Production Of Polyunsaturated Fatty Acids Research Articles

Omega-3 fatty acids-critical for the marine food web and for seabird productivity

Abstract Seabird reproductive success in northern latitudes is often linked with prey abundance, availability, size, or species. Causes are often broadly explained as changes in ocean currents/temperatures, or diets. Few studies trace diets down the food web to primary producers, thus missing what I propose as an underlying cause of seabird colony failure, dietary abundance of essential omega-3 polyunsaturated fatty acids (PUFA). PUFA differ from other nutrients because they are, in part, ligands—critical for reproduction and other physiological processes for the entire marine food web, not just for seabirds. Diatoms are one of the few life forms that can produce PUFA de novo, and a lack of PUFA in zooplankton and fish reduces their abundance and productivity, with consequences up the food web. Since the mid-1980s, historical prey of breeding seabirds has decreased, and frequency of colony failures has increased. In years of failure, prey often had less fat than historically preferred species. Likewise, proportions of alternate species’ PUFA, from published papers, show lower values than in historical prey. Production of PUFA by diatoms is depressed in warmer and more acidic environments, and I hypothesize that these warmer and more acidic seas have affected production of PUFA over the past 3–4 decades. Assuming this is true, I propose that these lower amounts of PUFA have negatively impacted the breeding success of all members of the marine food web, not just seabirds.

Production and Release of Proinflammatory Mediators by the Cockroach Allergen Bla g 1 via a Shared Membrane-Destabilization Mechanism.

The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1β). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.

PUFA-PLs biosynthesis enzymes contribute to pathogenic development of rice blast fungus Magnaporthe oryzae

ABSTRACT Rice blast is one of the most devastating diseases and a serious threat to global food security. It is caused by the ascomycetous fungus Magnaporthe oryzae. During the pathogenic development of M. oryzae, ferroptotic death of conidial cells is critical for appressorium formation and infection to host rice. In this study, we identified and functionally characterised orthologs of fatty acid desaturase (Fad2) and acyl-CoA synthetase long-chain family (Acsl4) in M. oryzae. Pathogenicity was impaired in the fad2Δ or acsl4Δ mutant and targeted lipidomics analysis demonstrated that Fad2 and Acsl4 were involved in the production of polyunsaturated fatty acids (PUFAs)-containing phospholipids (PUFA-PLs) potentially contributing to ferroptosis. Treatment with FeCl3, an oxidative agent to cause lipid peroxidation, could partially restore fad2Δ pathogenicity. Fad2 was also found to potentially interact with proteins involved in cellular redox homoeostasis. Overall, our results elucidate the role of PUFA-PLs biosynthesis in fungal cell death and fungal pathogenicity, providing a theoretical basis for the development of specific pesticides/drugs targeting ferroptosis caused by lipid peroxidation.

Cross-species metabolomic profiling reveals phosphocholine-mediated liver protection from cold and ischemia/reperfusion

Cold and ischemia/reperfusion (IR)-associated injuries are seemingly inevitable during liver transplantation and hepatectomy. Because Syrian hamsters demonstrate intrinsic tolerance to transplantation-like stimuli, cross-species comparative metabolomic analyses were conducted with hamster, rat, and donor liver samples to seek hepatic cold and IR-adaptive mechanisms. Lower hepatic phosphocholine contents were found in recipients with early graft-dysfunction and with virus-caused cirrhosis or high model for end-stage liver disease scores (≥30). Choline/phosphocholine deficiency in cultured human THLE-2 hepatocytes and animal models weakened hepatocellular cold tolerance and recovery of glutathione and ATP production, which was rescued by phosphocholine supplements. Among the biological processes impacted by choline/phosphocholine deficiency, 3 lipid-related metabolic processes were downregulated, whereas phosphocholine elevated the expression of genes in methylation processes. Consistently, in THLE-2, phosphocholine enhanced the overall RNA m6A methylation, among which the transcript stability of fatty acid desaturase 6 (FADS6) was improved. FADS6 functioned as a key phosphocholine effector in the production of polyunsaturated fatty acids, which may facilitate the hepatocellular recovery of energy and redox homeostasis. Thus, our study reveals the choline-phosphocholine metabolism and its downstream FADS6 functions in hepatic adaptation to cold and IR, which may inspire new strategies to monitor donor liver quality and improve recipient recovery from the liver transplantation process.

Transcriptional, post-transcriptional, and post-translational regulation of polyunsaturated fatty acid synthase genes in Aurantiochytrium limacinum strain BL10: Responses to nitrogen starvation

Under nitrogen deficient conditions, the Aurantiochytrium limacinum strain BL10 greatly increases the production of docosahexaenoic acid (DHA) and n-6 docosapentaenoic acid. Researchers have yet to elucidate the mechanism by which BL10 promotes the activity of polyunsaturated fatty acid synthase (Pfa), which plays a key role in the synthesis of polyunsaturated fatty acid (PUFA). Analysis in the current study revealed that in nitrogen-depleted environments, BL10 boosts the transcription and synthesis of proteins by facilitating the expression of pfa genes via transcriptional regulation. It was also determined that BL10 adjusts the lengths of the 5′- and 3′-untranslated regions (suggesting post-transcriptional regulation) and modifies the ratio of two Pfa1 isoforms to favor PUFA production via post-translational regulation (ubiquitination). These findings clarify the exceptional DHA production of BL10 and provide additional insights into the regulatory mechanisms of PUFA biosynthesis in Aurantiochytrium.

Enhancing the biosynthesis of polyunsaturated fatty acids by Rhodotorula mucilaginosa and Lodderomyces elongisporus

Single-cell oils (SCO) produced by oleaginous yeast hold promise as a sustainable alternative for producing nutritionally and pharmaceutically valuable lipids. However, the accumulation of oils varies substantially between yeast spp. Consequently, identifying well-suited producers with a high innate capacity for lipids biosynthesis is paramount. Equally important is optimizing culturing and processing conditions to realize the total lipids production potential of selected strains. The marine Rhodotorula mucilaginosa and Lodderomyces elongisporus yeast were investigated to explore their potential for polyunsaturated fatty acids (PUFAs) production on high glucose media (HGM) using two-stage culture mode. Both strains accumulated > 20% (w/w) of their dry cell weight as lipids when grown on HGM using a two-stage culture system. Both yeast isolates exhibited a maximal lipid/biomass coefficient (YL/X) of 0.58–0.66 mg/mg at 7 °C and 0.49–0.53 mg/mg at 26 °C when grown on 8% glucose and produced monounsaturated and PUFAs similar to that of Menhaden and Salmon marine oils. For the first time, significant amounts of Eicosapentaenoic acid (19%) and Eicosadienoic acid (19.6%) were produced by L. elongisporus and R. mucilaginosa, respectively. Thus, the SCO derived from these wild strains possesses significant potential as a substitute source for the industrial-scale production of long-chain PUFAs, making them a promising contender in the market.Graphical

Investigation of Crypthecodinium cohnii High-Cell-Density Fed-Batch Cultivations

Crypthecodinium cohnii is a marine microalga that can accumulate high amounts of polyunsaturated fatty acids (PUFAs) and thus replace conventional routes of fish oil production. They are associated with the destruction of marine resources and multiple downstream/purification complications. The major drawbacks of using C. cohnii for industrial-scale production are associated with low PUFA productivity. One of the means of increasing the PUFA synthesis rate is to maintain the medium component concentrations at optimal values throughout cultivation, thus increasing PUFA production efficiency, which can result in the successful transfer of the process to pilot and/or industrial scale. The goal of the present research was to develop techniques for increasing the efficiency of PUFA production via C. cohnii cultivation. Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen transfer factors. The biomass yields from individual components, yeast extract, sea salts, and glucose amounted to 5.5, 0.65, and 0.61 g·g−1, respectively. C. cohnii cell susceptibility to mechanical damage was experimentally evaluated. Power inputs of <276.5 W/m3 did not seem to promote cell destruction when Pitched-blade impellers were used. The obtained cultivation conditions were shown to be efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii. By using the applied methods, the maximal biomass productivity reached 8.0 g·L−1·day−1, while the highest obtained biomass concentration reached 110 g·L−1. A steady increase in the concentration of PUFAs during cultivation was observed from the FTIR data.

Metal-phenolic nanocatalyst rewires metabolic vulnerability for catalytically amplified ferroptosis

Ferroptosis is associated with excessive lipid peroxidation (LPO) accumulation, followed by membrane fragmentation. Although lipid metabolism is essential for regulating ferroptosis execution in tumors, there has been no use of nanomedicine to modulate the effect of metabolic vulnerability on ferroptosis. This study presents a metal-phenolic nanocatalyst (Fe@MT nanocatalyst), self-assembled using ferric ions (Fe3+) and coordinating with mitoxantrone (MT). This nanocatalyst accumulates lipid peroxides to strengthen ferroptotic vulnerability by initiating reactive oxygen species (ROS) storm and remodeling polyunsaturated fatty acids (PUFAs)-containing lipid metabolism. Fe@MT nanocatalyst catalytically converts endogenous hydrogen peroxide into highly active hydroxyl radical and depletes high intracellular levels of glutathione upon its internalization by U87MG cells, resulting in the release of MT, which in return promotes irreversible ferroptosis by elevating the intracellular production of ROS-sensitive PUFAs and inducing lethal LPO. Fe@MT nanocatalyst exhibits near-infrared photothermal property because of Fe3+ and MT self-assembly, causing photothermal-amplified catalytic ferroptosis therapy. This study sheds new light on promoting the sensitivity and execution of ferroptosis by reinforcing lethal lipid metabolism and accumulating excessive LPO and will establish an exploitable interaction between ferroptosis and lipid metabolic vulnerability in cancer.

Assessment of heavy metals and environmental stress conditions on the production potential of polyunsaturated fatty acids (PUFAs) in indigenous microalgae isolated from the Gulf of Mannar coastal waters.

The present study evaluated the effects of heavy metals, viz., lead, mercury, and cadmium, on growth, chlorophyll a, b, c, carotenoids, and PUFA content of marine microalgae Chlorella sp. and Cylindrotheca fusiformis. At 96-h exposure, the IC50 values for Hg2+, Pb2+, and Cd2+ were 0.85mg/L, 2.4mg/L, and 5.3mg/L respectively, in Chlorella sp. In C. fusiformis, IC50 values for Hg2+, Pb2+, and Cd2+ were 0.5mg/L, 1.2mg/L, and 3mg/L respectively. The pigment contents of both microalgae were significantly affected upon heavy metal exposure. In Chlorella sp. and C. fusiformis, the exposed concentrations of Hg2+ averagely decreased the PUFA content by 76.34% and 78.68%, respectively. Similarly, Pb2+-exposed concentrations resulted in 54.50% and 82.64% average reductions in PUFA content of Chlorella sp. and C. fusiformis, respectively. Cd2+-exposed concentrations showed 32.58% and 40.54% average reduction in PUFA content of Chlorella sp. and C. fusiformis, respectively. Among the environmental stress conditions, the dark treatment has increased total PUFA content by 6.63% in Chlorella sp. and 3.92% in C. fusiformis. It was observed that the 50% nitrogen starvation (two-stage) significantly improved the PUFA production from 26.47 ± 6.55% to 40.92 ± 10.74% in Chlorella sp. and from 11.23 ± 5.01 to 32.8 ± 14.17% in C. fusiformis. The toxicity for both microalgae was followed in the order Hg2+ > Pb2+ > Cd2+. Among the two species, Chlorella sp. has shown a high tolerance to heavy metals and can be effectively utilized in PUFA production.

Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB.Key points• This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189.• This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain.• This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.

Characterization of Three Types of Elongases from Different Fungi and Site-Directed Mutagenesis.

Fatty acid elongases play crucial roles in synthesizing long-chain polyunsaturated fatty acids. Identifying more efficient elongases is essential for enhancing oleaginous microorganisms to produce high yields of target products. We characterized three elongases that were identified with distinct specificities: McELO from Mucor circinelloides, PrELO from Phytophthora ramorum, and PsELO from Phytophthora sojae. Heterologous expression in Saccharomyces cerevisiae showed that McELO preferentially elongates C16 to C18 fatty acids, PrELO targets Δ6 polyunsaturated fatty acids, and PsELO uses long chain saturated fatty acids as substrates. McELO and PrELO exhibited more homology, potentially enabling fatty acid composition remodeling and enhanced LC-PUFAs production in oleaginous microorganisms. Site-directed mutagenesis of conserved amino acids across elongase types identified residues essential for activity, supported by molecular docking. Alanine substitution of conserved polar residues led to enzyme inactivation, underscoring their importance in the condensation reaction. Our findings offer promising elongase candidates for polyunsaturated fatty acid production, contributing to the bioindustry's sustainable development.

Screening for the production of polyunsaturated fatty acids and cerebrosides in fungi.

To investigate fatty acid, including polyunsaturated fatty acids (PUFA), and cerebroside production of a large diversity of fungi from the Ascomycota, Basidiomycota, and Mucoromycota phyla. Seventy-nine fungal strains were grown in Kavadia medium using a microcultivation system, i.e. Duetz microtiter plates. Following cultivation, fatty acid and cerebroside contents were analyzed by gas chromatography-flame ionization detection (GC-FID) and high performance thin-layer chromatography (HPTLC), respectively. Mucoromycota fungi appeared as the most promising candidates for omega-6 PUFA production. The best omega-6 producer, including γ-linolenic acid (GLA, 18:3n-6), was Mucor fragilis UBOCC-A109196 with a concentration of 647mg L-1 total omega-6 PUFA (representing 35% of total fatty acids) and 225mg L-1 GLA (representing 12% of total fatty acids). Arachidonic acid concentration (20:4n-6) was the highest in Mortierella alpina UBOCC-A-112046, reaching 255mg L-1 and 18.56% of total fatty acids. Interestingly, several fungal strains were shown to produce omega-7 monounsaturated fatty acids. Indeed, Torulaspora delbrueckii strains accumulated palmitoleic acid (16:1n-7) up to 20% of total fatty acids, reaching 114mg L-1 in T. delbrueckii UBOCC-A-214128, while C. elegans UBOCC-A-102008 produced mainly paullinic acid (20:1n-7) with concentrations up to 100mg L-1. Concerning cerebroside production, HPTLC appeared as a relevant approach for their detection and quantification. Promising candidates belonging to the Mucoromycota phylum were found, especially in the Absidia genus with A. spinosa UBOCC-A-101332 as the best producer (12.7mg L-1). The present study highlighted PUFA and cerebroside production in a large diversity of fungi and the fact that members of the Mucoromycota phylum are good producers of PUFA as well as cerebrosides.

Recycling Fermentation Strategy for Waste Cellular Residues in the Production of Polyunsaturated Fatty Acids

Cellular residues after lipid extraction are the primary waste by-products of microbial polyunsaturated fatty acid production. To mitigate the discharge of cellular residues and reduce the cost of DHA and ARA production, this study examined the utilization of enzymatically hydrolyzed Aurantiochytrium cellular residues (ACRs) and Mortierella alpina cellular residues (MCRs) as nitrogen sources. Results demonstrated that ACRs and MCRs could partially substitute yeast extract (YE) without adverse effects on DHA and ARA fermentation. Moreover, the implementation of a new fermentation medium incorporating ACRs, MCRs, and YE as mixed nitrogen sources resulted in DHA and ARA yields of 17.78 and 5.77 g/L, respectively. These values represented increases of 10.37% and 9.28% compared to traditional cultural methods, while simultaneously reducing the usage of YE by 80% and 60%, respectively. Therefore, the strategy of recycling waste cellular residues presents a novel approach for reducing the costs and environmental impact associated with microbial fermentations.

Evaluation of meat and meat product oxidation and off-flavor formation: Managing oxidative changes

One of the primary issues with processed foods during heat treatment and freezing storage is fat oxidation, which causes significant changes in fats due to their interaction with reactive oxygen species (ROS). This interaction leads to the creation of various aldehydes that have a high affinity for large molecules, such as proteins, leading to the formation of final products of advanced oxidation processes that contribute to food spoilage. Co-oxidation can also result in extensive damage. Another problem affecting the quality and nutritional value of meat products is protein oxidation, which can occur during storage via freezing and thawing or as a result of heat treatment. Heat treatment can cause physical and chemical changes, such as the loss of some essential amino acids and the transformation of certain amino acids into carbonyl compounds via various mechanisms. Protein oxidation is indicated by the accumulation of these carbonyl compounds, and the heat treatment can lead to the denaturation of myoglobin, which is responsible for the brown color of cooked meat and is influenced by several factors. Active protein aggregates can interact with the oxidation products of polyunsaturated fatty acids and with carbohydrate glycation or glycoxidation to produce Maillard products. It is critical to understand the oxidative changes that occur in fats and proteins in food, particularly in meat products, since these components are among the primary constituents of food.

Fine-tuning of key parameters to enhance biomass and nutritional polyunsaturated fatty acids production from Thraustochytrium sp.

The escalating demand for long-chain polyunsaturated fatty acids (PUFAs) due to their vital health effects has deepened the exploration of sustainable sources. Thraustochytrium sp. stands out as a promising platform for omega-3 and 6 PUFA production. This research strategically optimizes key parameters: temperature, salinity, pH, and G:Y:P ratio and the optimized conditions for maximum biomass, total lipid, and DHA enhancement were 28 °C, 50 %, 6, and 10:1:2 respectively. Process optimization enhanced 32.30 and 31.92 % biomass (9.88 g/L) and lipid (6.57 g/L) yield. Notably, DHA concentration experienced a substantial rise of 69.91 % (1.63 g/L), accompanied by notable increases in EPA and DPA by 82.69 % and 31.47 %, respectively. MANOVA analysis underscored the statistical significance of the optimization process (p < 0.01), with all environmental factors significantly influencing biomass and lipid data (p < 0.05), particularly impacting DHA production. Thraustochytrium sp. can be a potential source of commercial DHA production with the fine-tuning of these key process parameters.

Development of user-friendly protocols for single-cell arachidonic acid production in developing countries

Arachidonic acid (ARA) is a sorely needed nutrient and an effective and safe schistosomicide and tumoricide. Consumption levels are low in developing countries because of unawareness of its critical impact on health and limited commercial availability. The research work aims to encourage production in developing countries via providing user-friendly definitive protocols for polyunsaturated fatty acids production by the fungus Mortierella alpina and ARA isolation. Fatty acids content was determined by gas chromatography-flame ionization detector. The optimal cultivation conditions included culture medium to flask volume 1:5; shake-flask culture at 25 °C, then at 20 °C, and stationary aging at 10 °C, each over 7 days, while addition of trace minerals and vitamins was dispensable. The ARA yield attained >1.8 g per liter culture. Repeat experiments led to the selection of an optimal lipid extraction method from dried biomass, and efficacious urea complexation-based protocol for obtention of high ARA yield and purity.

Microbial Fermentation of the Water-Soluble Fraction of Brewers’ Spent Grain for the Production of High-Value Fatty Acids

Brewers’ spent grain (BSG) constitutes the primary by-product of the brewing industry. The valorization of BSG from a circular economy perspective is of high industrial interest. The objective of this study was the exploitation of BSG for the microbial production of branched-chain fatty acids (BCFAs) and polyunsaturated fatty acids (PUFAs), representing two different classes of high-value fatty acids (FAs). In the present study, this waste material underwent treatment with hot water in an autoclave and the resultant extract was utilized for the preparation of a novel liquid medium (BSG medium) to be employed for microbial fermentation. Screening and subsequent scaling-up experiments confirmed the suitability of the BSG medium to support the microbial production of various high-value FAs. In particular, Streptomyces jeddahensis and Conidiobolus heterosporus could be employed for BCFAs production, Pythium ultimum and Mortierella alpina could be used to provide cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and arachidonic acid (ARA), whereas Mucor circinelloides, when grown in a BSG medium, was able to accumulate γ-linolenic acid (GLA).

Application of ANN-MOGA for nutrient sequestration for wastewater remediation and production of polyunsaturated fatty acid (PUFA) by Chlorella sorokiniana MSP1

Chlorella bears excellent potential in removing nutrients from industrial wastewater and lipid production enriched with polyunsaturated fatty acids. However, due to the changing nutrient dynamics of wastewater, growth and metabolic activity of Chlorella are affected. In order to sustain microalgal growth in wastewater with concomitant production of PUFA rich lipids, RSM (Response Surface Methodology) followed by heuristic hybrid computation model ANN-MOGA (Artificial Neural Network- Multi-Objective Genetic Algorithm) were implemented. Preliminary experiments conducted taking one factor at a time and design matrix of RSM with process variables viz. Sodium chloride (1 mM–40 mM), Magnesium sulphate (100 mg–800 mg) and incubation time (4th day to 20th day) were validated by ANN-MOGA. The study reported improved biomass and lipid yield by 54.25% and 12.76%, along with total nitrogen and phosphorus removal by 21.92% and 18.72% respectively using ANN-MOGA. It was evident from FAME results that there was a significantly improved concentration of linoleic acid (19.1%) and γ-linolenic acid (21.1%). Improved PUFA content makes it a potential feedstock with application in cosmeceutical, pharmaceutical and nutraceutical industry. The study further proves that C. sorokiniana MSP1 mediated industrial wastewater treatment with PUFA production is an effective way in providing environmental benefits along with value addition. Moreover, ANN-MOGA is a relevant tool that could control microalgal growth in wastewater.

Effects of sunflower oil infusions of Asparagopsis taxiformis on in vitro ruminal methane production and biohydrogenation of polyunsaturated fatty acids

Asparagopsis taxiformis inhibits ruminal methane (CH4) production due to its bromoform (CHBr3) content. The immersion of A. taxiformis in edible vegetable oils allows the extraction and stabilization of the highly volatile CHBr3 in the oil phase. The objectives of this study were to explore the effects of adding sunflower oils with increasing concentrations of CHBr3 on in vitro ruminal methanogenesis and biohydrogenation. Five batches of 48-h in vitro incubations were performed in 14 fermentation bottles, using rumen inocula collected shortly after the slaughter of young crossbred bulls and 1 g of dry matter (DM) from a total diet of mixed feed without added oil (control) or with 60 μL of sunflower oil per gram of DM as the substrate. The treatments were the CHBr3 content in the oil added: 0 μg (B0), 25 μg (B25), 50 μg (B50), 75 μg (B75), 100 μg (B100), and 150 μg (B150) of CHBr3 per gram of substrate DM. Organic matter (OM) degradability, total gas, CH4, volatile fatty acids (VFA), long-chain fatty acids, and dimethyl acetals (DMA) were analyzed at the end of each incubation. Data were analyzed with a model considering the treatments as the fixed effect and the run as a random block and using orthogonal contrasts. Degradability of OM was higher in the control group and was unaffected by CHBr3 concentration. Total gas production per gram of degraded OM was unaffected by treatments and averaged 205 ± 29.8 mL/g. Methane (mL) production decreased linearly with increasing CHBr3 concentrations, with 33%, 47%, and 87% reductions for B75, B100, and B150, respectively. Total VFA concentration was unaffected by oil inclusion but was reduced by 20% in CHBr3-containing treatments, although without any dose-response pattern. The molar percentage of acetate decreased linearly, whereas propionate and butyrate increased linearly with the increasing CHBr3 dosage. Including oil in the diet decreased the branched-chain fatty acids and DMA content. Increasing CHBr3 concentrations did not affect branched-chain fatty acids, but linearly increased most of the identified DMA. Adding oil to the control diet increased the 18:2n-6, whereas increasing the concentration of CHBr3 had no effect on 18:2n-6 but decreased linearly the 18:0 and increased the trans-18:1 isomers. The results obtained provide evidence that oil immersions of A. taxiformis can successfully inhibit ruminal production of CH4 in vitro at doses of 100 and 150 μg/g DM, and simultaneously modulate biohydrogenation.

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.