Production Of Polyunsaturated Fatty Acids Research Articles

Draft genome sequence of Aurantiochytrium spp. strains L3W, 9LR, 8W, 10W, and 10LW, a fatty acid-producing thraustochytrids from food wastes.

We report draft genome sequences of Aurantiochytrium spp. strains L3W, 9LR, 8W, 10W, and 10LW isolated from coastal environments. The strains were able to produce polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), confirming the metabolic pathways retained in the genomes.

Study of Different Parameters Affecting Production and Productivity of Polyunsaturated Fatty Acids (PUFAs) and γ-Linolenic Acid (GLA) by Cunninghamella elegans Through Glycerol Conversion in Shake Flasks and Bioreactors.

Microbial cultures repurposing organic industrial residues for value-added metabolite production is pivotal for sustainable resource use. Highlighting polyunsaturated fatty acids (PUFAs), particularly gamma-linolenic acid (GLA), renowned for their nutritional and therapeutic value. Notably, Zygomycetes' filamentous fungi harbor abundant GLA-rich lipid content, furthering their relevance in this approach. In this study, the strain C. elegans NRRL Y-1392 was evaluated for its capability to metabolize glycerol and produce lipids rich in GLA under different culture conditions. Various carbon-to-nitrogen ratios (C/N = 11.0, 110.0, and 220.0 mol/mol) were tested in batch-flask cultivations. The highest GLA production of 224.0 mg/L (productivity equal to 2.0 mg/L/h) was observed under nitrogen excess conditions, while low nitrogen content promoted lipid accumulation (0.59 g of lipids per g of dry biomass) without yielding more PUFAs and GLA. After improving the C/N ratio at 18.3 mol/mol, even higher PUFA (600 mg/L) and GLA (243 mg/L) production values were recorded. GLA content increased when the fungus was cultivated at 12 °C (15.5% w/w compared to 12.8% w/w at 28 °C), but productivity values decreased significantly due to prolonged cultivation duration. An attempt to improve productivity by increasing the initial spore population did not yield the expected results. The successful scale-up of fungal cultivations is evidenced by achieving consistent results (compared to flask experiments under corresponding conditions) in both laboratory-scale (Working Volume-Vw = 1.8 L; C/N = 18.3 mol/mol) and semi-pilot-scale (Vw = 15.0 L; C/N = 110.0 mol/mol) bioreactor experiments. To the best of our knowledge, cultivation of the fungus Cunninghamella elegans in glycerol-based substrates, especially in 20 L bioreactor experiments, has never been previously reported in the international literature. The successful scale-up of the process in a semi-pilot-scale bioreactor illustrates the potential for industrializing the bioprocess.

Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.

Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).

Mycelial mass, microbial lipids and γ-linolenic acid (GLA) by Cunninghamella elegans cultivated on agro-industrial residues

In the current study, the Zygomycetes fungus Cunninghamella elegans NRRL Y-1392 was evaluated for its ability to grow in extracts derived from dried and ground agricultural residues, such as mushroom stalks and roots from hydroponically cultivated lettuces and produce poly-unsaturated fatty acids (PUFA) and γ-linolenic acid (GLA) rich lipids. Initially, the compositions of stalks and lettuce roots were analysed, and the fungus was batch-flask cultivated on six different commercial semi-defined substrates containing different sugars detected in stalks and roots to evaluate its catabolic ability. C. elegans was capable to assimilate all sugars, but at a lower rate in the case of arabinose. Subsequently, C. elegans was cultivated on tailor-made semi-defined commercial substrates, resembling hydrolysates containing carbohydrates found in mushroom stalks, under both nitrogen-excess and nitrogen-limited conditions, and resembling that of hydrolysates of roots, under nitrogen-excess conditions. Based on the results, under nitrogen-excess conditions, in the case of media resembling stalks hydrolysates, higher production values for biomass, PUFAs, and GLA were observed (20.3 g/L, 1906 mg/L, 668 mg/L), accompanied by high productivity values due to short cultivation periods, while under nitrogen limitation, high lipid accumulation (lipid in dry cell weight =48%, w/w) was presented, and lipids rich in oleic acid were produced. Finally, the fungus was cultivated on a medium derived from hot water-extraction applied to mushroom stalks, enriched with organic nitrogen sources. The fungus was successfully grown on the sugar-rich water-extract derived from mushroom stalks, resulting in dry biomass of 14.5 g/L, lipids of 1.8 g/L, with 15% (w/w) of GLA in cellular lipids.

Canola meal extract as a low-cost substrate in Mortierella alpina culture for the production of arachidonic acid

The demand for high-quality and cost-effective raw materials is growing across various industries. Canola meal (CM), a low-cost by-product of canola processing facilities, boasts an impressive nutritional profile comprising protein, carbohydrates, vitamins, and minerals, making it a potential candidate for microbial culture media. Saskatchewan leads globally in canola production and plans to expand its canola processing facilities. Commercial valorization of CM could not only optimize the management of this by-product but also offer additional incentives and financial returns to regional canola stakeholders. Efficient utilization of canola meal extract (CME) in producing polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA), holds promise as valuable ingredients or base chemicals for the food, feed, and nutraceutical industries. This study focused on incorporating pH 10.5 treated alkaline extract (AE) and temperature 160°C treated subcritical extract (SE) of CM into the culture medium of Mortierella alpina, an oleaginous fungus, by replacing conventional yeast extract (YE). Trials investigated the effect of aeration levels ranging from 0.5 to 2.0 vvm and revealed that increasing oxygen supply significantly boosted fungal growth rate and ARA content in biomass up to 0.39 day⁻¹ and 21.86 %, respectively. ARA production correlated positively with biomass accumulation and lipid content. Replacing YE with CME resulted in less significant differences (P < 0.05) in biomass and lipid accumulation. Additionally, AE- and SE-supplemented media exhibited higher ARA accumulation of 144.91 mg/L and 165.8 mg/L, respectively, compared to standard media (124.87 mg/L). Therefore, this study introduces canola meal extract (CME) as a novel nitrogen source in microbial media, with comparative demonstrations showing its efficiency to be similar to that of conventional extracts.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

Fads2 knockout mice reveal that ALA prevention of hepatic steatosis is dependent on delta-6 desaturase activity

The production of the omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) relies on the delta-6 desaturase (D6D) enzyme encoded by the Fads2 gene. While EPA and DHA reduce hepatic triacylglycerol (TAG) storage and regulate lipogenesis, the independent impact of ALA is less understood. To address this gap in knowledge, hepatic fatty acid metabolism was investigated in male wild-type (WT) and Fads2 knockout (KO) mice fed diets (16% kcal from fat) containing either lard (no n-3 LCPUFA), flaxseed oil (ALA-rich), or menhaden oil (EPA/DHA rich) for 21 weeks. Fat content and composition, as well as markers of lipogenesis, glyceroneogenesis, and TAG synthesis, were analyzed using histology, gas chromatography, and reverse transcription quantitative PCR (RT-qPCR). Mice fed the menhaden diet had significantly lower hepatic TAG compared to both lard- and flax-fed mice, concomitant with changes in n-3 and n-6 LCPUFA in both TAG and phospholipid (PL) fractions (all P < 0.05). Flax-fed WT mice had lower liver TAG content compared to their KO counterparts. Menhaden-fed mice had significantly lower expression of key lipogenic (Scd1, Srebp-1c, Fasn, Fads1, and Fads2), glyceroneogenic (Pck1), and TAG synthesis (Agpat3) genes compared to lard, with flax-fed mice showing some intermediate effects. Gene expression effects were independent of D6D activity, since no differences were detected between WT and KO mice fed the same diet. This study demonstrates that EPA/DHA and not ALA itself is critical for the prevention of hepatic steatosis.

Nutritional Value and Productivity Potential of the Marine Microalgae Nitzschia laevis, Phaeodactylum tricornutum and Isochrysis galbana.

Microalgae are considered promising sustainable feedstocks for the production of food, food additives, feeds, chemicals and various high-value products. Marine microalgae Phaeodactylum tricornutum, Isochrysis galbana and Nitzschia laevis are rich in fucoxanthin, which is effective for weight loss and metabolic diseases. The selection of microalgae species with outstanding nutritional profiles is fundamental for novel foods development, and the nutritional value of P. tricornutum, I. galbana and N. laevis are not yet fully understood. Hence, this study investigates and analyzes the nutritional components of the microalgae by chromatography and mass spectrometry, to explore their nutritional and industrial application potential. The results indicate that the three microalgae possess high nutritional value. Among them, P. tricornutum shows significantly higher levels of proteins (43.29%) and amino acids, while I. galbana has the highest content of carbohydrates (25.40%) and lipids (10.95%). Notwithstanding that P. tricornutum and I. galbana have higher fucoxanthin contents, N. laevis achieves the highest fucoxanthin productivity (6.21 mg/L/day) and polyunsaturated fatty acids (PUFAs) productivity (26.13 mg/L/day) because of the competitive cell density (2.89 g/L) and the advantageous specific growth rate (0.42/day). Thus, compared with P. tricornutum and I. galbana, N. laevis is a more promising candidate for co-production of fucoxanthin and PUFAs.

Nano-enhanced lipid and essential polyunsaturated fatty acid production by Thraustochytrium sp.: Boosting nutraceuticals and biodiesel industries

In recent years, microbial platforms for biofuels and nutraceuticals have gained recognition as sustainable alternatives. This study investigates titanium dioxide nanoparticles’ effect on lipid and omega fatty acid production in Thraustochytrium sp. Both titanium dioxide and nitrogen starvation independently enhance lipid content, with a synergistic effect observed when combined. Titanium dioxide increases lipid content by 37.69% (up to 78.84%), while nitrogen starvation achieves a 34.91% enhancement (total lipid 75.92 ± 2.43%). The combined approach yields the highest lipid content at 81.48 ± 3.14%, denoting a 46.19% increase. Additionally, titanium dioxide increases omega-6 (LA by 66.95%) and omega-9 (OA by 44.98%) levels, while nitrogen starvation enhances omega-3 (mainly EPA, by 91.83%) levels. This eco-friendly strategy has implications for bioenergy and nutraceuticals, offering a cost-effective means of enhancing oleaginous microorganisms for biodiesel production. The research outcomes contribute to addressing key obstacles in cost-efficient biodiesel production, impacting the economic landscape of the biodiesel industry.

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.

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.

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.

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 &lt;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.