d, respectively. These results, especially the EPA production and productivity, are superior to many literature reports. The present study provides new insights into the highly efficient co-production of fucoxanthin and EPA by marine microalgae and has guiding significance for commercial production in heterotrophic mode.

Environmental profiling microalgae-based eicosapentaenoic acid production along the technical advancement via life cycle assessment

Microalgae have attracted attention from various fields due to their potential ability to accumulate diverse value-added products, including polyunsaturated fatty acids (PUFAs). Many approaches have been used to enhance the productivity of microalgae-based PUFAs, such as eicosapentaenoic acid (EPA), but in many cases, these have only focused on economic competitiveness. However, it is imperative to simultaneously quantify and optimize the associated environmental impact at an early stage to guide future developments and sustainable scale-up. This study conducted Life Cycle Assessments (LCA) along the technical advancements of laboratory-scale EPA production by Phaeodactylum tricornutum. Results indicated that technical advancements enhanced the environmental performance associated with EPA production. Among these, metabolic engineering led to the most significant reductions (93.48% and 93.45%) in terms of global warming potential and cumulative energy demand compared with the wild-type scenario due to elevated microalgal growth and EPA yield. Electricity consumption remained the dominant contributor to the associated environmental impact, followed by extraction solvents, especially chloroform. In terms of processes, cultivation of the microalgae and harvesting of the biomass accounted for the majority of the impact attributed to the long cultivation period and the energy-intensive processes such as freeze drying. As guided by the sensitivity analysis, future directions should focus on further increasing EPA productivity, adopting alternative cultivation approaches with less energy input, and recovering EPA via environmentally friendly methods.

Altering autotrophic carbon metabolism of Nitzschia closterium to mixotrophic mode for high-value product improvement

An adaptive laboratory evolution (ALE) strategy was designed to evolve autotrophic Nitzschia closterium to mixotrophic growth for high productivity of essential amino acid (EAA), eicosapentaenoic acid (EPA) and fucoxanthin. The N. closterium growth was limited under glucose initially, but a red light emitting diode was innovatively applied to modify carbon metabolism and obtain mixotrophic strain of N. closterium GM. The N. closterium GM biomass concentration was improved by 65.07% comparing with wild type, but exhibited weak photosynthesis and strong glucose metabolism. At carbon metabolism levels, ALE promoted NADPH oxidase activity and induced protein degradation to lipid biosynthesis by elevating acetyl-CoA and pyruvate contents. It also improved carbon flux to TCA cycle, and elevated contents of glucose-6-phosphate, fructose-6-phosphate, glyceraldehyde-3-phosphate for providing sufficient ATP and NADPH. Productivities of EPA, EAA and fucoxanthin were increased by 41.0%, 18.8% and 20.4%, respectively. This ALE strategy was promising in microalgal production of high-value products.

Dietary Use of Methionine Sources and Bacillus amyloliquefaciens CECT 5940 Influences Growth Performance, Hepatopancreatic Histology, Digestion, Immunity, and Digestive Microbiota of Litopenaeus vannamei Fed Reduced Fishmeal Diets.

An 8-week feeding trial investigated the effect of Fishmeal (FM) replacement by soybean meal (SBM) and poultry by-product meal (PBM) in diets supplemented with DL-Met, MET-MET (AQUAVI®), Bacillus amyloliquefaciens CECT 5940 (ECOBIOL®) and their combinations on growth performance and health of juvenile Litopenaeus vannamei. A total of six experimental diets were formulated according to L. vannamei nutritional requirements. A total of 480 shrimp (0.30 ± 0.04 g) were randomly distributed into 24 tanks (4 repetitions/each diet, 20 shrimp/tank). Shrimp were fed with control diet (CD; 200 g/Kg fishmeal) and five diets with 50% FM replacement supplemented with different methionine sources, probiotic (B. amyloliquefaciens CECT 5940) and their combinations: D1 (0.13% DL-MET), D2 (0.06% MET-MET), D3 (0.19% MET-MET), D4 (0.13% DL-MET plus 0.10% B. amyloliquefaciens CECT 5940 and D5 (0.06% MET-MET plus 0.10% B. amyloliquefaciens CECT 5940). Shrimp fed D3 and D5 had significantly higher final, weekly weight gain, and final biomass compared to shrimp fed CD (p < 0.05). Shrimp fed D2 to D5 increased the hepatopancreas epithelial cell height (p < 0.05). Digestive enzymatic activities were significantly increased in shrimp hepatopancreas’ fed D3 (p < 0.05). Meanwhile, shrimp fed D1 had significant downregulation of immune-related genes (p < 0.05). Moreover, shrimp fed D3 and D5 increased the abundance of beneficial prokaryotic microorganisms such as Pseudoalteromonas and Demequina related to carbohydrate metabolism and immune stimulation. Also, shrimp fed D3 and D5 increased the abundance of beneficial eukaryotic microorganism as Aurantiochytrium and Aplanochytrium were related to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production which plays a role in growth promoting or boosting the immunity of aquatic organisms. Therefore, fishmeal could be partially substituted up to 50% by SBM and PBM in diets supplemented with 0.19% MET-MET (AQUAVI®) or 0.06% MET-MET (AQUAVI®) plus 0.10% B. amyloliquefaciens CECT 5940 (ECOBIOL®) and improve the productive performance, health, and immunity of white shrimp. Further research is necessary to investigate synergistic effects of amino acids and probiotics in farmed shrimp diets, as well as to evaluate how SBM and PBM influence the fatty acid composition of reduced fishmeal diets and shrimp muscle quality. Nevertheless, this information could be interesting to develop low fishmeal feeds for aquaculture without affecting the growth and welfare of aquatic organisms.

Efficient co-production of EPA and DHA by Schizochytrium sp. via regulation of the polyketide synthase pathway

Presently, the supply of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) traditionally produced by marine fisheries will be insufficient to meet their market demand in food industry. Thus a sustainable alternative source is urgently required. Schizochytrium sp. is an ideal producer of DHA; however, its ability to co-produce DHA and EPA has not yet been proved. Herein, we first described a cobalamin-independent methionine synthase-like (MetE-like) complex, which contains independent acyltransferase and 3-ketoacyl synthase domains, independent of the traditional polyketide synthase (PKS) system. When the MetE-like complex was activated, the EPA content was increased from 1.26% to 7.63%, which is 6.06-folds higher than that in the inactivated condition. Through lipidomics, we find that EPA is more inclined to be stored as triglyceride. Finally, the EPA production was enhanced from 4.19 to 29.83 (mg/g cell dry weight) using mixed carbon sources, and the final yield reached 2.25 g/L EPA and 9.59 g/L DHA, which means that Schizochytrium sp. has great market potential for co-production of EPA and DHA.