scholarly journals Genetic suppression of lethal mutations in fatty acid biosynthesis mediated by a secondary lipid synthase

2021 ◽  
Author(s):  
Marco N. Allemann ◽  
Eric E. Allen
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Biosynthetic Pathways ◽  
Omega 3 ◽  
Genetic Redundancy ◽  
Acid Biosynthesis ◽  
Malonyl Coa

The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes is a unique feature of certain marine Gammaproteobacteria inhabiting high-pressure and/or low temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated Type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as EPA (20:5n-3) and DHA (22:6n-3) are produced by a hybrid polyketide/fatty acid synthase – encoded by the pfa genes - also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by several-fold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD, malonyl CoA-acyl carrier protein transacylase, coding sequence. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory fatty acid biosynthetic pathways in select marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. Importance A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights have been gained into the interactions between these two biosynthetic pathways. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be non-essential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.

Effect of Thiolactomycin on de novo Fatty Acid Biosynthesis in Plants

1990 ◽  
Vol 45 (5) ◽  
pp. 518-520 ◽  
Author(s):  
Manfred Focke ◽  
Andrea Feld ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acetate Incorporation ◽  
Acid Biosynthesis ◽  
Acetyl Coa ◽  
Malonyl Coa ◽  
Total Fatty Acids

Thiolactomycin was shown to be a potent inhibitor of de novo fatty acid biosynthesis in intact isolated chloroplasts (measured as [14C]acetate incorporation into total fatty acids). In our attempt to further localize the inhibition site we confirmed the inhibition with a fatty acid synthetase preparation, measuring the incorporation of [14C]malonyl-CoA into total fatty acids. From the two proposed enzymic targets of the fatty acid synthetase by thiolactomycin we could exclude the acetyl-CoA: ACP transacetylase. It appears that the inhibition by thiolactomycin occurs on the level of the condensing enzymes, i.e. the 3-oxoacyl-ACP synthases. We also demonstrated that the two starting enzymes of de novo fatty acid biosynthesis, the acetyl-CoA synthetase and the acetyl-CoA carboxylase, are not affected by thiolactomycin.

scholarly journals A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism

Open Biology ◽  
2017 ◽  
Vol 7 (2) ◽  
pp. 160277 ◽  
Author(s):  
Matías Cabruja ◽  
Sonia Mondino ◽  
Yi Ting Tsai ◽  
Julia Lara ◽  
Hugo Gramajo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Biosynthesis ◽  
Mycolic Acids ◽  
Storage Lipids ◽  
Conditional Mutant

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo , we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C 12 to C 18 acyl-CoAs, but not of long-chain acyl-CoAs (C 19 to C 24 ). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

scholarly journals The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue

1975 ◽  
Vol 146 (2) ◽  
pp. 425-437 ◽  
Author(s):  
P J Weaire ◽  
R G Kekwick
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Gradient Centrifugation ◽  
Fatty Acid Synthetase ◽  
Synthetase Activity ◽  
Acid Biosynthesis ◽  
Avocado Fruit ◽  
Malonyl Coa ◽  
Fatty Acid Synthetase Activity

1. Plastid and mitochondrial preparations were obtained by density-gradient centrifugation of homogenates made by gentle disintergration of avocado fruit mesocarp and cauliflower bud tissue. 2. The mitochondrial preparations had respiratory activity but did not incorporate [1-14C]acetate into fatty acids. 3. The plastid preparations incorporated [1-14C]acetate into the range of fatty acids found in the parent tissue. No fatty acid synthetase activity could be detected in the 12000g supernatant of these homogenates. 4. Homogenates produced by rupture of the tissue in an Ato-Mix blender and plastid preparations disintegrated by ultrasonic treatment both had fatty acid synthetase activity which did not sediment at 105000g and which formed mainly [14-C]stearate from [2-14C]malonyl-CoA. 5. It is concluded that the plastids are the principal site of fatty acid biosynthesis in the tissues studied.

Notes: Inhibition of the Acetyl-CoA Carboxylase of Barley Chloroplasts by Cycloxydim and Sethoxydim

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1361-1363 ◽  
Author(s):  
Manfred Focke ◽  
Hartmut K. Lichtenthaler
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enzyme Preparation ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acetyl Coa Carboxylase ◽  
Acid Biosynthesis ◽  
Hordeum Vulgare L ◽  
Acetyl Coa ◽  
Malonyl Coa

The effect of the three cyclohexane-1,3-dione derivatives cycloxydim, sethoxydim and clethodim on the incorpora­tion of 14C-labelled acetate, malonate. acctyl-CoA or malonyl-CoA into fatty acids was studied in an enzyme preparation isolated from barley chloroplasts (Hordeum vulgare L. var. “Alexis”). The herbicides cycloxydim, clethodim and sethoxydim block the de novo fatty acid biosynthesis from [2-14C]acetatc and [1-14C]acetyl-CoA, whereas that of [2-14C]malonatc and [2-14C)malonyl-CoA is not affected. The data indicate that the mode of action of the cyclohexane-1,3-dione derivatives in the sensitive bar­ley plant consists in the inhibition of de novo fatty acid biosynthesis by blocking the acetyl-CoA carboxylase (EC 6.4.1.2.).

scholarly journals Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C20 Fatty Acids to C60-to-C90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

2008 ◽  
Vol 74 (16) ◽  
pp. 5078-5085 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipoic Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Synthesis ◽  
Acid Biosynthesis ◽  
Mycolic Acids

ABSTRACT We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C20 fatty acids to form C60-to-C90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C4 to C8) than was previously thought (>C12). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

scholarly journals Elongation of fatty acids by microsomal fractions from the brain of the developing rat

1975 ◽  
Vol 152 (3) ◽  
pp. 495-501 ◽  
Author(s):  
P J Brophy ◽  
D E Vance
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Rat Brain ◽  
Microsomal Fraction ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Soluble Fraction ◽  
Residual Activity ◽  
Acid Biosynthesis ◽  
Malonyl Coa

Elongation of fatty acids by microsomal fractions obtained from rat brain was measured by the incorporation of [2-14C]malonyl-CoA into fatty in the presence of palmitoyl-CoA or stearoyl-CoA. 2. Soluble and microsomal fractions were prepared from 21-day-old rats; density gradient centrifugation demonstrated that the stearoyl-CoA elongation system was localized in the microsomal fraction whereas fatty acid biosynthesis de novo from acetyl-CoA occurred in the soluble fraction. The residual activity de novo in the microsomal fraction was attributed to minor contamination by the soluble fraction. 3. The optimum concentration of [2-14C]malonyl-CoA for elongation of fatty acids was 25 mum for palmitoyl-CoA or stearoyl-CoA, and the corresponding optimum concentrations for the two primer acyl-CoA esters were 8.0 and 7.2 muM respectively. 4. Nadph was the preferred cofactor for fatty acid formation from palmitoyl-CoA or stearoyl-CoA, although NADH could partially replace it. 5. The stearoyl-CoA elongation system required a potassium phosphate buffer concentration of 0.075M for maximum activity; CoA (1 MUM) inhibited this elongation system by approx. 30%. 6. The fatty acids formed from malonyl-CoA and palmitoyl-CoA had a predominant chain length of C18 whereas stearoyl-CoA elongation resulted in an even distribution of fatty acids with chain lengths of C20, C22 and C24. 7. The products of stearoyl-CoA elongation were identified as primarily unesterified fatty acids. 8. The developmental pattern of fatty acid biosynthesis by rat brain microsomal preparations was studied and both the palmitoyl-CoA and stearoyl-CoA elongation systems showed large increases in activity between days 10 and 18 after birth.

Fatty acid biosynthesis in developing fetal lung

1989 ◽  
Vol 257 (4) ◽  
pp. L195-L201 ◽  
Author(s):  
S. A. Rooney
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Fetal Lung ◽  
Fetal Life ◽  
Acid Biosynthesis ◽  
Ample Evidence ◽  
Phosphatidylcholine Biosynthesis

Fatty acids are integral components of glycerolipids and hence of the phosphatidylcholine-rich pulmonary surfactant. There is ample evidence that the lung is able to synthesize fatty acids de novo. Toward the end of gestation as the fetus prepares for life outside the uterus, there is a surge in phosphatidylcholine synthesis. At the same time there is an increase in de novo fatty acid biosynthesis as well as in the activity of fatty acid synthase, the enzyme that catalyzes the final steps in fatty acid synthesis. Glucocorticoids have long been known to accelerate phosphatidylcholine biosynthesis in the fetal lung and they have also been found to stimulate fatty acid biosynthesis and fatty acid synthase activity. In fact, fatty acid synthase is the first, and so far the only, enzyme involved in lipid biosynthesis to be clearly identified as glucocorticoid inducible in fetal lung. De novo fatty acid biosynthesis may have two important roles relating to surfactant production during late fetal life. In addition to providing fatty acids for incorporation into surfactant phospholipids, recent data suggest that fatty acids may also directly regulate phosphatidylcholine biosynthesis by activation of choline-phosphate cytidylyltransferase, an enzyme catalyzing a rate-limiting step in its biosynthetic pathway.

scholarly journals Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway

2020 ◽  
Author(s):  
Marco N. Allemann ◽  
Eric E. Allen
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Genetic Regulation ◽  
Transcriptional Regulator ◽  
Acid Synthesis ◽  
Biosynthesis Pathway ◽  
Omega 3

AbstractA characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. Biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is accomplished using a polyketide/fatty acid synthase mechanism encoded by a set of five genes pfaABCDE. This unique fatty acid synthesis (FAS) pathway co-exists with the canonical Type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is down regulated in response to exogenous fatty acids, particularly long chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site.ImportanceThe production of polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid responsive transcriptional regulator specific to the PUFA biosynthesis pathway.

scholarly journals Serratia symbiotica Enhances Fatty Acid Metabolism of Pea Aphid to Promote Host Development

2021 ◽  
Vol 22 (11) ◽  
pp. 5951
Author(s):  
Xiaofei Zhou ◽  
Xiaoyu Ling ◽  
Huijuan Guo ◽  
Keyan Zhu-Salzman ◽  
Feng Ge ◽  
...  
Keyword(s):  
Body Weight ◽  
Fatty Acid ◽  
Myristic Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Pea Aphids ◽  
Serratia Symbiotica ◽  
Aphid Host ◽  
Host Development

Bacterial symbionts associated with insects are often involved in host development and ecological adaptation. Serratia symbiotica, a common facultative endosymbiont harbored in pea aphids, improves host fitness and heat tolerance, but studies concerning the nutritional metabolism and impact on the aphid host associated with carrying Serratia are limited. In the current study, we showed that Serratia-infected aphids had a shorter nymphal developmental time and higher body weight than Serratia-free aphids when fed on detached leaves. Genes connecting to fatty acid biosynthesis and elongation were up-regulated in Serratia-infected aphids. Specifically, elevated expression of fatty acid synthase 1 (FASN1) and diacylglycerol-o-acyltransferase 2 (DGAT2) could result in accumulation of myristic acid, palmitic acid, linoleic acid, and arachidic acid in fat bodies. Impairing fatty acid synthesis in Serratia-infected pea aphids either by a pharmacological inhibitor or through silencing FASN1 and DGAT2 expression prolonged the nymphal growth period and decreased the aphid body weight. Conversely, supplementation of myristic acid (C14:0) to these aphids restored their normal development and weight gain. Our results indicated that Serratia promoted development and growth of its aphid host through enhancing fatty acid biosynthesis. Our discovery has shed more light on nutritional effects underlying the symbiosis between aphids and facultative endosymbionts.

scholarly journals The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis

2002 ◽  
Vol 364 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Sabine D'ANDREA ◽  
Hervé GUILLOU ◽  
Sophie JAN ◽  
Daniel CATHELINE ◽  
Jean-Noël THIBAULT ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Biosynthesis ◽  
Chain Polyunsaturated Fatty Acid ◽  
Acid Biosynthesis ◽  
Highly Unsaturated Fatty Acids ◽  
Long Chain ◽  
Δ6 Desaturase

The recently cloned Δ6-desaturase is known to catalyse the first step in very-long-chain polyunsaturated fatty acid biosynthesis, i.e. the desaturation of linoleic and α-linolenic acids. The hypothesis that this enzyme could also catalyse the terminal desaturation step, i.e. the desaturation of 24-carbon highly unsaturated fatty acids, has never been elucidated. To test this hypothesis, the activity of rat Δ6-desaturase expressed in COS-7 cells was investigated. Recombinant Δ6-desaturase expression was analysed by Western blot, revealing a single band at 45kDa. The putative involvement of this enzyme in the Δ6-desaturation of C24:5n-3 to C24:6n-3 was measured by incubating transfected cells with C22:5n-3. Whereas both transfected and non-transfected COS-7 cells were able to synthesize C24:5n-3 by elongation of C22:5n-3, only cells expressing Δ6-desaturase were also able to produce C24:6n-3. In addition, Δ6-desaturation of [1-14C]C24:5n-3 was assayed invitro in homogenates from COS-7 cells expressing Δ6-desaturase or not, showing that Δ6-desaturase catalyses the conversion of C24:5n-3 to C24:6n-3. Evidence is therefore presented that the same rat Δ6-desaturase catalyses not only the conversion of C18:3n-3 to C18:4n-3, but also the conversion of C24:5n-3 to C24:6n-3. A similar mechanism in the n-6 series is strongly suggested.

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