Academic literature on the topic 'Cyclopropane fatty acids (CFA)'

Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under Pi starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under Pi starvation. The cfa2 gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

The formation of cyclopropane fatty acid (CFA) and its role in the acid shock response in Salmonella enterica serovar Typhimurium (S. typhimurium) was investigated. Data obtained by GC/MS demonstrated that the CFA level in S. typhimurium increased upon its entry to the stationary phase, as in other bacteria. The cfa gene encoding CFA synthase was cloned, and mutants of the cfa gene were constructed by allelic exchange. A cfa mutant could not produce CFA and was sensitive to low pH. Introduction of a functional cfa gene into a cfa mutant cell made the mutant convert all unsaturated fatty acids to CFAs and partially restored resistance to low pH. Interestingly, the alternative sigma factor RpoS, which was induced during the stationary phase, affected the production of C19 CFA but not C17 CFA. Western blotting analysis showed that the increase in expression of CFA synthase at early stationary phase was due to the alternative sigma factor RpoS.

It has been known for several decades that cyclopropane fatty acids (CFAs) occur in the phospholipids of many species of bacteria. CFAs are formed by the addition of a methylene group, derived from the methyl group of S-adenosylmethionine, across the carbon-carbon double bond of unsaturated fatty acids (UFAs). The C1 transfer does not involve free fatty acids or intermediates of phospholipid biosynthesis but, rather, mature phospholipid molecules already incorporated into membrane bilayers. Furthermore, CFAs are typically produced at the onset of the stationary phase in bacterial cultures. CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is noteworthy in several respects. It is catalyzed by a soluble enzyme, although one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. The enzyme, CFA synthase, discriminates between phospholipid vesicles containing only saturated fatty acids and those containing UFAs; it exhibits no affinity for vesicles of the former composition. These and other properties imply that topologically novel protein-lipid interactions occur in the biosynthesis of CFAs. The timing and extent of the UFA-to-CFA conversion in batch cultures and the widespread distribution of CFA synthesis among bacteria would seem to suggest an important physiological role for this phenomenon, yet its rationale remains unclear despite experimental tests of a variety of hypotheses. Manipulation of the CFA synthase of Escherichia coli by genetic methods has nevertheless provided valuable insight into the physiology of CFA formation. It has identified the CFA synthase gene as one of several rpoS-regulated genes of E. coli and has provided for the construction of strains in which proposed cellular functions of CFAs can be properly evaluated. Cloning and manipulation of the CFA synthase structural gene have also enabled this novel but extremely unstable enzyme to be purified and analyzed in molecular terms and have led to the identification of mechanistically related enzymes in clinically important bacterial pathogens.

AbstractThe lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.

ABSTRACT Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation is unaffected by mutations in the lon, clpP, andgroEL genes or by depletion of the intracellular ATP pools. It seems likely that CFA synthase is the target of an unidentified energy-independent heat shock regulon protease. This seems to be the first example of heat shock-dependent degradation of a normal biosynthetic enzyme.

ABSTRACTDespite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera:Bacillus,Lysinibacillus,Rummeliibacillus,Brevibacillus,Coprothermobacter,Caloribacterium,Enterococcus,Hydrogenoanaerobacterium, andCellulosimicrobium, within the phylaFirmicutesandActinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed inEscherichia coli. The recombinantE. colishowed relatively higher butanol and isobutanol tolerance thanE. coliwithout thecfagene, suggesting thatcfacan confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

ABSTRACT The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.

ABSTRACT Strains of Escherichia coli carrying mutations at therelA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase. RelA protein catalyzes the synthesis of guanosine-3′,5′-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis. The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification. We report that the effect of RelA on CFA synthesis is indirect. In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity. TherelA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase.

ABSTRACT Fatty acid compositions in growing and resting cells of several strains of Pseudomonas putida (P8, NCTC 10936, and KT 2440) were studied, with a focus on alterations of the saturation degree, cis-trans isomerization, and cyclopropane formation. The fatty acid compositions of the strains were very similar under comparable growth conditions, but surprisingly, and contrary to earlier reports, trans fatty acids were not found in either exponentially growing cells or stationary-phase cells. During the transition from growth to the starvation state, cyclopropane fatty acids were preferentially formed, an increase in the saturation degree of fatty acids was observed, and larger amounts of hydroxy fatty acids were detected. A lowered saturation degree and concomitant higher membrane fluidity seemed to be optimal for substrate uptake and growth. The incubation of cells under nongrowth conditions rapidly led to the formation of trans fatty acids. We show that harvesting and sample preparation for analysis could provoke the enzyme-catalyzed formation of trans fatty acids. Freeze-thawing of resting cells and increased temperatures accelerated the formation of trans fatty acids. We demonstrate that cis-trans isomerization only occurred in cells that were subjected to an abrupt disturbance without having the possibility of adapting to the changed conditions by the de novo synthesis of fatty acids. The cis-trans isomerization reaction was in competition with the cis-to-cyclopropane fatty acid conversion. The potential for the formation of trans fatty acids depended on the cyclopropane content that was already present.

Les acides linoléiques conjugués (CLA) et les acides gras cyclopropanes (CFA) sont deux types d’acides gras à haute valeur ajoutée. Ils présentent un grand intérêt dans divers domaines agroalimentaire ou industriel. Les CLA sont connus pour leurs effets bénéfiques pour la santé, alors que les CFA constituent une bonne source pour la fabrication de lubrifiants, plastiques…après leur hydrogénation. Les CLA sont synthétisés par voie chimique qui génère différents types d’isomères. Les CFA sont produits par les plantes dépendantes du climat et les bactéries présentant naturellement de faibles productivités. Il semble donc intéressant de chercher des alternatives pour leurs productions. Y. lipolytica constitue une bonne alternative. Ceci passe par des modifications génétiques pour permettre ces synthèses et l’étude de l’influence des conditions de culture. Pour cela, les voies de synthèse de ces deux acides gras ainsi que les éléments qui peuvent influencer leurs productions sont à acquérir. Dans ce contexte, nous avons testé plusieurs souches possédant différentes modifications génétiques en erlenmeyer et en fermenteur sur le milieu de néo-synthèse et de bioconversion. Une souche a été sélectionnée permettant la diminution de la dégradation des CLA par le blocage de la voie de la β-oxydation. La meilleure production de CLA en fermenteur a été obtenue en présence d’huile de soja et grâce à la surexpression du gène FAD2 (302 mg. L-1). L’influence de la composition et des paramètres de culture a été étudiée. Les résultats ont montré une meilleure assimilation de la peptone de Soja Dynamis riche en acides aminés libres par la levure qui conduit à une productivité améliorée. Ceci par son effet positif sur la croissance et les productions. De plus, la stratégie Fed-batch en fermenteur a permis la production optimale de CLA (0,4 g.L-1), la limitation en azote et phosphore semble affecter leur accumulation. D’autres souches dites de premières générations productrices de CFA grâce à l’expression du gène de la CFAs d’E.coli ont été testées sur différents milieux de culture. Deux souches (GY1005 et GY1070) ont été retenues pour leur production maximale d’huile et/ou de CFA sur le milieu de néo-synthèse en fermenteur. Ceci grâce à la surexpression des gènes DGA2 et GPD1 permettant une forte accumulation de lipides et l’absence de remobilisation et de dégradation des acides gras. Les paramètres et la composition du milieu de culture tels qu’une forte agitation (1 000 rpm) et une stratégie Fed-batch en utilisant une solution de même composition que le milieu de départ ont permis de maximiser la production de CFA (1,2 g.L-1 pour GY1070 et 0,9 g.L-1 pour GY1005). Une souche dite de deuxième génération (JMY5578) a été optimisée génétiquement (expression du gène de la CFAs sous le contrôle d’un promoteur plus fort et l’expression du gène LRO1). Cette souche a été testée en erlenmeyer et en fermenteur dans le but d’évaluer l’influence des composés du milieu de culture sur la production de CFA. Les résultats ont montré un gain de 30% de CFA et 6% de biomasse en présence des chlorures de magnésium et des vitamines dans le milieu de culture. De plus, un ajout en continu de dextrose et un milieu de départ riche en azote et phosphore a permis une production de 3 g.L-1 de CFA. Nous avons étudié l’impact des promoteurs sur l’expression du gène de la CFAs dans des souches de 2éme génération. L’expression du gène sous le contrôle du promoteur php8d a donné le meilleur résultat en termes d’accumulation des CFA dans la souche JMY6068 (46% dans les lipides totaux) ; il semble être le promoteur le plus fort en comparaison avec TEF
Conjugated linoleic acids (CLA) and cyclopropane fatty acids (CFA) are two high value added products. They are of a great interest in various agri-food or industrial fields. CLAs are known for their health benefits, while CFAs, after their hydrogenation are a good source for the manufacture of lubricants, plastics.... CLAs are synthesized chemically which generates different types of isomers. CFAs are produced by climate-dependent plants and by bacteria with low productivity. It seems therefore interesting to look for alternatives for their production. Y. lipolytica is a good alternative. This involves genetic modifications to enable these syntheses and the study of the influence of culture conditions. For this purpose, synthetic pathways of these two fatty acids as well as the elements that can influence their production are to be acquired. In this context, we tested several strains with different genetic background in flask and fermenter on neo-synthesis and bioconversion media. One strain was selected allowing reduction of CLA degradation by blocking the β-oxidation pathway and the better production of CLAs in fermenter with soybean oil by overexpression of FAD2 (302 mg. L-1). The influence of media composition was studied. The results showed better assimilation of Dynamis soy peptone rich in free amino acids by this yeast. This has a positive effect on growth and productions. In addition, the Fed-batch fermenter strategy allowed the optimal production of CLA (0.4 g. L-1), and nitrogen and phosphorus limitations seems to affect their accumulation. First generation strains producing CFA by expression of the CFA gene of E. coli were studied. They were tested on different culture media. Two strains (GY1005 and GY1070) were retained for their maximum oil production and/or CFA accumulation on neosynthesis medium in fermenter. This is due to DGA2 and GPD1 genes overexpression allowing a strong lipid accumulation and in the absence of remobilization and degradation of fatty acids. Media composition and culture parameters, such as strong stirring (1 000 rpm) and a Fed-batch strategy using a solution with the same composition as the starting medium, made it possible to maximize CFA production (1.2 g. L-1 for GY1070 and 0.9 g. L-1 for GY1005). A so-called second generation strain (JMY5578) was genetically optimized (CFA gene was expressed under the control of a stronger promoter and LRO1 over-expression gene). This strain was tested in flask and fermenter in order to evaluate the influence of culture medium compounds on CFA production. The results showed a gain of 30% CFA and 6% biomass in the presence of magnesium chloride and vitamins in the culture medium. In addition, a continuous addition of dextrose in fermenter and a nitrogen and phosphorus-rich starting medium allowed production of 3 g.L-1 of CFA. We also looked at the effect of promoters for the CFA gene expression in secondgeneration strains. Expression of the gene under the control of the php8d promoter gave the best result in terms of CFA accumulation in JMY6068 strain (46% in total lipids) and appears to be the strongest promoter in comparison with TEF

La tuberculose est l’une des maladies les plus mortelles dans le monde. L'apparition de souches de Mycobacterium tuberculosis résistantes aux antibiotiques actuellement utilisés, impose de trouver de nouvelles cibles et de nouveaux antituberculeux. Les enzymes responsables de la biosynthèse des acides mycoliques, composants essentiels de la paroi des mycobactéries, constituent de très bonnes cibles thérapeutiques, et en particulier, les Cyclopropane Synthase (CS) de Mycobacterium tuberculosis. La Cyclopropane Fatty Acid Synthase (CFAS) d’Escherichia coli est une enzyme homologue de ces CS et constitue un très bon modèle d’étude pour élucider le mécanisme enzymatique des CS et pour trouver de nouveaux agents antituberculeux. En effet, les CS de M. Tuberculosis ne sont pas fonctionnelles in vitro. La CFAS transfère le méthyle de la S-adénosyl-L-méthionine vers une double liaison non activée de phospholipides insaturés pour former le motif cyclopropane. Les travaux réalisés, au cours de cette thèse, ont permis de mieux comprendre le mécanisme réactionnel de la CFAS : En utilisant diverses approches (mutagenèse dirigée, échanges isotopiques, mesures d'effets isotopiques cinétiques…) nous avons pu préciser quelques points concernant le mécanisme réactionnel de cette réaction. En ce qui concerne la recherche d’inhibiteurs, grâce à un test colorimétrique mis au point au laboratoire nous avons pu cribler la chimiothèque de l’ICSN, et nous avons identifié quelques inhibiteurs de faibles IC50. Le mode d'action de certain de ces inhibiteurs a été précisé. D'autre part, nous avons montré qu'un des inhibiteur inhibe la cyclopropanation in vivo chez E. Coli. C'est le premier inhibiteur actif contre la CFAS in vivo. Une méthode de synthèse de cet inhibiteur et d'analogues a été mise au point et permettra de faire des études de structure-activité afin d'identifier des inhibiteurs plus puissants de la CFAS.

Ce travail de recherche portait sur l'extraction d'huile microbienne, notamment de certains actifs inhabituels comme les acides gras cyclopropanes, à partir de la levure oléagineuse Yarrowia lipolytica. L'objectif était de développer un itinéraire d'extraction en voie humide afin de réduire le coût global de production de cette huile microbienne. Pour cela, des pré-traitements de la matière ont été mis en œuvre (pressage, champs électriques pulsés, décharges électriques de haute tension, ultrasons et homogénéisation haute pression) afin de lyser les cellules. L'impact de ces technologies sur le rendement en huile extraite et le profil en acides gras a été étudié. Le criblage, en voie sèche, de ces différents procédés a permis de sélectionner les deux techniques les plus efficaces à approfondir : les ultrasons et l'homogénéisation haute pression (HHP). Afin d'améliorer leur efficacité, les ultrasons ont été appliqués directement en voie humide en utilisant une suspension de levures dans un solvant. Les paramètres clés du traitement ont été optimisés. Ainsi, le même rendement d'extraction d'huile a pu être obtenu en voie humide comparativement à la voie sèche. L'HHP a tout d'abord été étudiée en voie sèche afin de déterminer les valeurs optimales des paramètres opératoires. Par la suite, la faisabilité d'une extraction en voie humide a été démontrée. Une suspension a subi un pré-traitement à 150 MPa à température ambiante pendant 5 passages puis a été extraite dans un solvant à l'aide d'un disperseur à haute vitesse. Le rendement maximal obtenu par cette méthode était très proche de celui de l'extraction voie sèche qui permettait de récupérer la totalité de l'huile
This research work was focused on microbial oil extraction, especially unusual molecules such as cyclopropane fatty acids, from the oleaginous yeast Yarrowia lipolytica. The aim was to develop a wet extraction route in order to reduce the overall cost of microbial oil production. Therefore, biomass pretreatments were studied (mechanical pressing, pulsed electric fields, high voltage electrical discharges, ultrasounds and high pressure homogenization) in order to disrupt the cell wall. The effect of these technologies on oil extraction and fatty acid profile was investigated. Screening of these technologies in dry route allowed the selection of two effective techniques; ultrasounds and high pressure homogenization, which were further studied. ln order to enhance their efficiency, ultrasounds were directly applied in a wet route (yeast suspension in an extracting solvent). The key parameters were optimized and, thus, the same yield was obtained for both wet and dry routes. High pressure homogenization was studied in dry route in order to determine the optimum values for pressure, the number of passes and the temperature. The feasibility of a wet route extraction has been then demonstrated. A yeast suspension was treated by high pressure homogenization at 150 MPa, room temperature, and for 5 passes. Oil was then extracted in a solvent using a high speed disperser. Following this methodology, the maximum oil recovery yield was very close to the yield reached in the dry route

Chapter 2 In solution, py(mi)ridinols 1.33, 1.34 and 1.35 are 2-, 5- and 28-fold more reactive antioxidants, respectively, than α-TOH (the most potent lipid-soluble antioxidant in nature). In order to develop a highly-reactive fluorescent indicator of lipid peroxidation in cells, we sought to couple these antioxidants with boron-dipyrro- methene (BODIPY) dyes, such that the resulting conjugates will display a significant fluorecence enhancement upon oxidation. This chapter details efforts towards the synthesis of these compounds. Chapter 3 Lipoxygenases are a family of important enzymes that catalyze the dioxygenation of arachidonic acid to yield a variety of potent lipid mediators that have been implicated in the pathogenesis of numerous degenerative conditions. We have undertaken a preliminary study of the effect of replacing the unsaturation in the related polyunsaturated lipid linoleic acid with cyclopropane rings on both the oxidizability of the lipid, as well as lipoxygenase’s ability to utilize it as a substrate. We anticipate that these analogs will be useful in co-crystallization studies with the enzyme that will provide unique insight into substrate acquisition, binding and the necessary conformation for catalysis.
Thesis (Master, Chemistry) -- Queen's University, 2011-11-10 16:15:05.643