Production Of Unsaturated Fatty Acids Research Articles

Mechanism of Guishao Yigong decoction in treating colorectal cancer based on network pharmacology and experimental validation.

To explore the effective components of Guishao Yigong decoction (GYD) in the treatment of colorectal cancer and reveal its potential mechanism of action. Through network pharmacology, the main target and signaling pathway of GYD therapy for colorectal cancer (CRC) were found. Subsequently, the effect of GYD was verified by in vitro cell viability measurements, colony formation, and scratch healing tests. The effects of GYD on metabolic pathways in vivo were found through plasma metabolomics. Finally, flow cytometry and qPCR experiments were used to verify the cycle-blocking effect of GYD on CRC cells. Based on the network pharmacological analysis and molecular docking technology, it was found that GYD could restrain the growth of CRC cells by affecting lipid metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathways. A series of cell experiments showed that GYD could inhibit the proliferation, migration and clonogenic ability of CRC cells. Furthermore, the plasma metabolomics results showed that GYD could affect the production of unsaturated fatty acids in mice. Flow cytometry and qPCR experiments further proved that GYD blocked the CRC cells in the G1 phase and modulated the expression of cell cycle-related targets, such as AKT, TP53, CDKN1A, and CDK2. All the results indicated that GYD could regulate the related metabolism of unsaturated fatty acids. Thus, the cell cycle was blocked and the expressions of the key proteins such as AKT and TP53 were regulated, which achieved the purpose of intervention in colorectal cancer.

Untangling the adaptive strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1 under low temperature.

The present study investigates the low temperature tolerance strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1, which grows optimally at 55°C , by subjecting it to a temperature down-shift of 10°C (45°C) for 4 and 6h followed by studying its growth, morphophysiological, molecular and proteomic responses. Results suggested that although TPH1 experienced increased growth inhibition, ROS production, protein oxidation and membrane disruption after 4h of incubation at 45°C yet maintained its DNA integrity and cellular structure through the increased expression of DNA damage repair and cell envelop synthesizing proteins and also progressively alleviated growth inhibition by 20% within two hours i.e., 6h, by inducing the expression of antioxidative enzymes, production of unsaturated fatty acids, capsular and released exopolysaccharides and forming biofilm along with chemotaxis proteins. Conclusively, the adaptation of Anoxybacillus rupiensis TPH1 to lower temperature is mainly mediated by the synthesis of large numbers of defense proteins and exopolysaccharide rich biofilm formation.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

An artificial neuronal network coupled with a genetic algorithm to optimise the production of unsaturated fatty acids in Parachlorella kessleri

In this study, an Artificial Neural Network-Genetic Algorithm (ANN-GA) approach was successfully applied to optimise the physicochemical factors influencing the synthesis of unsaturated fatty acids (UFAs) in the microalgae P. kessleri UCM 001. The optimized model recommended specific cultivation conditions, including glucose at 29 g/L, NaNO3 at 2.4 g/L, K2HPO4 at 0.4 g/L, red LED light, an intensity of 1000 lx, and an 8:16-h light-dark cycle. Through ANN-GA optimisation, a remarkable 66.79% increase in UFAs production in P. kessleri UCM 001 was achieved, compared to previous studies. This underscores the potential of this technology for enhancing valuable lipid production. Sequential variations in the application of physicochemical factors during microalgae culture under mixotrophic conditions, as optimized by ANN-GA, induced alterations in UFAs production and composition in P. kessleri UCM 001. This suggests the feasibility of tailoring the lipid profile of microalgae to obtain specific lipids for diverse industrial applications. The microalgae were isolated from a high-mountain lake in Colombia, highlighting their adaptation to extreme conditions. This underscores their potential for sustainable lipid and biomaterial production. This study demonstrates the effectiveness of using ANN-GA technology to optimise UFAs production in microalgae, offering a promising avenue for obtaining valuable lipids. The microalgae's unique origin in a high-mountain environment in Colombia emphasises the importance of exploring and harnessing microbial resources in distinctive geographical regions for biotechnological applications.

Pharmaceutical composition of 1,2,4-trioxolanes with betulin in fish oil. Effect on oxidative and energy metabolism in the treatment of burns in rats

Background. Ozonation products of unsaturated fatty acids in fats and oils – 1,2,4-trioxolanes – which contribute to the normalization of oxidative and energy metabolism in various diseases, may be a promising substance for the creation of new combined anti-burn drugs.The aim. To study the effect of 1,2,4-trioxolanes in a pharmaceutical composition with betulin, which exhibits wound-healing properties, on indicators of oxidative stress and on energy metabolism in the treatment of burns in an experiment onrats. Methods. The experiments were carried out on 50 male Wistar rats with seconddegree skin burns. We assessed the activity of glutathione reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, aldehyde dehydrogenase, superoxide dismutase and catalase, as well as the level of lipid peroxidation indicators.Results. The properties of 1,2,4-trioxolanes were studied and a pharmaceutical composition of 1,2,4-trioxolanes with betulin from birch bark in fish oil was developed. The effect of this composition on oxidative and energy metabolism in rats during the treatment of burns was studied. The activation of NADP/H and NAD/H dependent enzymes (glutathione reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, aldehyde dehydrogenase), as well as superoxide dismutase and catalase, and the normalization of lipid peroxidation parameters under the influence of the studied composition in the treatment of burns were revealed. The effect of the composition on the energy metabolism of erythrocytes was noted, which was assessed by changes in the ratio of lactate dehydrogenase activity in the reverse and direct reactions. Morphohistological studies showed that using the composition under normal conditions and in case of a burn had no effect on the structure of the stomach wall, and also improved the condition of the burn wound on the day 10 compared to the wound condition in animals without treatment.Conclusion. The proposed composition of 1,2,4-trioxolanes and betulin in fish oil, which allows normalizing oxidative and energy metabolism and improving the condition of a burn wound without toxic effects on the stomach wall, can be used as an element of additional therapy in the treatment of burns.

Comparative Genome-Wide Identification of the Fatty Acid Desaturase Gene Family in Tea and Oil Tea.

Camellia oil is valuable as an edible oil and serves as a base material for a range of high-value products. Camellia plants of significant economic importance, such as Camellia sinensis and Camellia oleifera, have been classified into sect. Thea and sect. Oleifera, respectively. Fatty acid desaturases play a crucial role in catalyzing the formation of double bonds at specific positions of fatty acid chains, leading to the production of unsaturated fatty acids and contributing to lipid synthesis. Comparative genomics results have revealed that expanded gene families in oil tea are enriched in functions related to lipid, fatty acid, and seed processes. To explore the function of the FAD gene family, a total of 82 FAD genes were identified in tea and oil tea. Transcriptome data showed the differential expression of the FAD gene family in mature seeds of tea tree and oil tea tree. Furthermore, the structural analysis and clustering of FAD proteins provided insights for the further exploration of the function of the FAD gene family and its role in lipid synthesis. Overall, these findings shed light on the role of the FAD gene family in Camellia plants and their involvement in lipid metabolism, as well as provide a reference for understanding their function in oil synthesis.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) caused by deletion of yhdP and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, but not by ΔtamB ΔfadR or ΔydbH ΔfadR,. Deletion of tamB suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is related to phospholipid transport. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Although indirect effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.

Development of Novel Genomewide Simple Sequence Repeat Markers for Acer truncatum Bunge and Assessment of Their Transferability to Other Closely Related Species

Acer truncatum Bunge is a versatile woody tree species with high economic and medicinal value in the production of bioactive substances and unsaturated fatty acids (especially nervonic acid). However, the exploitation and evaluation of A. truncatum germplasm resources are limited owing to a lack of sound molecular marker systems. In this study, a large set of genomewide simple sequence repeat (SSR) markers of A. truncatum was developed based on its whole-genome sequences. A total of 462,331 SSR loci were identified in the genome sequences, 99.3% (459,193) of which were located on 13 chromosomes. The chromosome length was significantly positively correlated with the number of SSR loci on the chromosome (r = 0.977, p < 0.001). The (A/T)n, (AT/TA)n, and (AAT/ATT/TAA/TTA/TAT/ATA)n were the most frequent motifs for mono-, di-, and trinucleotide repeat motifs, respectively, showing A/T-base bias. After BLASTN and electronic polymerase chain reaction (PCR) analyses, 199,990 loci with specific physical positions were screened. Most of the SSR loci were located in the intergenic regions and fewest in the coding sequences (CDSs). The frequency of loci with tri- and hexanucleotide repeat motifs was the highest in the CDSs, potentially serving to maintain the stability of gene function and structure. In randomly selected 105 SSR markers, 82 (78.1%) showed allelic polymorphism, with polymorphism information content (PIC) values of 0.032–0.926 (0.481 on average). The SSRs in the noncoding regions exhibited significantly higher PIC values than those in the CDSs. The transferability of the 105 markers was 48.6%–59.0% to seven other Acer species. The large set of valid SSR markers provides a powerful tool for studies on population genetics, conservation genetics, linkage mapping, comparative genomics, and marker-assisted breeding of the genus Acer.

Unraveling the Genetic Adaptations in Cell Surface Composition and Transporters of Lactiplantibacillus plantarum for Enhanced Acid Tolerance.

This study employed adaptive laboratory evolution to improve the acid tolerance of Lactiplantibacillus plantarum, a vital strain in food fermentation and a potential probiotic. Phenotype and genomic analyses identified the overexpression of stress response proteins, ATP synthases, and transporters as pivotal in conferring acid tolerance to the evolved strains. These adaptations led to a shorter lag phase, improved survival rates, and higher intracellular pH values compared to the wild-type strain under acid stress conditions. Additionally, the evolved strains showed an increased expression of genes in the fatty acid synthesis pathway, resulting in a higher production of unsaturated fatty acids. The changes in cell membrane composition possibly prevented H+ influx, while mutant genes related to cell surface structure contributed to observed elongated cells and thicker cell surface. These alterations in cell wall and membrane composition, along with improved transporter efficiency, were key factors contributing to the enhanced acid tolerance in the evolved strains.

Urea-N influences biomass yield, neutral lipids accumulation, and unsaturated fatty acid production in photoautotrophically grown microalga Chlorella sp

Microalgal cultivation using cost-effective nutrients is found to decrease the biodiesel production cost significantly. Therefore, in this study, various nitrogen sources namely sodium nitrate, ammonium nitrate, potassium nitrate, and fertilizer-grade urea, were tested to evaluate their impact on the biomass, lipids, and fatty acid content of microalga Chlorella sp. For biodiesel production. Onset of the study, the highest biomass yield, about 1.35g/L, was obtained in urea-N, potassium nitrate, and sodium nitrate. For lipid extraction, different methods such as sonication, stirring, Soxhlet, homogenization, and sonication + Soxhlet were compared, and the highest efficiency (19.86% lipid content) was noticed in the combined sonication + Soxhlet method with chloroform: methanol (2:1). Regarding the lipid content, 19.86, 19.91, 18.44, and 19.13% were extracted from sodium nitrate, ammonium nitrate, potassium nitrate, and fertilizer grade urea-grown cultures, respectively. Further optimization of urea-N revealed that, of the 0.0, 0.75, 1.5, 2.25, and 3.0 g/L urea-N concentrations, total lipid content and neutral lipid was decreased when increasing the urea-N level in the medium and maximum content of 23.6%, and 14.7 %, were observed, respectively in 0.0 g/L urea-N. Biodiesel yield with respect to urea-N concentration showed a maximum of 64.7% biodiesel from 0 g/L grown microalgal cells. The fatty acid fingerprinting of the biodiesel by gas chromatography showed higher levels of UFAs (57.18% w/w lipids) in nil urea-N concentration (0 g/L) and higher levels of SFAs (39.84% w/w lipids) in higher urea-N (3.0 g/L) grown culture.

Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer.

DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301-induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Research on the metabolic regulation mechanism of Yangyin Qingfei decoction plus in severe pneumonia caused by Mycoplasma pneumoniae in mice.

Introduction: With amazing clinical efficacy, Yangyin Qingfei Decoction Plus (YQDP), a well-known and age-old Chinese compound made of ten Chinese botanical drugs, is utilized in clinical settings to treat a range of respiratory conditions. This study examines the impact of Yangyin Qingfei Decoction (YQDP) on lung tissue metabolic products in severe Mycoplasma pneumoniae pneumonia (SMPP) model mice and examines the mechanism of YQDP in treating MP infection using UPLC-MS/MS technology. Methods: YQDP's chemical composition was ascertained by the use of Agilent 1260 Ⅱ high-performance liquid chromatography. By using a nasal drip of 1010 CCU/mL MP bacterial solution, an SMPP mouse model was created. The lung index, pathology and ultrastructural observation of lung tissue were utilized to assess the therapeutic effect of YQDP in SMPP mice. Lung tissue metabolites were found in the normal group, model group, and YQDP group using UPLC-MS/MS technology. Using an enzyme-linked immunosorbent test (ELISA), the amount of serum inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α), was found. Additionally, the protein expression of PI3K, P-PI3K, AKT, P-AKT, NF-κB, and P-NF-κB was found using Western blot. Results: The contents of chlorogenic acid, paeoniflorin, forsythrin A, forsythrin, and paeonol in YQDP were 3.480 ± 0.051, 3.255 ± 0.040, 3.612 ± 0.017, 1.757 ± 0.031, and 1.080 ± 0.007mg/g respectively. YQDP can considerably lower the SMPP mice's lung index (p < 0.05). In the lung tissue of YQDP groups, there has been a decrease (p < 0.05) in the infiltration of inflammatory cells at varying concentrations in the alveoli compared with the model group. A total of 47 distinct metabolites, including choline phosphate, glutamyl lysine, L-tyrosine, 6-thioinosine, Glu Trp, 5-hydroxydecanoate, etc., were linked to the regulation of YQDP, according to metabolomics study. By controlling the metabolism of porphyrins, pyrimidines, cholines, fatty acids, sphingolipids, glycerophospholipids, ferroptosis, steroid hormone biosynthesis, and unsaturated fatty acid biosynthesis, enrichment analysis suggested that YQDP may be used to treat SMPP. YQDP can lower the amount of TNF-α and IL-6 in model group mice as well as downregulate P-PI3K, P-AKT, and P-NF-κB expression (p < 0.05). Conclusion: A specific intervention effect of YQDP is observed in SMPP model mice. Through the PI3K/Akt/NF-κB signaling pathways, YQDP may have therapeutic benefits by regulating the body's metabolism of α-Linoleic acid, sphingolipids, glycerophospholipids, arachidonic acid, and the production of unsaturated fatty acids.

In vitro assessment of ruminal biohydrogenation of polyunsaturated fatty acids in diets with different types and levels of protected fat and diverse sources of fibre.

The objective was to assess the in vitro rumen fermentation characteristics, methane production, and biohydrogenation of unsaturated fatty acids of diets with two protected fat (PF) sources from soybean or linseed oil, two levels of PF (0 and 6%) and two forage sources (canola silage (CS) or alfalfa hay (AH)) in a factorial 2x2x2 completely randomised design. Only fatty acids content at final incubation was affected (P<0.05) by triple interaction, where C18:2 was highest with AH plus 6% soybean PF (4.41mg/g DM), while C18:3 was with CS plus 6% linseed oil protected (1.98mg/g DM). C18:2 cis-9 trans-11 had high concentration (308 mg/g DM; P<0.05) with AH plus 6% PF regardless PF type, and C18:1 trans-11 was higher with 6% PF than without PF (13.41 vs 7.89 mg/g DM). Cumulative methane production was not affected by treatments (0.9973 ± 0.1549 mmol/g DM; P>0.05). Gas production and in vitro NDF digestibility were lower with 6% PF of linseed than soybean (160.88 vs 150.97 ml; and 69.28vs 62.89 %, respectively P<0.05). With linseed PF the NH3-N concentration was highest in CS than AH (41.27 vs 27.95 mg/dL; P<0.05) but IVDMD had the opposite result (78.54 vs 85.04). In conclusion, although methane production was not affected and in vitro digestibility and gas production were reduced with linseed PF, the concentration of C18:3 and C18:1 trans-11 was increased, which could improve the lipid profile of milk. The negative effects on digestibility were less with AH than of CS regardless of PF type and level.

Oleic Acid Metabolism in Response to Glucose in C. elegans.

A key response to glucose stress is an increased production of unsaturated fatty acids to balance the increase in saturated fatty acids in the membrane. The C. elegans homolog of stearoyl-CoA desaturase, FAT-7, introduces the first double bond into saturated C18 fatty acids yielding oleic acid, and is a critical regulatory point for surviving cold and glucose stress. Here, we incorporated 13C stable isotopes into the diet of nematodes and quantified the 13C-labelled fatty acid using GC-MS and HPLC/MS-MS to track its metabolic response to various concentrations of glucose. Previous work has analyzed the membrane composition of C. elegans when responding to mild glucose stress and showed few alterations in the overall fatty acid composition in the membrane. Here, in nematodes exposed to higher concentrations of glucose, a specific reduction in oleic acid and linoleic acid was observed. Using time courses and stable isotope tracing, the response of fatty acid metabolism to increasing levels of glucose stress is characterized, revealing the funneling of monounsaturated fatty acids to preserve the abundance of polyunsaturated fatty acids. Taken together, higher levels of glucose unveil a specific reduction in oleic and linolenic acid in the metabolic rewiring required to survive glucose stress.

Effects of Individual Circulating FFAs on Plasma and Hepatic FFA Epoxides, Diols, and Epoxide-Diol Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Oxylipins, oxidation products of unsaturated free fatty acids (FFAs), are involved in various cellular signaling systems. Among these oxylipins, FFA epoxides are associated with beneficial effects in metabolic and cardiovascular health. FFA epoxides are metabolized to diols, which are usually biologically less active, by soluble epoxide hydrolase (sEH). Plasma epoxide-diol ratios have been used as indirect measures of sEH activity. This study was designed to examine the effects of acute elevation of individual plasma FFAs on a variety of oxylipins, particularly epoxides, diols, and their ratios. We tested if FFA epoxide-diol ratios are altered by circulating FFA levels (i.e., substrate availability) independent of sEH activity. Wistar rats received a constant intravenous infusion of olive (70% oleic acid (OA)), safflower seed (72% linoleic acid (LA)), and fish oils (rich in ω-3 FFAs) as emulsions to selectively raise OA, LA, and ω-3 FFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), respectively. As expected, olive, safflower seed, and fish oil infusions selectively raised plasma OA (57%), LA (87%), EPA (70%), and DHA (54%), respectively (p < 0.05 for all). Raising plasma FFAs exerted substrate effects to increase hepatic and plasma epoxide and diol levels. These increases in epoxides and diols occurred to similar extents, resulting in no significant changes in epoxide-diol ratios. These data suggest that epoxide-diol ratios, often used as indices of sEH activity, are not affected by substrate availability or altered plasma FFA levels and that epoxide-diol ratios may be used to compare sEH activity between conditions of different circulating FFA levels.

Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

Immobilization of lipase on hydrophobic MOF synthesized simultaneously with oleic acid and application in hydrolysis of natural oils for improving unsaturated fatty acid production

The hydrolysis of natural oils (vegetable oils and fats) by lipase has significant applications in food and medicine. However, free lipases are usually sensitive to temperature, pH and chemical reagents in aqueous solutions, which hinders their widespread industrial application. Excitingly, immobilized lipases have been widely reported to overcome these problems. Herein, inspired by lipase interface activation, a hydrophobic Zr-MOF (UiO-66-NH2-OA) with oleic acid was synthesized for the first time in an emulsion consisting of oleic acid and water, and the Aspergillus oryzae lipase (AOL) was immobilized onto the UiO-66-NH2-OA through hydrophobic interaction and electrostatic interaction to obtain immobilized lipase (AOL/UiO-66-NH2-OA). 1H NMR and FT-IR data indicated that oleic acid was conjugated with the 2-amino-1,4-benzene dicarboxylate (BDC-NH2) by amidation reaction. As a result, the Vmax and Kcat values of AOL/UiO-66-NH2-OA were 179.61 μM﹒min−1 and 8.27 s−1, which were 8.56 and 12.92 times higher than those of the free enzyme, respectively, due to the interfacial activation. After treated at 70 °C for 120 min, the immobilized lipase maintained 52 % of its original activity, but free AOL only retained 15 %. Significantly, the yield of fatty acids by the immobilized lipase reached 98.3 % and still exceeded 82 % after seven times of recycling.

AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity

The human AdipoR2 and its C. elegans homolog PAQR-2 are multi-pass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labelled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified co-immunoprecipitated proteins using mass spectroscopy. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation, and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.

Comparative transcriptome and metabolite survey reveal key pathways involved in the control of the chilling injury disorder superficial scald in two apple cultivars, 'Granny Smith' and 'Ladina'.

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in 'Granny Smith' and 'Ladina' apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in 'Granny Smith'. 'Ladina', more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in 'Granny Smith' apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder.

Metabolite and Proteomic Profiling of Serum Reveals the Differences in Molecular Immunity between Min and Large White Pig Breeds.

Pig diseases seriously threaten the health of pigs and the benefits of pig production. Previous research has indicated that Chinese native pigs, such as the Min (M) pig, has a better disease resistance ability than Large White (LW) pigs. However, the molecular mechanism of this resistance is still unclear. In our study, we used serum untargeted metabolomics and proteomics, interrogated to characterize differences in the molecular immunities between six resistant and six susceptible pigs raised in the same environment. A total of 62 metabolites were identified as being significantly exhibited in M and LW pigs. Ensemble feature selection (EFS) machine learning methods were used to predict biomarkers of metabolites and proteins, and the top 30 were selected and retained. Weighted gene co-expression network analysis (WGCNA) confirmed that four key metabolites, PC (18:1 (11 Z)/20:0), PC (14:0/P-18: 0), PC (18:3 (6 Z, 9 Z, 12 Z)/16:0), and PC (16:1 (9 Z)/22:2 (13 Z, 16 Z)), were significantly associated with phenotypes, such as cytokines, and different pig breeds. Correlation network analysis showed that 15 proteins were significantly correlated with the expression of both cytokines and unsaturated fatty acid metabolites. Quantitative trait locus (QTL) co-location analysis results showed that 13 of 15 proteins co-localized with immune or polyunsaturated fatty acid (PUFA)-related QTL. Moreover, seven of them co-localized with both immune and PUFA QTLs, including proteasome 20S subunit beta 8 (PSMB8), mannose binding lectin 1 (MBL1), and interleukin-1 receptor accessory protein (IL1RAP). These proteins may play important roles in regulating the production or metabolism of unsaturated fatty acids and immune factors. Most of the proteins could be validated with parallel reaction monitoring, which suggests that these proteins may play an essential role in producing or regulating unsaturated fatty acids and immune factors to cope with the adaptive immunity of different pig breeds. Our study provides a basis for further clarifying the disease resistance mechanism of pigs.