Biomanufacturing ofγ-linolenic acid-enriched galactosyldiacylglycerols:Challenges in microalgae and potential in oleaginous yeasts

γ-Linolenic acid-enriched galactosyldiacylglycerols(GDGs-GLA),as the natural form ofγ-linolenic acid in microalgae,have a range of functional activities,including anti-inflammatory,antioxidant,and anti-allergic properties.The low abundance of microalgae and the structural stereoselectivity complexity impede microalgae extraction or chemical synthesis,resulting in a lack of supply of GDGs-GLA with a growing demand.At present,there is a growing interest in engineering oleaginous yeasts for mass production of GDGs-GLA based on their ability to utilize a variety of hydrophobic substrates and a high metabolic flux toward fatty acid and lipid(triacylglycerol,TAG)production.Here,we first introduce the GDGs-GLA biosynthetic pathway in microalgae and challenges in the engineering of the native host.Subsequently,we describe in detail the applications of oleaginous yeasts with Yarrowia lipolytica as the representative for GDGs-GLA biosynthesis,including the development of synthetic biology parts,gene editing tools,and metabolic engineering of lipid biosynthesis.Finally,we discuss the development trend of GDGs-GLA biosynthesis in Y.lipolytica.

Biotechnology ofα-linolenic acid in oilseed rape(Brassica napus)using FAD2 and FAD3 from chia(Salvia hispanica)

α-Linolenic acid(ALA,18:3Δ9,12,15)is an essential fatty acid for humans since it is the precursor for the biosynthesis of omega-3 long-chain polyunsaturated fatty acids(LC-PUFA).Modern people generally suffer from deficiency of ALA because most staple food oils are low or lack ALA content.Biotechnological enrichment of ALA in staple oil crops is a promising strategy.Chia(Salvia hispanica)has the highest ALA content in its seed oil among known oil crops.In this study,the FAD2 and FAD3 genes from chia were engineered into a staple oil crop,oilseed rape(Brassica napus),via Agrobaterium tumefaciens-mediated transformation of their LP4-2A fusion gene construct driven by the seed-specific promoter P_(NapA).In seeds of T0,T1,and T2 lines,the average ALA contents were 20.86,23.54,and 24.92%,respectively,which were 2.21,2.68,and 3.03 folds of the non-transformed controls(9.42,8.78,and 8.22%),respectively.The highest seed ALA levels of T0,T1,and T2 plants were 38.41,35.98,and 39.19%respectively,which were 4.10-4.77 folds of the respective controls.FA-pathway enzyme genes(BnACCD,BnFATA,BnSAD,BnSCD,BnDGAT1,BnDGAT2,and BnDGAT3)and positive regulatory genes(BnWRI1,BnLEC1,BnL1L,BnLEC2,BnABI3,BnbZIP67,and BnMYB96)were all significantly up-regulated.In contrast,BnTT1,BnTT2,BnTT8,BnTT16,BnTTG1,and BnTTG2,encoding negative oil accumulation regulators but positive secondary metabolism regulators,were all significantly down-regulated.This means the foreign ShFAD2-ShFAD3 fusion gene,directly and indirectly,remodeled both positive and negative loci of the whole FA-related network in transgenic B.napus seeds.

Formulation ofα-linolenic acid microemulsion free of co-surfactant

A novel class ofα-linolenic acid-in-water microemulsion free of co-surfactant was investigated as potential food delivery systems.Rough demarcation within the transparent region was deduced from the results of conductivity and polarizing optical microscopy.The microemulsion mean hydrodynamic diameter and characterization were determined by dynamic light scattering and negative-staining TEM.The location of ALA molecules in the microemulsion formulations was determined by ~1H NMR spectroscopy.

α-Linolenic acid alleviates aluminium chloride-induced toxicity in PC12 cells by activation of PKA-CREB-BDNF signaling pathway

Aluminum has been associated with neurodegenerative diseases.ALA(α-linolenic acid),an essential dietary component for human health,possesses prominent biological activities.Herein,we aim to explore the neuroprotective effects of ALA on aluminum toxicity and reveal the underlying mechanism.Results show that aluminum chloride(denoted as Al)enabled cell viability decline and apoptosis with oxidative stress and mitochondrial damage in differentiated rat pheochromocytoma cells(PC12)for 24 h incubation.Compared with Al(10 mmol/L)treatment alone,ALA(50μmol/L)pretreatment for 24 h significantly enhanced cell viability by 28.40%,and hindered cell apoptosis by 12.35%,together with recovering redox state balance and alleviating mitochondrial damage.It was measured that ALA treatment upregulated Bcl-2 expression and down-regulated Bax level,accompanied with an expression decline of caspase-3 and caspase-9.Meanwhile,ALA pretreatment was proved to increase protein kinase A(PKA)expression and to promote phosphorylation of cAMP response element-binding protein(p-CREB),resulting in elevation on the level of brain-derived neurotrophic factor(BDNF).The above results showed that ALA attenuated Al toxicity in PC12 cells by mediating the PKA-CREBBDNF signaling pathway.

Feeding Calcium Salts of Linseed Oil on Metabolic Status and Reproductive Traits in Grazing Dairy Cows

This experiment aimed to evaluate the effect of calcium salts of linseed oil (rich in alpha-linolenic acid (ALA, C18:3n-3) on metabolic and reproductive traits in high-producing dairy cows under grazing. Thirty-six Holstein dairy cows were randomly assigned, in a complete block design, to receive ALA supplementation (0.85 kg∙day−1 of calcium salts of linseed oil) or to remain as untreated control (CON). The concentrate was formulated to offer the same amount of energy across treatments (CON cows received an extra kg of corn to compensate for the higher energy density of ALA treatment). A PMR + Alfalfa pasture was offered to all cows at the same time. A fixed time artificial insemination (FTAI) at 80 DIM, preceded by a Presynch plus Ovsynch protocol was implemented for the first service and later, on return to estrus, heat detection and artificial insemination (AI) were performed. Pregnancy diagnosis was checked at 30, 42, 60, and 90 d after AI. Blood and milk samples were taken biweekly. Treatment affected plasma cholesterol concentration (160.36 vs. 186.70 mg∙dl−1, p = 0.03, for ALA and CON, respectively) and on size of corpus luteum (CL, 17.6 vs. 13.7 mm, p = 0.02, for ALA and CON, respectively). Supplementation tended (p = 0.136) to increase conception rate by 200 DIM (81.69% vs. 55.43% in ALA and CON cows, respectively). However, treatment had no effect (p > 0.05) on body weight (BW), body condition score (BCS), and circulating levels of beta-hydroxybutyrate (BHBA), glucose, insulin, growth hormone (GH) and insulin-like growth factor (IGF-I). Our results suggest that supplementation with calcium salts of linseed oil could enhance ovarian function without affecting energy metabolism in early lactation dairy cows.

The Role of Jasmonates as Antibulbing Substances in the Bulb Formation of Onion

Onion plants form spherical bulbs under long-day conditions.Substances regulating bulb formation remain unknown.In the course of chemical studies on the bulb formation,α-linolenic acid was isolated from onion extracts as an antibulbing substance,the amount of which was synchronized with the bulb formation.Since allene oxide synthase inhibitor canceled the antibulbing activity ofα-linolenic acid,it was disclosed that jasmonic acid concerns this regulation.Structure-activity-relationship study revealed that its(3R,7S)stereochemistry is necessary for showing its antibulbing activity.It is concluded that(3R,7S)-jasmonate derived fromα-linolenic acid actually participates in the regulation of bulb formation.

Evaluation of anti-tubercular activity of linolenic acid and conjugatedlinoleic acid as effective inhibitors against Mycobacterium tuberculosis

Objective: To evaluate a new pharmacological activity/effect of linolenic acid(α- and γ-form) and conjugated-linoleic acid(CLA) causing antibacterial activity against Mycobacterium tuberculosis(Mtb). Methods: The anti-Mtb activity/effect of linolenic acid and CLA were determined using different anti-Mtb indicator methods such as resazurin microtiter assay(REMA) and MGIT 960 system assay. The Mtb was incubated with various concentrations(12.5–200) μg/m L of the compounds and anti-Mtb first-line drugs for 5 d in the REMA, and for 3 wk in MGIT 960 system assay. Results: Linolenic acid and CLA obviously indicated their anti-Mtb activity/effect by strongly inhibiting the growth/proliferation of Mtb in a dosedependent manner in the REMA and the MGIT 960 system assay. Interestingly, linolenic acid and CLA consistently induced anti-Mtb activity/effect by effectively inhibiting the growth/proliferation of Mtb in MGIT 960 system for 21 d with a single-treatment, and their minimum inhibitory concentrations were measured as 200 μg/m L respectively. Conclusions: These results demonstrate that linolenic acid and CLA not only have effective anti-Mtb activity/properties, but also induce the selective-anti-Mtb effects by strongly inhibiting and blocking the growth/proliferation of Mtb through a new pharmacological activity/action. Therefore, this study provides novel perspectives for the effective use of them and the potential that can be used as potent anti-Mtb candidate drugs, as well as suggests the advantage of reducing the cost and/or time for developing a new/substantive drug by effectively repurposing the existing drugs or compounds as one of new strategies for the global challenge of tuberculosis.

Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

The promoter region (BCSP666) of b-conglycinin a-subunit gene from the genomic DNA of soybean Jilin 43 was isolatedby PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure to the soybeanseed-specific promoter b-conglycinin a'-subunit gene promoter and b-conglycinin b-subunit gene promoter, and it alsocontains many motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, wededuced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constructed the seed-specific expression vector pBMI666 with the promoter fragment BCSP666 and D6-fatty acid desaturase gene fromMortierella isabellina. The D6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in theproduction of a human essential fatty acid, g-linolenic acid(GLA). The production of g-linolenic acid(GLA) was observedin soybean callus cells, which were transformed with this vector. This confirmed the activity of the activity fragmentBCSP666.

Fatty acid analysis in the seeds of 50 Paeonia ostii individuals from the same population

Tree peony seeds are rich in α-linolenic acid(ALA), and the peony seed oil is now being produced in China. Paeonia ostii is the most widely used tree peony species for oil extraction, which is commercially called Fengdan and treated as a single cultivar. Here, 50 P. ostii individuals from the same population in northern China were randomly selected for fatty acids(FAs) analysis. Thirteen FAs were isolated, and the most abundant five were palmitic acid(5.31–6.99%), stearic acid(1.22–2.76%), oleic acid(18.78–28.15%), linoleic acid(11.86–26.10%), and ALA(41.11–57.51%). There were significant individual differences of plants in FA quality and quantity and the linoleic acid content in Plant No. 48 even exceeded the scope of 1–99%. Further statistical analysis indicated that most of the individual FAs, saturated FAs, unsaturated FAs, and total FAs levels showed significant positive correlations to each other, whereas the seed yield per plant was independent and not correlated to the factors mentioned above. Ward’s hierarchical clustering results grouped the 50 plants into four clusters based on FA contents and seed yield, and the seven plants in Cluster IV were identified as good candidates for oil production. Our results confirmed that the individual differences did occur in P. ostii and Fengdan cannot be simply treated as one uniform cultivar. Also, these results may help simplify the selection of plants for oil peony breeding and accelerate the development of the oil peony industry.

Relationship between high dietary fat intake and Parkinson’s disease risk: a meta-analysis

OBJECTIVE: To assess whether dietary fat intake influences Parkinson’s disease risk. DATA SOURCES: We systematically surveyed the Embase and PubMed databases, reviewing manuscripts published prior to October 2018. The following terms were used:(“Paralysis agitans” OR “Parkinson disease” OR “Parkinson” OR “Parkinson’s” OR “Parkinson’s disease”) AND (“fat” OR “dietary fat” OR “dietary fat intake”). DATA SELECTION: Included studies were those with both dietary fat intake and Parkinson’s disease risk as exposure factors. The Newcastle-Ottawa Scale was adapted to investigate the quality of included studies. Stata V12.0 software was used for statistical analysis. OUTCOME MEASURES: The primary outcomes included the relationship between high total energy intake, high total fat intake, and Parkinson’s disease risk. The secondary outcomes included the relationship between different kinds of fatty acids and Parkinson’s disease risk. RESULTS: Nine articles met the inclusion criteria and were incorporated into this meta-analysis. Four studies scored 7 and the other five studies scored 9 on the Newcastle-Ottawa Scale, meaning that all studies were of high quality. Meta-analysis results showed that high total energy intake was associated with an increased risk of Parkinson’s disease (P = 0.000, odds ratio (OR)= 1.49, 95% confidence interval (CI): 1.26–1.75);in contrast, high total fat intake was not associated with Parkinson’s disease risk (P = 0.123, OR = 1.07, 95% CI: 0.91–1.25). Subgroup analysis revealed that polyunsaturated fatty acid intake (P = 0.010, OR = 1.03, 95% CI: 0.88–1.20) reduced the risk of Parkinson’s disease, while arachidonic acid (P = 0.026, OR = 1.15, 95% CI: 0.97–1.37) and cholesterol (P = 0.002, OR = 1.09, 95% CI: 0.92–1.29) both increased the risk of Parkinson’s disease. Subgroup analysis also demonstrated that, although the results were not significant, consumption of n-3 polyunsaturated fatty acids (P = 0.071, OR = 0.88, 95% CI: 0.73–1.05),α-linolenic acid (P = 0.06, OR = 0.86, 95% CI: 0.72–1.02), and the n-3 to n-6 ratio (P = 0.458, OR = 0.89, 95% CI: 0.75–1.06) were all linked with a trend toward reduced Parkinson’s disease risk. Monounsaturated fatty acid (P = 0.450, OR = 1.06, 95% CI: 0.91–1.23), n-6 polyunsaturated fatty acids (P = 0.100, OR = 1.15, 95% CI: 0.96–1.36) and linoleic acid (P = 0.053, OR = 1.11, 95% CI: 0.94–1.32) intakes were associated with a non-significant trend toward higher PD risk. Saturated fatty acid (P = 0.619, OR = 1.01, 95% CI: 0.87–1.18) intake was not associated with Parkinson’s disease. CONCLUSION: Dietary fat intake affects Parkinson’s disease risk, although this depends on the fatty acid subtype. Higher intake of polyunsaturated fatty acids may reduce the risk of Parkinson’s disease, while higher cholesterol and arachidonic acid intakes may elevate Parkinson’s disease risk. However, further studies and evidence are needed to validate any link between dietary fat intake and Parkinson’s disease.

Practical Application of Diets Rich in Ground Linseed to Enhance Omega-3 Milk Production in Holstein Dairy Farms Located in Northeast Iran

In an attempt to enhance organic omega-3 milk production, a practical study was performing in 7 Holstein dairy farms in Northeast Iran. The experiment was conducted at the ＂ShafashirToos industrial dairy farmers production and distribution cooperative＂ located in Northeast Iran, between June 2010 and December 2010. In Golshid-Mashhad Co., cows were fed with no linseed containing diets （NO-Lin）, while in the other farms （Hanaei Co., Ghasemi Co., Taheri Co., Teliseh Co., Arasp Co. and Navingholshid-Khorasan Co.）, cows were fed diets containing 9% ground linseed （Lin）. Cows were used in a randomized complete block experimental design. Diets were provided as TMR. Concentration of fat, protein and lactose in milk was measured. Milk fat extraction and fatty acid separation were determined. There were no differences in the most of the milk fatty acid profiles of cows in farms that received ground linseed but feeding Lin diets compared with NO-Lin, significantly decreased （P 〈 0.05） C8：0-C16：0 concentration and it increased those of C 18：1 n9trans, C 18：1 n9cis, C 18：2 trans and C 18：3 n3. As a result, farms with Lin diets had better milk fatty acid profiles with doubled in a-Linolenic acid concentration and increased in mono and polyunsaturated fatty acids and decrease in medium chain and saturated fatty acids compared with those of the NO-Lin diet. These results clearly demonstrate the possibility for sustainable production of organic omega-3 milk with levels ofC18：3 FA up to 2.4 times higher than typical levels in dairy fat. The manufacture of omega-3 enriched milk and milk products could supply dietary fatty acids at levels that may henefit health, without the need for unrealistic changes to eating habits.

Feeding Calcium Salts of Linseed Oil on Productive Performance and Milk Fatty Acid Profile in Grazing Dairy Cows

The objective of this study was to determine the effect of supplying calcium salts of linseed oil (Ca-FA) rich in omega-3 (α-linolenic acid) on the production and chemical composition of milk and its nutraceutical value in dairy cows in early lactation. The trial lasted 12 weeks (2 weeks for adaptation to lipids and 10 weeks of data collection). A total of 36 Holstein dairy cows with 58.0 ± 17.0 days in milk (DIM), 594.1 ± 92.4 kg BW, 2.6 ± 1.5 parity and 38.9 ± 9.3 kg milk day-1 were used in a randomized complete block design. The treatments were: 1) Omega-3 (O3): 5.2 kg DM day-1 of concentrate including 0.7 kg DM of Ca-FA + 13.5 kg DM day-1 of partial mixed ration (PMR) + 12 kg DM day-1 of alfalfa pasture (Medicago sativa) and 2) Control (C): diet similar to O3 but lipid supplementation was replaced by cracked corn grain so that the diets were isoenergetic. No treatment effect was detected (P > 0.05) for any milk production and composition variables, except for urea in milk that was slightly higher in O3 (P = 0.02). The treatment × week interaction was significant (P < 0.05) for fat yield and content, with differences (P < 0.01) only in the 3rd week of the data collection period in favor of group C (1.39 vs. 1.13 kg·day-1 and 3.86% vs. 3.23% for fat yield and content, respectively). Total DMI and PMR were similar (P > 0.05) between treatments. Concentrate intake was higher (P < 0.01) in C compared with O3. Pasture DMI tended (P = 0.06) to be greater for cows that received the O3 treatment compared with C. Total metabolizable energy (ME) intake was similar (P = 0.44) between treatments. No treatment effect was detected (P > 0.05) in rumen environment parameters. Supplementation with Ca-FA reduced (P < 0.05) the hypercholesterolemic fraction of milk (C12:0, C14:0 and C16:0, -13.6%, -7.4% and -9.0%, respectively). The concentration of α-linolenic acid (C18:3n-3) increased (108%, P < 0.01) in O3 group compared with group C. The absence of negative effects of lipids on the fat content of milk and ruminal fermentation suggests that protection by saponification was effective. The supplementation with Ca-FA (0.85 kg·day-1) improved the healthy value of the milk.

Preliminary Research on the Effect of Linolenic Acid on Human Oocyte Maturation

Objective:To investigate the effects of exogenousα-linolenic acid(ALA)on in vitro maturation(IVM)and developmental competence of human oocytes.Methods:Experiment 1 examined the effects of ALA at different concentrations(0[control],10,50,100,and 200μmol/L)in the IVM medium on oocyte maturation.Treatment with 50μmol/L ALA significantly accelerated oocyte maturation(P<0.05)and resulted in significantly higher mitochondrial DNA(mtDNA)copy number compared to the control.Hence,50μmol/L ALA was selected for combination with follicular fluid(FF)to investigate oocyte developmental potential.mtDNA of the matured oocyte was analyzed by real-time polymerase chain reaction.Experiment 2 investigated the effects of FF with optimal ALA concentration(Group A:ALA+FF+IVM medium)or without ALA(Group B:FF+IVM medium)on oocyte maturation,fertilization,2 pronuclear cleavage,and embryo and blastocyst development.Malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were measured for maturation medium from Group A,Group B,and Group C(control group,IVM medium only).Results:Treatment with 50μmol/L ALA obviously accelerated oocyte maturation(P<0.05)and resulted in significantly higher mtDNA copy number(P<0.05)in the matured oocytes compared to the control(0μmol/L ALA).Supplementation of 50μmol/L ALA and FF(Group A)obviously increased the total maturation rate than FF-treated group(Group B)which has higher(P<0.05)total maturation rate than that of Group C.However,no significant differences were observed in fertilization,embryo availability,and blastocyst production among Group A,B,and C.Treatment with 50μmol/L ALA decreased the level of MDA(P<0.05),but had no effect on the activity of SOD in the IVM medium.Conclusions:Our results suggested that the treatment with 50μmol/L ALA during IVM improves maturation in human oocytes.It is also likely to improve embryo availability and blastocyst formation.This might be associated with the alteration of mtDNA replication(increased mtDNA copy number)and the reduction of oxidative stress(reduced MDA level).