Agronomic performance of high oleic,low linolenic soybean in Tennessee

Soybean oil hydrogenation alters the linolenic acid molecule to prevent the oil from becoming rancid, however, health reports have indicated trans-fat caused by hydrogenation, is not generally regarded as safe. Typical soybeans contain approximately 80 g kg⁻¹ to 120 g kg⁻¹ linolenic acid and 240 g kg⁻¹ of oleic acid. In an effort to accommodate the need for high-quality oil, the United Soybean Board introduced an industry standard for a high oleic acid greater than 750 g kg⁻¹ and linolenic acid less than 30 g kg⁻¹ oil. By combing mutations in the soybean plant at four loci, FAD2-1A and FAD2-1B, oleate desaturase genes and FAD3A and FAD3C, linoleate desaturase genes, and seed oil will not require hydrogenation to prevent oxidation and produce high-quality oil. In 2017 and 2018, a study comparing four near-isogenic lines across multiple Tennessee locations was performed to identify agronomic traits associated with mutations in FAD3A and FAD3C loci, while holding FAD2-1A and FAD2-1B constant in the mutant (high oleic) state. Soybean lines were assessed for yield and oil quality based on mutations at FAD2-1 and FAD3 loci. Variations of wild-type and mutant genotypes were compared at FAD3A and FAD3C loci. Analysis using a generalized linear mixed model in SAS 9.4, indicated no yield drag or other negative agronomic traits associated with the high oleic and low linolenic acid genotype. All four mutations of fad2-1A, fad2-1B, fad3A, and fad3C were determined as necessary to produce a soybean with the new industry standard (>750 g kg⁻¹ oleic and <30 g kg⁻¹ linolenic acid) in a maturity group-IV-Late cultivar for Tennessee growers.     

Genetic Improvement of the Fatty Acid Biosynthesis System to Alter the ω-6/ω-3 Ratio in the Soybean Seed

A high ω‐6/ω‐3 fatty acid ratio in the soybean seed adversely affects human health. The objective of the present study was to improve the fatty acid biosynthesis to reduce the ω‐6/ω‐3 ratio by combining the FAD2‐1A and FAD2‐1B mutant alleles with α‐linolenic acid (ω‐3) related alleles from wild soybean. The F₂ population comprising 2320 F_2:3 lines developed from S08‐14717 × PI 483463 cross exhibited significant variation for fatty acid components. Of these, 114 lines were advanced to the F_5:6 generation and genotyped for FAD2‐1A and FAD2‐1B alleles. The lines carrying mutant FAD2‐1A and FAD2‐1B alleles showed ~ 761 g kg^?1 oleic, and ~ 50 g kg^?1 linoleic acids, which reduced ω‐6/ω‐3 ratios to ~ 0.6. Conversely, the lines carrying FAD2‐1A or FAD2‐1B mutant alleles had 267 or 399 g kg^?1 oleic, 327 or 471 g kg^?1 linoleic, and 120 or 130 g kg^?1 α‐linolenic acids concentration, respectively. The elevated α‐linolenic acid resulted in the reduction of ω‐6/ω‐3 ratios in the range 2.5–3.9. The present study demonstrated that combining FAD2 mutant alleles with α‐linolenic acid‐related alleles from wild soybean reduces the seed ω‐6/ω‐3 ratio.     

Combination of the Elevated Stearic Acid Trait with Other Fatty Acid Traits in Soybean

Stearic acid is one of five major fatty acids found in soybean oil. It is a fully saturated lipid and is known for neutral or positive effects on LDL cholesterol when consumed by humans. Unfortunately, stearic acid only accounts for about 4% of the total seed oil produced in commodity soybean. Previous work has shown that stearic acid can reach levels as high as 28% of the total oil fraction when the SACPD-C gene, encoding the delta-9-stearoyl-acyl carrier protein desaturase responsible for most of the stearic acid variation in soybean seed, is ablated in combination with other loci. In order to increase stearic acid content and create soybeans with improved utility based on fatty acid composition, we combined mutations in SACPD-C with other mutations in the fatty acid biosynthetic pathway. Soybean plants carrying mutant alleles of both SACPD-C and FAD2-1A produce seed with stearic acid levels from 14% to 21%, and with elevated levels of oleic acid. Soybeans carrying mutations in both SACPD-C and FAD3A or FAD3C have both statistically significantly elevated levels of stearic acid (from 15–21%) and statistically reduced linolenic acid levels. Neither mutant combination appears to affect other agronomic properties such as plant morphology or seed protein levels making this a potentially viable trait.     

Effect of a Mutant Danbaekkong Allele on Soybean Seed Yield,Protein, and Oil Concentration

There is a known negative correlation between soybean [Glycine max [L.] Merr.] seed protein and oil and between protein and yield. This challenges breeders to increase protein concentration while maintaining oil concentration and yield. The objective of this study was to determine if marker-assisted selection for the Danbaekkong (Dan) protein allele on chromosome 20 influences seed yield and quality traits in near isogenic genetic backgrounds. A population of 24 F₇-derived near isogenic lines (NIL) of soybean was created by crossing G03-3101 × LD00-2817P. The 24 NIL consisted of 12 wild type (WT) and 12 mutant Dan type lines. These NIL were grown in 2016 and 2017 field seasons in replicated field trials in nine environments, with six in Tennessee and one each in Arkansas, Missouri, and North Carolina. There were significant (P < 0.05) differences in yield, protein, and oil concentrations between the two experimental groups. The Dan group had significantly (P < 0.05) more protein (421 g kg⁻¹), less oil (192 g kg⁻¹), and lower yield (3143 kg ha⁻¹) than the WT group (390 g kg⁻¹ protein, 210 g kg⁻¹ oil, and 3281 kg ha⁻¹ yield). These results support previous research and corroborate the overall negative genetic correlations. Nevertheless, seed yield of several higher-protein Dan lines MC-13, MC-16, MC-19, and MC-24 exceeded seed yield of lower protein WT lines MC-2, MC-3, MC-6, and MC-10. The higher-protein lines represent genetic resources for reducing the negative correlation between protein and yield.     

Peanut FAD2 Genotype and Growing Location Interactions Significantly Affect the Level of Oleic Acid in Seeds

The level of oleic acid is an important parameter in determining seed nutritional quality and oil stability. The level of oleic acid in peanut is genetically controlled by a pair of fatty acid desaturase genes (FAD2A and FAD2B), but the environmental conditions of the production sites can also have a significant effect. To investigate the effect of gene and environment interaction, 45 accessions were grown at three locations for 2 years. Environmental data were collected; individual plants were genotyped with functional SNP markers from FAD2A and FAD2B; and seed level of oleic acid was determined by gas chromatography. Three FAD2A/FAD2B genotypes (448G/no insertion 442A, 448A/no insertion 442A, and 448A/insertion 442A) were identified and designated as G/W, A/W, and A/A, respectively. A/A genotype averaged the highest level of oleic acid (80.0%), followed by A/W (56.0%), and then G/W (40.7%). Analysis of gene and environment interaction revealed that oleic acid phenotype plasticity could be explained by the interaction of FAD2 genotype and photothermal time, which quantified environmental conditions. The A/W genotype was the most sensitive to photothermal time changes. The oleic acid plasticity revealed in this study would be useful for breeders, farmers, and product processors.     

Efficient Production of 1,3-Dioleoyl-2-Palmitoylglycerol through Rhodococcus opacus Fermentation

1,3-Dioleoyl-2-palmitoylglycerol (OPO) is the main triacylglycerol species in human milk-fat substitute. The production of OPO is of considerable research interest. In this study, a new strategy for producing OPO by fermentation with R. opacus is proposed. Chemically Interesterified fat (high oleic acid sunflower oil/hydrogenated palm oil 1.73:1 w/w), or a mixture of ethyl oleate/ethyl palmitate 2:1 (w/w) as a starting material. The highest biomass and oil content obtained were 3.3 g L⁻¹ and 40.2% (dry cell weight), respectively. The yield of OPO was 0.62 g L⁻¹. The fatty acid composition of produced OPO was 55.7–59.7% 18:1 and 28.3–29.8% 16:0, and the sn-2 position was predominantly 16:0 (64.7–74.5%). ¹³C-nuclear magnetic resonance analysis showed that the sn-1,3 and sn-2 positions were predominately esterified by 18:1 and 16:0, respectively. OPO (47.1%), OPL (13.9%), PPO (13.1%), and PPoO (16:0–16:1–18:1) (10.3%) were the most abundant triacylglycerol species.     

Bacterial Over-Expression and Purification of the 3′phosphoadenosine 5′phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ_1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ_1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ_1-328-hFADS enzyme shows a slight, but not significant, change of K_m values (0.24 and 6.23 μM for FMN and ATP, respectively) and of k_cat (4.2 × 10⁻² s⁻¹) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ_1-328-hFADS versus wild-type protein.     

Differential temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms overexpressed in Saccharomyces cerevisiae

The oxygen‐independent temperature regulation of three sunflower microsomal oleate desaturase (FAD2) isoforms has been investigated using Saccharomyces cerevisiae cells expressing each FAD2 gene. Yeast cells transformed with the FAD2‐1 gene showed the highest percentage of dienoic acids when they were grown at 10–15 °C. In contrast, the maximal level of dienoic acids for S. cerevisiae cells expressing the FAD2‐2 and FAD2‐3 genes were obtained at 30 and 35 °C, respectively. Temperature shifts in the phase of exponential growth, from 30 to 15 °C or from 15 to 30 °C, produced changes in the final percentage of dienoic acids, mostly in yeast cells transformed with the FAD2‐1 gene, to reach the content corresponding to the new temperature. Low temperature (15 °C) increased the amount of neutral lipids in all transformed yeast cells, mainly because it favored triacylglycerol accumulation. In addition, the FAD2‐expressing yeast cells showed a higher polar lipid content than those transformed with the empty vector. Dienoic acids were present in all lipids, although high temperature (30 °C) favored their accumulation in neutral lipids. As the main conclusion, the low thermal stability observed for the major and seed specific isoform (FAD2‐1) is the key factor controlling the direct temperature regulation in sunflower seeds.     

Transgenic cotton plants with increased seed oleic acid content

Cottonseed typically contains about 15% oleic acid. Here we report the development of transgenic cotton plants with higher seed oleic acid levels. Plants were generated by Agrobacterium-mediated transformation. A binary vector was designed to suppress expression of the endogenous cottonseed †-12 desaturase (fad2) by subcloning a mutant allele of a rapeseed fad2 gene downstream from a heterologous, seedspecific promoter (phaseolin). Fatty acid profiles of total seed lipids from 43 independent transgenic lines were analyzed by gas chromatography. Increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in 22 of the primary transformants. The increase in oleic acid content was at the expense of linoleic acid, consistent with reduced activity of cottonseed FAD2. Progeny of some lines yielded oleic acid content as high as 47% (three times that of standard cottonseed oil). Molecular analyses of nuclear DNA from transgenics confirmed the integration of the canola transgene into the cotton genome. Collectively, our results extend the metabolic engineering of vegetable oils to cottonseed and should provide the basis for the development of a family of novel cottonseed oils.     

Electrocatalytic properties of NDGA and NDGA/FAD hybrid film modified electrodes for NADH/NAD redox reaction

The fabrication of monolayers composed of nordihydroguaiaretic acid (NDGA), and hybrid films composed of NDGA-flavin adenine dinucleotide (FAD) adsorbed films was performed in neutral aqueous solutions to produce electrochemically active thin films exhibiting one and two redox couples, respectively. An electrochemical quartz crystal microbalance and cyclic voltammetry were used to study the in situ growth of the NDGA and hybrid NDGA/FAD film monolayers. The NDGA modified film electrocatalytically oxidized NADH, ascorbic acid, dopamine, and N₂H₄ in neutral aqueous solutions. Well-separated voltammetric peaks were observed for dopamine and uric acid mixtures, and also for ascorbic acid and uric acid mixtures using the NDGA/GC modified electrode. When transferred to various aqueous buffered solutions, the two redox couples of the NDGA/FAD hybrid film and their formal potentials were observed to be pH-dependent. The electrocatalytic oxidation and reduction of NADH and NAD⁺ by a NDGA/FAD hybrid film in neutral aqueous solutions was carried out, and the electrocatalytic oxidation of NADH was performed using a NDGA/FAD hybrid film.     

Evaluation of Agronomic and Seed Characteristics in Elevated Oleic Acid Soybean Lines in the South-Eastern US

Increasing oleic acid, a monounsaturated fatty acid, is reported to strike the best balance between cold flow properties and oxidative stability in soybean seed oil to enhance biodiesel and produce a better burning fuel. In addition, it is important that elevated oleic acid soybeans have the agronomic traits of local cultivars and maintain oleic acid stability across environments. Research was conducted in 2007–2008 to evaluate six Roundup Ready^® soybean recombinant inbred lines exhibiting enhanced levels of oleic acid. The six elevated oleic lines averaged a 55% increase in oleic acid and a 43% decrease in linolenic acid over the two commercial cultivars (AG3906 and AG4103). Some elevated oleic acid genotypes fulfilled the linear regression definition of a stable genotype. TN03-93RR was the best genotype because of its oleic acid content (397 g kg^?1) and desirable regression estimates for stability. Iodine value (IV), peroxide value (PV), and induction period (IP) were used to evaluate the fuel properties of the two lines with the highest oleic acid content and the two commercial cultivars. The elevated oleic acid lines had significantly better IP, PV and IV for better biodiesel properties and oxidative stability than the two commercial cultivars.     

The Interaction of the Soybean Seed High Oleic Acid Oil Trait With Other Fatty Acid Modifications

Oil value is determined by the functional qualities imparted from the fatty acid profile. Soybean oil historically had excellent use in foods and industry; the need to increase the stability of the oil without negative health consequences has led to a decline in soybean oil use. One solution to make the oil stable is to have high oleic acid (>70%) and lower linolenic acid content in the oil. Other fatty acid profile changes are intended to target market needs: low‐saturated fatty acid and high stearic acid content in the oil. The objective of this study is to determine the interaction of the high oleic acid oil trait with other alleles controlling fatty acid profiles. Soybean lines containing high oleic acid allele combinations plus other fatty acid modifying alleles were produced, and the seed was produced in multiple field environments over 2 years. Stable high oleic acid with low linolenic acid (<3.0%) was achieved with a 4‐allele combination. The target of >20% stearic acid in the seed oil was not achieved. Reducing total saturated fatty acids below 7% in a high oleic acid background was possible with mutant alleles of both an acyl‐ACP thioesterase B and a β‐ketoacyl‐[acyl‐carrier‐protein] synthase III gene. The results identified allele combinations that met the target fatty acid profile thresholds and were most stable across environments.     

Computed Thermodynamic and Explosive Properties of 1‐Azido‐2‐nitro‐2‐azapropane (ANAP)

Some thermodynamic and explosive properties of the recently reported 1‐azido‐2‐nitro‐2‐azapropane (ANAP) have been determined in a combined computational ab initio (MP2/aug‐cc‐pVDZ) and EXPLO5 (Becker–Kistiakowsky–Wilson's equation of state, BKW EOS) study. The enthalpy of formation of ANAP in the liquid phase was calculated to be Δ_fH°, ANAP(l)=+297.1 kJ mol⁻¹. The heat of detonation (Q_v), the detonation pressure (P), and the detonation velocity of ANAP were calculated to be Q_v=−6088 kJ kg⁻¹, P=23.8 GPa, D=8033 m s⁻¹. A mixture of ANAP and tetranitromethane (TNM) was investigated in an attempt to tailor the impact sensitivity of ANAP, but results obtained indicate that the mixture is almost as sensitive as pure ANAP. On the other hand, ANAP and TNM were found to be chemically compatible (¹H, ¹³C, ¹⁴N NMR; DSC) and a 1 : 1 mixture (by weight) of both components was calculated to have superior explosive properties than either of the individual components: Q_v=−6848 kJ kg⁻¹, P=27.0 GPa, D=8284 m s⁻¹.     

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1–3 steps with moderate yields (10–57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.     

Accumulation of 2-tert-Butyl-1,4-Benzoquinone in Frying Oil and Fried Food during Repeated Deep Fat Frying Processes

2-tert-Butyl-1,4-benzoquinone (TBBQ), the main oxidation product of tert-butyl-hydroquinone (TBHQ) during frying, is cytotoxic and its residual levels in frying oils and foods are unknown. In this study, TBBQ residues have been evaluated during the preparation of french fries. Results showed that frying at 140 °C resulted in the highest TBBQ peak concentration (48.42 mg kg⁻¹) compared with frying at 190 or 170 °C. This unexpected finding can be attributed to more extensive hydrolytic reaction when frying at the lower temperature, generating more peroxyl radicals. TBBQ concentrations proved to be independent of the oil type among various unsaturated oils. However, higher TBBQ levels were observed in saturated palm oil and crude soybean oil than in unsaturated oil or refined oil. Continuous frying leads to the accumulation of a large amount of TBBQ in fried food. After frying 1–5 batches, TBBQ levels in both the frying oil and fries were above 10 mg kg⁻¹, exceeding its critical cytotoxic concentration (IC₅₀ value of 10.71 mg kg⁻¹ for RAW 246.7 cells in our previous study), warranting concern with respect to the safety of fried food. FTIR has been utilized as an effective tool for visually monitoring the degree of oxidation in the frying medium with respect to its hydrogen peroxide level, which contributes to the increased level of TBBQ derived from TBHQ therein.     

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing⁺ (TbyS⁺), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS⁺ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS⁺, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.     

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products (“normal” and “abnormal”) when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k_cat/K_M) 8.9-fold to 484 s⁻¹ mM⁻¹ for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO_C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L⁻¹ d⁻¹. This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.     

Physicochemical Characteristics and Technological Properties of Lupinus mutabilis Oil

The aim of the presented study is to examine the physicochemical parameters of the lipids present in Lupinus mutabilis seed and to compare the results with the available data for other commonly used vegetable oils. The oil quality indexes, oxidative stability index (OSI), and melting characteristics are examined. Andean lupin oil has remarkably high oxidative stability (OSI = 65 h) comparable to high-oleic oils counterparts. Quality parameters meet commonly accepted standards, including peroxide value (3.95 meq O₂ kg⁻¹) and p-anisidine value (1.25). The acid number value is 1.85 mg KOH g⁻¹. The iodine value is 110.27 g/100 g, while the enthalpy required to increase the temperature of the sample from −60 to 80 °C is equal to 57.41 kJ kg⁻¹. The beginning of the melting event (T_onset) and the phase transition temperature (T_peak) values for L. mutabilis seed oil are −29.46 and −22.63 °C, respectively. The presented results indicate the unusually high oxidative stability of the oil obtained from L. mutabilis seeds, which opens up a whole spectrum of application possibilities, e.g., designing blends with other commonly used vegetable oils to enhance their low stability. Practical Applications: The presented results provide insight into physicochemical parameters of the lipid fraction isolated from Lupinus mutabilis seeds. Andean lupin oil has very high oxidative stability, comparable to high-oleic rapeseed and sunflower oils. Therefore, the identified potential use of the studied oils is, e.g. an additive that can increase the stability of commercial vegetable oils characterized by much lower oxidative stability.