Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

The coexpression of two desaturases provides an optimized reduction of saturates in camelina oil.

Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16 : 0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16 : 0/18 : 0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.

Metabolic engineering to produce palmitic acid or palmitoleic acid in an oleic acid-producing Corynebacterium glutamicum strain.

Focusing on the differences in the catalytic properties of two type I fatty acid synthases FasA and FasB, the fasA gene was disrupted in an oleic acid-producing Corynebacterium glutamicum strain. The resulting oleic acid-requiring strain whose fatty acid synthesis depends only on FasB exhibited almost exclusive production (217 mg/L) of palmitic acid (C16:0) from 1% glucose under the conditions supplemented with the minimum concentration of sodium oleate for growth. Plasmid-mediated amplification of fasB led to a 1.47-fold increase in palmitic acid production (320 mg/L), while fasB disruption resulted in no fatty acid production, with excretion of malonic acid (30 mg/L). Next, aiming at conversion of the palmitic acid producer to a producer of palmitoleic acid (POA, C16:1Δ9), we introduced the Pseudomonas nitroreducens Δ9-desaturase genes desBC into the palmitic acid producer. Although this resulted in failure, we noticed the emergence of suppressor mutants that exhibited the oleic acid-non-requiring phenotype. Production experiments revealed that one such mutant M-1 undoubtedly produced POA (17 mg/L) together with palmitic acid (173 mg/L). Whole genomic analysis and subsequent genetic analysis identified the suppressor mutation of strain M-1 as a loss-of-function mutation for the DtxR protein, a global regulator of iron metabolism. Considering that DesBC are both iron-containing enzymes, we investigated the conditions for increased iron availability to improve the DesBC-dependent conversion ratio of palmitic acid to POA. Eventually, supplementation of both hemin and the iron chelator protocatechuic acid in the engineered strain dramatically enhanced POA production to 161 mg/L with a conversion ratio of 80.1%. Cellular fatty acid analysis revealed that the POA-producing cells were really equipped with unnatural membrane lipids comprised predominantly of palmitic acid (85.1% of total cellular fatty acids), followed by non-native POA (12.4%).

Phenotypic variation in milk fatty acid composition and its association with stearoyl-CoA desaturase 1 (SCD1) gene polymorphisms in Gir cows.

Individual variation in milk fatty acid (FA) composition has been partially attributed to stearoyl-CoA desaturase 1 (SCD1) gene polymorphisms in taurine breeds, but much less is known for Zebu breeds. This study investigated the phenotypic variation in milk FA composition, and the influence of SCD1 variants on this trait and on milk fat desaturase indices (DI) in Gir cows. The functional impact of SCD1 variants was predicted using bioinformatics tools. Milk and blood samples were collected from 312 cows distributed in 10 herds from five states of Brazil. SCD1 variants were identified through target sequencing, and milk FA composition was determined by gas chromatography. Phenotypic variation in milk FA composition fell within the range reported for taurine breeds, with SCD18 index showing the lowest variation among the DI. Fourteen SCD1 variants were identified, six of which not previously described. Regarding the A293V polymorphism, all cows were homozygous for the C allele (coding for alanine), whereas all genotypes were detected for the second SNP affecting the 293 codon (G > A), with compelling evidence for functional effects. Significant associations (based on raw p-values) were found between this SNP and C12:0, cis-9, trans-11 CLA and short-chain FA, and between another SNP (rs523411937) and C15:0 and odd-chain linear FA. A new SNP on Chr26:21277069 was associated with trans-11 C18:1, cis-9, trans-11 CLA, C18:3 n-3 and n-3 FA. These findings indicate that SCD1 polymorphisms also contributes to the phenotypic variation in milk FA composition of Gir cows, with potential use in their breeding programmes.

Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum.

The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, particularly in galactolipids such as mono- and di-galactosyldiacylglycerol. LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analyzed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of triacylglycerol. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. KEY POINTS: • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Insight into Arthrospira platensis Δ9 desaturase: a key enzyme in poly-unsaturated fatty acid synthesis.

Membrane-bound Δ9 desaturase perform oxygenated desaturation reactions to insert the first double bonds within fatty acyl chains between C9 and C10 positions of most saturated substrates. Arthrospira platensis, a blue green microalga, is an important source of polyunsaturated fatty acids (PUFA) such as oleic, linoleic and linolenic acids lending benefits and functions in dietetics and therapeutic uses. In this paper, we report homology modeling and docking studies of a Δ9 desaturase from Arthrospira platensis strain. The protein model showed high topology resemblance compared to membrane-bound desaturases with a cytoplasmic core displaying the catalytic site and a transmembrane domain created by four α-helices. The cytoplasmic cap contained the three conserved-histidine boxes typical for all membrane bound desaturases. The protein model was used to perform protein-protein docking and the dimer structure was generated. The two monomers are tightly related with hydrophobic interactions between the transmembrane domain helices. The study highlighted also the potent role of a particular 53 residues sequence located at the N terminal end of the enzyme.

Distinct correlations between lipogenic gene expression and fatty acid composition of subcutaneous fat among cattle breeds.

BACKGROUND: The fatty acid (FA) composition of adipose tissue influences the nutritional quality of meat products. The unsaturation level of FAs is determined by fatty acid desaturases such as stearoyl-CoA desaturases (SCDs), which are under control of the transcription factor sterol regulatory element-binding protein (SREBP). Differences in SCD genotype may thus confer variations in lipid metabolism and FA content among cattle breeds. This study investigated correlations between FA composition and lipogenic gene expression levels in the subcutaneous adipose tissue of beef cattle breeds of different gender from the Basque region of northern Spain. Pirenaica is the most important beef cattle breed in northern Spain, while Salers cattle and Holstein-Friesian cull cows are also an integral part of the regional beef supply. RESULTS: Pirenaica heifers showed higher monounsaturated FA (MUFA) and conjugated linoleic acid (CLA) contents in subcutaneous adipose tissue than other breeds (P < 0.001). Alternatively, Salers bulls produced the highest oleic acid content, followed by Pirenaica heifers (P < 0.001). There was substantial variability in SCD gene expression among breeds, consistent with these differences in MUFA and CLA content. Correlations between SCD1 expression and most FA desaturation indexes (DIs) were positive in Salers (P < 0.05) and Pirenaica bulls, while, in general, SCD5 expression showed few significant correlations with DIs. There was a significant linear correlation between SCD1 and SRBEP1 in all breeds, suggesting strong regulation of SCD1 expression by SRBEP1. Pirenaica heifers showed a stronger correlation between SCD1 and SREBP1 than Pirenaica bulls. We also observed a opposite relationship between SCD1 and SCD5 expression levels and opposite associations of isoform expression levels with the ∆9 desaturation indexes. CONCLUSIONS: These results suggest that the relationships between FA composition and lipogenic gene expression are influenced by breed and sex. The opposite relationship between SCD isoforms suggests a compensatory regulation of total SCD activity, while opposite relationships between SCD isoforms and desaturation indexes, specially 9c-14:1 DI, previously reported as an indicator of SCD activity, may reflect distinct activities of SCD1 and SCD5 in regulation of FA content. These findings may be useful for beef/dairy breeding and feeding programs to supply nutritionally favorable products.

Plasma Fatty Acid Composition and Estimated Desaturase Activities Reflect Dietary Patterns in Subjects with Metabolic Syndrome.

Changes in plasma fatty acid (FA) composition and desaturase activities are observed in metabolic syndrome (MS). However, whether these changes are a reflection of dietary intakes of fats and FAs is not well established. The current study was aimed at assessing plasma FA composition and desaturase enzyme activities as biomarkers of dietary intakes in subjects with MS. Case control study was done on 41 MS patients and was compared with age matched 45 controls. Dietary intakes, anthropometric and clinical parameters were measured. FA composition was analysed using gas chromatography-flame ionisation detector and desaturase enzyme activities were estimated as ratios of product to precursor FAs. Higher levels of 14:0, 16:0, 16:1, 18:1, D9D-18 activity and lower levels of 18:0 and 18:2 n-6 were seen in MS group when compared to controls (p < 0.05). Strong positive correlations were seen between plasma triglyceride (TG) levels and 14:0, 16:0, 16:1, 18:1, total saturated fatty acid, total monounsaturated fatty acid, and D9D activities, while 18:0, 18:2 n-6 and total polyunsaturated fatty acid were negatively correlated with TG. Positive correlations were seen between plasma 14:0, 18:1 and D9D-18 activity with total energy intake and carbohydrate (CHO) intakes but not with fat intake. Plasma FA profile appears to be a better index of total energy intake and CHO intake than fat intake, suggesting it might be a good reflection of endogenous FA metabolism. Changes in FA composition may therefore serve as an early index of dysregulation of FA metabolism, resulting in increased risk of MS.

Myostatin deficiency is associated with lipidomic abnormalities in skeletal muscles.

Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and ß-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle.

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae.

The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans. Saccharomyces cerevisiae is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene, OLE1, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of OLE1 was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for OLE1). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of OLE1, leading to cadmium resistance. Such regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. IMPORTANCE: A S. cerevisiae gene encoding a delta-9 desaturase, OLE1, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of OLE1 involvement in membrane stability may indicate a novel defense strategy against cadmium stress.

Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering.

OBJECTIVE: To construct a clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system and use this system to obtain a recombinant Escherichia coli strain possessing the fatty acid metabolism genes from a lipid-rich marine bacterium. RESULTS: The fatty acid regulatory transcription factor (fadR), delta9 (Δ(9) desaturase) and acetyl-CoA carboxylase (acc) genes were cloned from Shewanella frigidimarina. The fatty acid regulatory transcription factor (fadD) and phosphoenolpyruvate carboxylase inactivated strains were used to construct the fadR/delta9 and acc knock-in strains, which are both markerless and "scar"-less, and identified the change in fatty acid composition in the recombinant strains. There was no change in fatty acid composition between the wild-type strain and recombinant strains. All strains had 11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:1, 17:0 and 18:0 fatty acids, with 16:0 and 18:0 fatty acids being dominant. The total lipid content of each recombinant strain was higher than the wild-type strain, with a maximum of 13.1 %, nearly 5.3 % higher than wild-type strain. CONCLUSION: The CRISPR/cas9 system, in conjunction with λ-Red recombinases, can rapidly and efficiently edit the E. coli genome. The CRISPR/cas9 recombineering machinery can be modified to select biotechnologically-relevant bacteria other than E. coli.

Associated factors of estimated desaturase activity in the EPIC-Potsdam study.

BACKGROUND AND AIMS: Altered activity of desaturase enzymes may be involved in the development of metabolic diseases like type 2-diabetes. Desaturase activities might be modifiable by diet and lifestyle-related factors, but no study has systematically investigated such factors so far. We aimed to evaluate the association of demographic, anthropometric, dietary and lifestyle characteristics with estimated Δ5-, Δ6- and Δ9-desaturase activity. METHODS AND RESULTS: A subsample (n = 1782) of the EPIC-Potsdam study was used for a cross-sectional analysis, involving men and women, mainly aged 35-65 years. Fatty acid (FA) product-to-precursor ratios, derived from the FA composition of erythrocyte membrane phospholipids, were used to estimate desaturase activities. Multiple linear regression models were used with estimated Δ5-, Δ6- and Δ9-desaturase activity as outcome and demographic (age, sex), anthropometric (BMI, WHR), dietary intake (FAs, carbohydrates) and lifestyle (physical activity, smoking, alcohol consumption) factors as exposure variables. Alcohol intake was positively associated with estimated Δ6- (explained variance in desaturase activity: 1.52%) and estimated Δ9-desaturase activity (explained variance: 5.53%). BMI and WHR showed a weak inverse association with estimated Δ5-desaturase activity (explained variance: BMI: 1.07%; WHR: 1.02%) and weak positive associations with estimated Δ6-(explained variance: BMI: 1.17%; WHR: 1.19%) and estimated Δ9-desaturase activities (explained variance: BMI: 0.70%; WHR: 0.96%). Age, sex, physical activity, smoking and dietary factors were only weakly associated with the estimated desaturase activities. CONCLUSION: Our findings suggest that alcohol intake as well as obesity measures are associated with the FA ratios reflecting desaturase activity.

Oral administration of cobalt acetate alters milk fatty acid composition, consistent with an inhibition of stearoyl-coenzyme A desaturase in lactating ewes.

Previous investigations have shown that cobalt (Co) modifies milk fat composition in cattle, consistent with an inhibition of stearoyl-coenzyme A desaturase (SCD) activity, but it remains unclear whether other ruminant species are also affected. The present study examined the effects of oral administration of Co acetate on intake, rumen function, and milk production and fatty acid (FA) composition in sheep. Twenty lactating Assaf ewes were allocated into 1 of 4 groups and used in a continuous randomized block design that involved a 15-d adaptation, a 6-d treatment, and a 10-d posttreatment period. During the treatment period, animals received an oral drench supplying 0 (control), 3 (Co3), 6 (Co6), and 9 (Co9) mg of Co/kg of BW per day, administered in 3 equal doses at 8-h intervals. Cobalt acetate had no influence on intake or milk fat and protein concentrations, whereas treatments Co6 and Co9 tended to lower milk yield. Results on rumen parameters showed no effects on rumen fermentation, FA composition, or bacterial community structure. Administration of Co acetate decreased milk concentrations of FA containing a cis-9 double bond and SCD product:substrate ratios, consistent with an inhibition of SCD activity in the ovine mammary gland. Temporal changes in milk fat composition indicated that the effects of treatments were evident within 3d of dosing, with further changes being apparent after 6d and reverting to pretreatment values by d 6 after administration. Effect on milk FA composition did not differ substantially in response to incremental doses of Co acetate. On average, Co decreased milk cis-9 10:1/10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, and cis-9,trans-11 18:2/trans-11 18:1 concentration ratios by 30, 32, 38, 33, 21, 24, and 25%, respectively. Changes in milk fat cis-9 10:1, cis-9 12:1, and cis-9 14:1 concentrations to Co treatment indicated that 51% of cis-9 18:1 and cis-9,trans-11 18:2 secreted in milk originated from Δ(9)-desaturation. In conclusion, results demonstrated the potential of oral Co administration for the estimation of endogenous synthesis of FA containing a cis-9 double bond in the mammary gland of lactating ruminants. Indirect comparisons suggest that the effects of Co differ between sheep and cattle.

Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae.

The seed oil of tung tree is rich in a-eleostearic acid (ESA), which endows tung oil with the characteristic of an excellently dry oil. The stearoyl-acyl carrier protein δ9 desaturase (SAD) is a rate-limiting enzyme that converts the stearic acid to the oleic acid, the substrate for the production of the α-ESA. However, the function of the two predicted VfSAD1 and VfSAD2 genes in the tung tree has not been determined. In this study, quantitative real-time PCR (qRT-PCR) analysis showed that VfSAD1 and VfSAD2 were expressed in multiple organs of tung tree but were highly expressed in the seed during the oil rapid accumulation period. Heterologous expression of VfSAD1 and VfSAD2 could promote the production of oleic acid and its derivatives in Arabidopsis thaliana and yeast BY4741, indicating that VfSAD1 and VfSAD2 possess the stearoyl-ACP desaturases function. Furthermore, both VfSAD1 and VfSAD2 could significantly improve seed oil accumulation in Arabidopsis. VfSAD1 could also significantly promote the oil accumulation in the yeast BY4741 strain. In addition, overexpression of VfSAD1 and VfSAD2 enhanced the tolerance of yeast and Arabidopsis seedlings to low temperature stress. This study indicates that the two VfSAD genes play a vital role in the process of oil accumulation and fatty acid biosynthesis in the tung tree seed, and both of them could be used for molecular breeding in tung tree and other oil crops.

Understanding desaturation/hydroxylation activity of castor stearoyl Δ9-Desaturase through rational mutagenesis.

A recently proposed reaction mechanism of soluble Δ9 desaturase (Δ9D) allowed us to identify auxiliary residues His203, Asp101, Thr206 and Cys222 localized near the di-iron active site that are supposedly involved in the proton transfer (PT) to and from the active site. The PT, along with the electron transfer (ET), seems to be crucial for efficient desaturation. Thus, perturbing the major PT chains is expected to impair the native reaction and (potentially) amplify minor reaction channels, such as the substrate hydroxylation. To verify this hypothesis, we mutated the four residues mentioned above into their counterparts present in a soluble methane monooxygenase (sMMO), and determined the reaction products of mutants. We found that the mutations significantly promote residual monohydroxylation activities on stearoyl-CoA, often at the expense of native desaturation activity. The favored hydroxylation positions are C9, followed by C10 and C11. Reactions with unsaturated substrate, oleoyl-CoA, yield erythro-9,10-diol, cis-9,10-epoxide and a mixture of allylic alcohols. Additionally, using 9- and 11-hydroxystearoyl-CoA, we showed that the desaturation reaction can proceed only with the hydroxyl group at position C11, whereas the hydroxylation reaction is possible in both cases, i.e. with hydroxyl at position C9 or C11. Despite the fact that the overall outcome of hydroxylation is rather modest and that it is mostly the desaturation/hydroxylation ratio that is affected, our results broaden understanding of the origin of chemo- and stereoselectivity of the Δ9D and provide further insight into the catalytic action of the NHFe2 enzymes.

SCD1 is nutritionally and spatially regulated in the intestine and influences systemic postprandial lipid homeostasis and gut-liver crosstalk.

Stearoyl-CoA desaturase-1 is an endoplasmic reticulum (ER)-membrane resident protein that inserts a double bond into saturated fatty acids, converting them into their monounsaturated counterparts. Previous studies have demonstrated an important role for SCD1 in modulating tissue and systemic health. Specifically, lack of hepatic or cutaneous SCD1 results in significant reductions in tissue esterified lipids. While the intestine is an important site of lipid esterification and assimilation into the body, the regulation of intestinal SCD1 or its impact on lipid composition in the intestine and other tissues has not been investigated. Here we report that unlike other lipogenic enzymes, SCD1 is enriched in the distal small intestine and in the colon of chow-fed mice and is robustly upregulated by acute refeeding of a high-sucrose diet. We generated a mouse model lacking SCD1 specifically in the intestine (iKO mice). These mice have significant reductions not only in intestinal lipids, but also in plasma triacylglycerols, diacylglycerols, cholesterol esters, and free cholesterol. Additionally, hepatic accumulation of diacylglycerols is significantly reduced in iKO mice. Comprehensive targeted lipidomic profiling revealed a consistent reduction in the myristoleic (14:1) to myristic (14:0) acid ratios in intestine, liver, and plasma of iKO mice. Consistent with the reduction of the monounsaturated fatty acid myristoleic acid in hepatic lipids of chow fed iKO mice, hepatic expression of Pgc-1α, Sirt1, and related fatty acid oxidation genes were reduced in chow-fed iKO mice. Further, lack of intestinal SCD1 reduced expression of de novo lipogenic genes in distal intestine of chow-fed mice and in the livers of mice fed a lipogenic high-sucrose diet. Taken together, these studies reveal a novel pattern of expression of SCD1 in the intestine. They also demonstrate that intestinal SCD1 modulates lipid content and composition of not only intestinal tissues, but also that of plasma and liver. Further, these data point to intestinal SCD1 as a modulator of gut-liver crosstalk, potentially through the production of novel signaling lipids such as myristoleic acid. These data have important implications to understanding how intestinal SCD1 may modulate risk for post-prandial lipemia, hepatic steatosis, and related pathologies.

The effect of α-linolenic acid enrichment in perinatal diets in preventing high fat diet-induced SCD1 increased activity and lipid disarray in adult offspring of low density lipoprotein receptor knockout (LDLRKO) mice.

The present study examined the effects of maternal perinatal dietary ALA enrichment on the high fat diet (HFD)-induced lipid disarray in the adult offspring of low density lipoprotein receptor knock-out (LDLRKO) mice. Female LDLRKO mice received, during pregnancy and lactation, isocaloric diets with either corn oil, RD, or flax oil, ALA. The weaning offspring was given a regular chow diet for a washout period of eight weeks, which was followed by HFD for eight weeks. Plasma and liver lipids and SCD1 activity were then analyzed. The HFD-fed RD adult offspring had substantially higher plasma cholesterol levels than the HFD-fed ALA offspring (15.7 versus 9.7 mmole/l, p<0.00001) and non-alcoholic fatty liver disease (NAFLD) (65.0 versus 23.9 mg/g lipids, p<0.00001). Liver lipids oleic acid (OA) content and monounsaturated to saturated fatty acids (MUFA/SAT) ratio, were two times lower in RD compared to ALA (p<0.0001). The threefold HFD-induced SCD1 raised activity (p<0.00001), and OA produced from SA, observed in RD adult offspring were prevented by perinatal ALA. In conclusion, the resilience of SCD1 to HFD- induced increased activity may account for the beneficial effects of perinatal ALA dietary enrichment in preventing NAFLD and hypercholesterolemia from occurring in adult LDLRKO offspring mice.