Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.

BACKGROUND: Mammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids. RESULTS: Exogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1. SIGNIFICANCE: The NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Knockout of the delta11-desaturase SfruDES1 disrupts sex pheromone biosynthesis, mating and oviposition in the fall armyworm, Spodoptera frugiperda.

Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda.

Characterization and Functional Analysis of Fads Reveals Δ5 Desaturation Activity during Long-Chain Polyunsaturated Fatty Acid Biosynthesis in Dwarf Surf Clam Mulinia lateralis.

Fatty acid desaturases (Fads), as key enzymes in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), catalyze the desaturation between defined carbons of fatty acyl chains and control the degree of unsaturation of fatty acids. In the present study, two Fads genes, designated MulFadsA and MulFadsB, were identified from the genome of the dwarf surf clam Mulinia lateralis (Mollusca, Mactridae), and their spatiotemporal expression was examined. MulFadsA and MulFadsB contained the corresponding conserved functional domains and clustered closely with their respective orthologs from other mollusks. Both genes were expressed in the developmental stages and all tested adult tissues of M. lateralis, with MulFadsA exhibiting significantly higher expression levels in adult tissues than MulFadsB. Subsequently, the effects of dietary microalgae on Fads expressions in the dwarf surf clam were investigated by feeding clams with two types of unialgal diets varying in fatty acid content, i.e., Chlorella pyrenoidosa (Cp) and Platymonas helgolandica (Ph). The results show that the expressions of MulFads were significantly upregulated among adult tissues in the Cp group compared with those in the Ph group. In addition, we observed the desaturation activity of MulFadsA via heterologous expression in yeasts, revealing Δ5 desaturation activity toward PUFA substrates. Taken together, these results provide a novel perspective on M. lateralis LC-PUFA biosynthesis, expanding our understanding of fatty acid synthesis in marine mollusks.

Identification of Conjugated Dienes of Fatty Acids in Vischeria sp. IPPAS C-70 under Oxidative Stress.

The microalgae Vischeria sp. IPPAS C-70 produces eicosapentaenoic acid. Several stresses cause the formation of fatty acid peaks that resemble hexadecadienoic acids. We used the integrated technique including TLC, HPLC, and GC-MS to search and determine these fatty acids. Double bond positioning in these fatty acids indicated that they were conjugated dienes and allenes. We identified and described natural nine isomers of C16 polyunsaturated fatty acids, including common methylene-interrupted dienes (Δ6,9-16:2, Δ7,10-16:2, Δ9,12-16:2), and unusual conjugated dienes (Δ6,8-, Δ7,9-, Δ8,10-, Δ9,11-, and Δ10,12-16:2), as well as allenic diene (Δ9,10-16:2). We hypothesize that the formation of conjugated dienes and allenes among fatty acids is the result of oxidative stress caused by H2O2. Hydrogen peroxide also caused an increase in saturated at the expense of unsaturated fatty acids, suggesting inhibition either fatty acid desaturases activities or the corresponding gene expression.

Lipid-Restricted Culture Media Reveal Unexpected Cancer Cell Sensitivities.

Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimic the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example, on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provide more realistic levels of lipids. Here, we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. The mechanism of action studies indicates that ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets delta 4-desaturase, sphingolipid 1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies suggest ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and reveal a general screening strategy for identifying additional cancer dependencies masked by the superabundance of medium lipids.

The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

Fatty acid desaturase 1/2 (FADS1 and FADS2), fatty acid desaturase indices, and their relationships with metabolic syndrome in female adults with first-episode schizophrenia after antipsychotic medications.

OBJECTIVE: Although antipsychotics constitute the best treatment for patients with schizophrenia, this treatment class carries a high risk of metabolic disarrangements thus developing metabolic syndrome (MetS). Altered fatty acid (FA) composition and desaturase indices have been associated with several metabolic diseases, including MetS. Herein, we determined fatty acid desaturase 1 (FADS1) and FADS2 gene expressions, serum delta-5 desaturase (D5D) and D6D indices in female adults with first-episode schizophrenia after olanzapine medication, as well as their relationship with the incidence of MetS. METHODS: This study prospectively recruited 120 female patients with first-episode schizophrenia who completed 6-month olanzapine medication. Among these female patients, 31 patients developed MetS and 89 patients did not. RESULTS: The mRNA expression levels of FADS1 and FADS2 in patients were analyzed according to the presence of MetS and evaluation times with results of two-way ANOVAs (FADS1: PMetS = 0.0006, Ptime = 0.004, Pinteraction = 0.010; FADS2: PMetS = 0.012, Ptime < 0.0001, Pinteraction = 0.001). The D5D and D6D indices in patients were analyzed according to the presence of MetS and evaluation times with results of two-way ANOVAs (D5D: PMetS = 0.002, Ptime = 0.009, Pinteraction = 0.014; D6D: PMetS = 0.011, Ptime = 0.006, Pinteraction = 0.0001). The SCD-16 and SCD-18 indices in patients were analyzed according to the presence of MetS and evaluation times (SCD-16: PMetS = 0.005, Ptime = 0.009, Pinteraction = 0.016; SCD-18: PMetS = 0.037, Ptime = 0.382, Pinteraction = 0.163). The following multiple comparisons test showed the MetS exhibited reduced FADS1 mRNA expression and D5D index, increased FADS2 mRNA expression and D6D index, concomitant with an enhanced SCD-16 index, compared to the non-MetS did not after 6-month olanzapine medication. CONCLUSION: The study suggests changes of FADS1, FADS2 expressions, and fatty acid desaturase indices including D5D, D6D, and SCD-16 may be associated with the development of MetS in female adults with first-episode schizophrenia after olanzapine medication.

Brain tissue- and cell type-specific eQTL Mendelian randomization reveals efficacy of FADS1 and FADS2 on cognitive function.

Epidemiological studies suggested an association between omega-3 fatty acids and cognitive function. However, the causal role of the fatty acid desaturase (FADS) gene, which play a key role in regulating omega-3 fatty acids biosynthesis, on cognitive function is unclear. Hence, we used two-sample Mendelian randomization (MR) to estimate the gene-specific causal effect of omega-3 fatty acids (N = 114,999) on cognitive function (N = 300,486). Tissue- and cell type-specific effects of FADS1/FADS2 expression on cognitive function were estimated using brain tissue cis-expression quantitative trait loci (cis-eQTL) datasets (GTEx, N ≤ 209; MetaBrain, N ≤ 8,613) and single cell cis-eQTL data (N = 373), respectively. These causal effects were further evaluated in whole blood cis-eQTL data (N ≤ 31,684). A series of sensitivity analyses were conducted to validate MR assumptions. Leave-one-out MR showed a FADS gene-specific effect of omega-3 fatty acids on cognitive function [ß = -1.3 × 10-2, 95% confidence interval (CI) (-2.2 × 10-2, -5 × 10-3), P = 2 × 10-3]. Tissue-specific MR showed an effect of increased FADS1 expression in cerebellar hemisphere and FADS2 expression in nucleus accumbens basal ganglia on maintaining cognitive function, while decreased FADS1 expression in nine brain tissues on maintaining cognitive function [colocalization probability (PP.H4) ranged from 71.7% to 100.0%]. Cell type-specific MR showed decreased FADS1/FADS2 expression in oligodendrocyte was associated with maintaining cognitive function (PP.H4 = 82.3%, respectively). Increased FADS1/FADS2 expression in whole blood showed an effect on cognitive function maintenance (PP.H4 = 86.6% and 88.4%, respectively). This study revealed putative causal effect of FADS1/FADS2 expression in brain tissues and blood on cognitive function. These findings provided evidence to prioritize FADS gene as potential target gene for maintenance of cognitive function.

Reconstruction of Long-Chain Polyunsaturated Acid Synthesis Pathways in Marine Red Microalga Porphyridium cruentum Using Lipidomics and Transcriptomics.

The marine red microalga Porphyridium can simultaneously synthesize long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5, EPA) and arachidonic acid (C20:4, ARA). However, the distribution and synthesis pathways of EPA and ARA in Porphyridium are not clearly understood. In this study, Porphyridium cruentum CCALA 415 was cultured in nitrogen-replete and nitrogen-limited conditions. Fatty acid content determination, transcriptomic, and lipidomic analyses were used to investigate the synthesis of ARA and EPA. The results show that membrane lipids were the main components of lipids, while storage lipids were present in a small proportion in CCALA 415. Nitrogen limitation enhanced the synthesis of storage lipids and ω6 fatty acids while inhibiting the synthesis of membrane lipids and ω3 fatty acids. A total of 217 glycerolipid molecular species were identified, and the most abundant species included monogalactosyldiglyceride (C16:0/C20:5) (MGDG) and phosphatidylcholine (C16:0/C20:4) (PC). ARA was mainly distributed in PC, and EPA was mainly distributed in MGDG. Among all the fatty acid desaturases (FADs), the expressions of Δ5FAD, Δ6FAD, Δ9FAD, and Δ12FAD were up-regulated, whereas those of Δ15FAD and Δ17FAD were down-regulated. Based on these results, only a small proportion of EPA was synthesized through the ω3 pathway, while the majority of EPA was synthesized through the ω6 pathway. ARA synthesized in the ER was likely shuttled into the chloroplast by DAG and was converted into EPA by Δ17FAD.

Contiguous identity between entire coding regions of transgenic and native genes rather than special regions is essential for a strong co-suppression.

The discovery of co-suppression in plants has greatly boosted the study of gene silencing mechanisms, but its triggering mechanism has remained a mystery. In this study, we explored its possible trigger mechanism by using Fatty acid desaturase 2 (FAD2) and Fatty acid elongase 1 (FAE1) strong co-suppression systems. Analysis of small RNAs in FAD2 co-suppression lines showed that siRNAs distributed throughout the coding region of FAD2 with an accumulated peak. However, mutations of the peak siRNA-matched site and siRNA derived site had not alleviated the co-suppression of its transgenic lines. Synthetic FAD2 (AtFAD2sm), which has synonymous mutations in the entire coding region, failed to trigger any co-suppression. Furthermore, 5' and 3' portions of AtFAD2 and AtFAD2sm were swapped to form two hybrid genes, AtFAD2-3sm and AtFAD2-5sm. 80 % and 92 % of their transgenic lines exhibited co-suppression, respectively. Finally, FAE1s with different degrees of the continuous sequence identity compared with AtFAE1 were tested in their Arabidopsis transgenic lines, and the results showed the co-suppression frequency was reduced as their continuous sequence identity stepped down. This work suggests that contiguous identity between the entire coding regions of transgenic and native genes rather than a special region is essential for a strong co-suppression.

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism.

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%-80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

The Specificities of Lysophosphatidic Acid Acyltransferase and Fatty Acid Desaturase Determine the High Content of Myristic and Myristoleic Acids in Cyanobacterium sp. IPPAS B-1200.

The cyanobacterial strain Cyanobacterium sp. IPPAS B-1200 isolated from Lake Balkhash is characterized by high relative amounts of myristic (30%) and myristoleic (10%) acids. The remaining fatty acids (FAs) are represented mainly by palmitic (20%) and palmitoleic (40%) acids. We expressed the genes for lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) and Δ9 fatty acid desaturase (FAD; EC 1.14.19.1) from Cyanobacterium sp. IPPAS B-1200 in Synechococcus elongatus PCC 7942, which synthesizes myristic and myristoleic acids at the level of 0.5-1% and produces mainly palmitic (~60%) and palmitoleic (35%) acids. S. elongatus cells that expressed foreign LPAAT synthesized myristic acid at 26%, but did not produce myristoleic acid, suggesting that Δ9-FAD of S. elongatus cannot desaturate FAs with chain lengths less than C16. Synechococcus cells that co-expressed LPAAT and Δ9-FAD of Cyanobacterium synthesized up to 45% palmitoleic and 9% myristoleic acid, suggesting that Δ9-FAD of Cyanobacterium is capable of desaturating saturated acyl chains of any length.

A mutation in CsDWF7 gene encoding a delta7 sterol C-5(6) desaturase leads to the phenotype of super compact in cucumber (Cucumis sativus L.).

KEY MESSAGE: A novel super compact mutant, scp-3, was identified using map-based cloning in cucumber. The CsDWF7 gene encoding a delta7 sterol C-5(6) desaturase was the candidate gene of scp-3. Mining dwarf genes is important in understanding stem growth in crops. However, only a small number of dwarf genes have been cloned or characterized. Here, we characterized a cucumber (Cucumis sativus L.) dwarf mutant, super compact 3 (scp-3), which displays shortened internodes and dark green leaves with a wrinkled appearance. The photosynthetic rate of scp-3 is significantly lower than that of the wild type. The dwarf phenotype of scp-3 mutant can be partially rescued by the exogenous brassinolide (BL) application, and the endogenous brassinosteroids (BRs) levels in the scp-3 mutant were significantly lower compared to the wild type. Microscopic examination revealed that the reduced internode length in scp-3 resulted from a decrease in cell size. Genetic analysis showed that the dwarf phenotype of scp-3 was controlled by a single recessive gene. Combined with bulked segregant analysis and map-based cloning strategy, we delimited scp-3 locus into an 82.5 kb region harboring five putative genes, but only one non-synonymous mutation (A to T) was discovered between the mutant and its wild type in this region. This mutation occurred within the second exon of the CsGy4G017510 gene, leading to an amino acid alteration from Leu156 to His156. This gene encodes the CsDWF7 protein, an analog of the Arabidopsis DWF7 protein, which is known to be involved in the biosynthesis of BRs. The CsDWF7 protein was targeted to the cell membrane. In comparison to the wild type, scp-3 exhibited reduced CsDWF7 expression in different tissues. These findings imply that CsDWF7 is essential for both BR biosynthesis as well as growth and development of cucumber plants.

Catalytic mechanism of trans-2-enoyl-CoA reductases in the fatty acid elongation cycle and its cooperative action with fatty acid elongases.

The fatty acid (FA) elongation cycle produces very-long-chain FAs with ≥C21, which have unique physiological functions. Trans-2-enoyl-CoA reductases (yeast, Tsc13; mammals, TECR) catalyze the reduction reactions in the fourth step of the FA elongation cycle and in the sphingosine degradation pathway. However, their catalytic residues and coordinated action in the FA elongation cycle complex are unknown. To reveal these, we generated and analyzed Ala-substituted mutants of 15 residues of Tsc13. An in vitro FA elongation assay showed that nine of these mutants were less active than WT protein, with E91A and Y256A being the least active. Growth complementation analysis, measurement of ceramide levels, and deuterium-sphingosine labeling revealed that the function of the E91A mutant was substantially impaired in vivo. In addition, we found that the activity of FA elongases, which catalyze the first step of the FA elongation cycle, were reduced in the absence of Tsc13. Similar results were observed in Tsc13 E91A-expressing cells, which is attributable to reduced interaction between the Tsc13 E91A mutant and the FA elongases Elo2/Elo3. Finally, we found that E94A and Y248A mutants of human TECR, which correspond to E91A and Y256A mutants of Tsc13, showed reduced and almost no activity, respectively. Based on these results and the predicted three-dimensional structure of Tsc13, we speculate that Tyr256/Tyr248 of Tsc13/TECR is the catalytic residue that supplies a proton to trans-2-enoyl-CoAs. Our findings provide a clue concerning the catalytic mechanism of Tsc13/TECR and the coordinated action in the FA elongation cycle complex.

Exogenous alpha-linolenic acid and Vibrio parahaemolyticus induce EPA and DHA levels mediated by delta-6 desaturase to enhance shrimp immunity.

Globally, penaeid shrimp are the most farmed and traded aquatic organisms, although they are easily susceptible to microbial pathogens. Moreover, there is a desire to increase the nutritional value of shrimp, especially the levels of n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which also possess immunomodulatory and anti-inflammatory properties. Some aquatic animals can synthesize EPA and DHA from dietary plant-sourced alpha-linolenic acid (ALA), but penaeid shrimps' ability to synthesize these n-3 PUFAs is unknown. Here, molecular biology techniques, including gas chromatography-mass spectrometry, qPCR, ELISA, etc., were used to demonstrate that exogenous ALA or Vibrio parahaemolyticus could modulate EPA and DHA levels and immune genes in Penaeus vannamei by inducing key enzymes involved in n-3 PUFAs biosynthesis, such as delta desaturases and elongation of very long-chain fatty acid (ELOVLs). Most importantly, knockdown or inhibition of ∆6 desaturase significantly decreased EPA and DHA levels and immune gene expression even with exogenous ALA treatment, consequently affecting shrimp antibacterial immunity and survival. This study provides new insight into the potential of P. vannamei to synthesize n-3 PUFAs from exogenous ALA or upon bacteria challenge, which could be leveraged to increase their nutritional content and antimicrobial immunity.

Engineering the synthesis of unsaturated fatty acids by introducing desaturase improved the stress tolerance of yeast.

BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild-type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry.

Loss of function mutations at the tomato SSI2 locus impair plant growth and development by altering the fatty acid desaturation pathway.

The stearoyl-ACP desaturase (SACPD) is a key enzyme in the regulation of saturated to unsaturated fatty acid ratio, playing a crucial role in regulating membrane stability and fluidity, as well as photosynthesis efficiency, which makes it an important research focus in crop species. This study reports the characterization and molecular cloning of pale dwarf (pad), a new tomato (Solanum lycopersicum L.) T-DNA recessive mutant, which exhibits a dwarf and chlorotic phenotype. Functional studies of the T-DNA tagged gene were conducted, including phylogenetic analysis, expression and metabolomic analyses, and generation of CRISPR/Cas9 knockout lines. The cloning of T-DNA flanking genomic sequences and a co-segregation analysis found the pad phenotype was caused by a T-DNA insertion disrupting the tomato homologue of the Arabidopsis SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY 2 (SlSSI2), encoding a plastid localized isoform of SACPD. The phenotype of CRISPR/Cas9 SlSSI2 knockout lines confirmed that the morphological abnormalities in pad plants were due to SlSSI2 loss of function. Functional, metabolomic and expression analyses proved that SlSSI2 disruption causes deficiencies in 18:1 fatty acid desaturation and leads to diminished jasmonic acid (JA) content and increased salicylic acid (SA) levels. Overall, these results proved that SSI2 plays a crucial role in the regulation of polyunsaturated fatty acid profiles in tomato, and revealed that SlSSI2 loss of function results in an inhibited JA-responsive signalling pathway and a constitutively activated SA-mediated defence signalling response. This study lays the foundation for further research on tomato SACPDs and their role in plant performance and fitness.

Unsaturated fatty acid synthesis in bacteria: Mechanisms and regulation of canonical and remarkably noncanonical pathways.

Unsaturated phospholipid acyl chains are required for membrane function in most bacteria. The double bonds of the cis monoenoic chains arise by two distinct pathways depending on whether oxygen is required. The oxygen-independent pathway (traditionally called the anaerobic pathway) introduces the cis double bond by isomerization of the trans double bond intermediate of the fatty acid elongation cycle. Double bond isomerization occurs at an intermediate chain length (e.g., C10) and the isomerization product is elongated to the C16-C18 chains that become phospholipid monoenoic acyl chains. This pathway was first delineated in Escherichia coli and became the paradigm pathway. However, studies of other bacteria show deviations from this paradigm, the most exceptional being reversal of the fatty acid elongation cycle by a reaction paralleling the initial step in the ß-oxidative degradation of fatty acids. In the oxygen-dependent pathway diiron enzymes called desaturases introduce a double bond into a saturated acyl chain by regioselective cis dehydrogenation through activation of molecular oxygen with an active-site diiron cluster. This difficult hydrogen abstraction from a methylene group often occurs at the midpoint of a saturated fatty acyl chain. In bacteria the acyl chain is a phospholipid acyl chain, and the desaturase is membrane bound. Both the oxygen-independent oxygen-dependent pathways are transcriptionally regulated by repressor and activator proteins that respond to small molecule ligands such as acyl-CoAs. However, in Bacillus subtilis the desaturase is synthesized only at low growth temperatures, a process controlled by a signal transduction regulatory pathway dependent on membrane lipid properties.

The influence of first desaturase subfamily genes on fatty acid synthesis, desiccation tolerance and inter-caste nutrient transfer in the termite Coptotermes formosanus.

Desaturase enzymes play an essential role in the biosynthesis of unsaturated fatty acids (UFAs). In this study, we identified seven "first desaturase" subfamily genes (Cfor-desatA1, Cfor-desatA2-a, Cfor-desatA2-b, Cfor-desatB-a, Cfor-desatB-b, Cfor-desatD and Cfor-desatE) from the Formosan subterranean termite Coptotermes formosanus. These desaturases were highly expressed in the cuticle and fat body of C. formosanus. Inhibition of either the Cfor-desatA2-a or Cfor-desatA2-b gene resulted in a significant decrease in the contents of fatty acids (C16:0, C18:0, C18:1 and C18:2) in worker castes. Moreover, we observed that inhibition of most of desaturase genes identified in this study had a negative impact on the survival rate and desiccation tolerance of workers. Interestingly, when normal soldiers were reared together with dsCfor-desatA2-b-treated workers, they exhibited higher mortality, suggesting that desaturase had an impact on trophallaxis among C. formosanus castes. Our findings shed light on the novel roles of desaturase family genes in the eusocial termite C. formosanus.

Association of the Arabidopsis oleoyl Δ12-desaturase FAD2 with pre-cis-Golgi stacks at endoplasmic reticulum-Golgi-exit sites.

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12-desaturase FAD2 converts oleic (18:1Δ9 ) to linoleic acid (18:2Δ9,12 ) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid-associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence-tagged FAD2 variants in flattened donut-shaped structures of ~0.5-1 µm diameter, in a pattern not resembling mere ER association. High-resolution imaging of coexpressed organellar markers showed fluorescence-tagged FAD2 in a ring-shaped pattern surrounding ER-proximal Golgi particles, colocalizing with pre-cis-Golgi markers. This localization required the unusual C-terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre-cis-Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.