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We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the ω3/6 Δ 8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Δ 9 -specific elongating activity from Isochrysis galbana , a Δ 8 -desaturase from Euglena gracilis and a Δ 5 -desaturase from Mortierella alpina . Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Δ 9 -elongating activity, which may bypass rate-limiting steps present in the conventional Δ 6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

Biosynthesis of free fatty acids (FFAs) in mammals is pivotal for metabolic homeostasis, yet a comprehensive understanding of tissue‐specific biosynthesis and inter‐tissue crosstalk of FFAs remains incomplete. In vivo stable‐isotope tracing metabolomics is utilized to comprehensively measure FFA biosynthesis and inter‐tissue crosstalk in mice. Systematically assessing tissue‐specific biosynthesis of 13 FFAs across 15 tissues unveils dynamic spatial and temporal accumulation and redistribution of FFAs throughout the body. Employing an analytical framework to deconvolve mass isotopologue patterns, inter‐tissue crosstalk is explored for saturated, polyunsaturated, and monounsaturated FFAs, and quantify communications of FFA (16:0) and FFA (18:0) between the liver and other tissues. Then, a decline in fatty acid biosynthesis in peripheral tissues but not in the brain of aged mice is observed, particularly evident in palmitic acid and monounsaturated fatty acids. These findings illuminate the complex interplay between tissue‐specific fatty acid biosynthesis and the maintenance of metabolic homeostasis. Biosynthesis rates of 13 FFAs are quantitatively calculated in 15 tissues from glucose and an analytical framework to quantitatively calculate inter‐tissue communications of fatty acids between the liver and other tissues is developed. Moreover, the approach is extended to aging condition and a system‐wide decline of fatty acid synthesis in peripheral tissues but not in the brain of aged mice is revealed.

ObjectiveIncreased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC).DesignWe investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies.ResultsAblation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture.ConclusionOur study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

Fatty acids (FAs) are a class of diverse biomolecules that are essential for a wide variety of biological processes. Recently, ω-3 and ω-6 polyunsaturated FAs (PUFAs) have received a lot of attention for their benefit and essentiality to human health. Their demand is constantly increasing alongside the ever-decreasing availability of sources of natural PUFAs, such as some plants and marine ecosystems, which are now endangered by agribusiness and climate change. Most of the novel microbial synthetic platforms to produce ω-3 and ω-6 PUFAs (e.g. yeast, microalgae, oleaginous microorganisms, bacteria etc.) are costly. On the other hand, industrial processes based on chemical total synthesis of PUFAs is often challenging. Here, we propose an innovative and bio-sustainable method to make ω-3 and ω-6 PUFAs by building a cell-factory in Crithidia fasciculata , using genetic manipulation of endogenous FA desaturases and elongases, supplementation of the culture media with cheap sources of fats (such as used cooking oils), and culturing temperature reduction. In these unusual environments, C. fasciculata show high adaptability: the cells grow rapidly producing large biomass from small volumes of culture, scavenge and remodel unusual fat sources, as well as biosynthesising high value FAs and PUFAs de novo . By genetic manipulation, allowing overexpression of either the endogenous Δ6-desaturases or Δ4-desaturases, in conjunction with the endogenous elongase Elo4, high value ω-6 and ω-3 PUFAs were yielded to a maximum of 50% of the total FA content of the cells. These bioengineered C. fasciculata provide a low-cost and bio-sustainable platform to increase the production of essential PUFAs which can be introduced into the food chain at lower costs.

Identification of specific and therapeutically actionable vulnerabilities, ideally present across multiple mutational backgrounds, is needed to improve acute myeloid leukemia (AML) patients’ outcomes. We identify stearoyl-CoA desaturase (SCD), the key enzyme in fatty acid (FA) desaturation, as prognostic of patients' outcomes and, using the clinical-grade inhibitor SSI-4, show that SCD inhibition (SCDi) is a therapeutic vulnerability across multiple AML models in vitro and in vivo. Multiomic analysis demonstrates that SCDi causes lipotoxicity, which induces AML cell death via pleiotropic effects. Sensitivity to SCDi correlates with AML dependency on FA desaturation regardless of mutational profile and is modulated by FA biosynthesis activity. Finally, we show that lipotoxicity increases chemotherapy-induced DNA damage and standard chemotherapy further sensitizes AML cells to SCDi. Our work supports developing FA desaturase inhibitors in AML while stressing the importance of identifying predictive biomarkers of response and biologically validated combination therapies to realize their full therapeutic potential.

The utilization of natural products in food preservation represents a promising strategy for the dual benefits of controlling foodborne pathogens and enhancing the nutritional properties of foods. Among the phytonutrients, flavonoids have been shown to exert antibacterial effects by disrupting bacterial cell membrane functionality; however, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of quercetin on the cell membrane permeability of Staphylococcus aureus ATCC 27217. A combined metabolomic and transcriptomic approach was adopted to examine the regulatory mechanism of quercetin with respect to the fatty acid composition and associated genes. Kinetic analysis and molecular docking simulations were conducted to assess quercetin’s inhibition of β-ketoacyl-acyl carrier protein reductase (FabG), a potential target in the bacterial fatty acid biosynthesis pathway. Metabolomic and transcriptomic results showed that quercetin increased the ratio of unsaturated to saturated fatty acids and the levels of membrane phospholipids. The bacteria reacted to quercetin-induced stress by attempting to enhance fatty acid biosynthesis; however, quercetin directly inhibited FabG activity, thereby disrupting bacterial fatty acid biosynthesis. These findings provide new insights into the mechanism of quercetin’s effects on bacterial cell membranes and suggest potential applications for quercetin in bacterial inhibition.

In the present two-year study, an attempt was made to estimate the grain yield, grain nutrient uptake, and oil quality of three commonly grown maize (Zea mays L.) hybrids fertilized with varied levels of nitrogen (N), phosphorus (P) and potassium (K). Results obtained from both the experimental years indicated that application of 125% of recommended dose of fertilizer (RDF) recorded maximum grain yield (10.37 t ha-1; 124% higher than control). When compared with 100% RDF, grain yield reduction with nutrient omission was 44% for N omission, 17% for P omission, and 27% for K omission. Nitrogen uptake was increased with increasing NPK levels up to 150% RDF that was statistically at par (p ≥ 0.01) with 125% RDF. Increasing trend in P and K uptake was observed with successive increase in NPK levels up to 125% RDF, above which it declined. The protein content was significantly higher in grains of var. P 3396 with 125% RDF. Nutrient management has significant (p ≤ 0.01) role in the grain oil content. Saturated fatty acids (palmitic, stearic and arachidic acid) content decreased, and unsaturated fatty acid (oleic, linoleic and linolenic acid) increased with increasing NPK levels. The average oleic acid desaturation and linoleic acid desaturation ratios were increased with increasing NPK levels up to 100 and 125% RDF, respectively. However, average monounsaturated fatty acids (MUFA): poly-unsaturated fatty acids (PUFA), saturated: unsaturated as well as linoleic: linolenic acid ratios were increased on receiving 75% RDF, and beyond that it showed decreasing trend. The omission of K had the highest inhibitory effect on corn oil quality followed by N and P omission.

Fatty acid biosynthesis in α- and γ-proteobacteria requires two functionally distinct dehydratases, FabA and FabZ. Here, mechanistic cross-linking facilitates the structural characterization of a stable hexameric complex of six Escherichia coli FabZ dehydratase subunits with six AcpP acyl carrier proteins. The crystal structure sheds light on the divergent substrate selectivity of FabA and FabZ by revealing distinct architectures of the binding pocket. Molecular dynamics simulations demonstrate differential biasing of substrate orientations and conformations within the active sites of FabA and FabZ such that FabZ is preorganized to catalyze only dehydration, while FabA is primed for both dehydration and isomerization.

Doc number: 113 Abstract Background: High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs. Results: Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response. Conclusions: A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of Gram-negative bacteria, and its levels are tightly co-regulated with phospholipid (PL) amounts. This homeostatic regulation necessitates the involvement of numerous genes, including lapD in a poorly defined manner. To understand the function of LapD, we took advantage of the synthetic lethal phenotype conferred by the concomitant absence of LapD and myristoyltransferase LpxM or heptosyltransferase WaaC and isolated extragenic suppressors that could bypass this lethality. Suppressor analyses of Δ(lapD lpxM) bacteria identified five single amino acid exchanges in AccA and two in each of AccC and AccD. These proteins comprise different subunits of the acetyl-CoA carboxylase complex, which catalyzes the rate-limiting step in the initiation of fatty acid synthesis, mediating the conversion of acetyl-CoA to malonyl-CoA. Fatty acid analysis revealed that these mutations restored the ratio of saturated to unsaturated fatty acids and repressed elevated PL levels. Suppressor analyses of Δ(lapD waaC) identified a single amino acid substitution in LptD, which is required for LPS assembly in the OM, and in NlpI, which regulates the amount of peptidoglycan hydrolase MepS. These results posit LapD as the point of critical regulation of homeostatic control of three essential cell envelope components.