In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli

Abstract Background Hydroxy fatty acids are widely used in food, chemical and cosmetic industries. A variety of dihydroxy fatty acids have been synthesized so far; however, no studies have been done on the synthesis of 9,10-dihydroxyhexadecanoic acid. In the present study recombinant E. coli has been used for the heterologous expression of fatty acid hydroxylating enzymes and the whole cell lysate of the induced culture was used for in vitro production of 9,10-dihydroxyhexadecanoic acid. Results A first of its kind proof of principle has been successfully demonstrated for the production of 9,10-dihydroxyhexadecanoic acid using three different enzymes viz. fatty acid desaturase (FAD) from Saccharomyces cerevisiae, epoxide hydrolase (EH) from Caenorhabditis elegance and epoxygenase (EPOX) from Stokasia laevis. The genes for these proteins were codon-optimised, synthesised and cloned in pET 28a (+) vector. The culture conditions for induction of these three proteins in E. coli were optimised in shake flask. The induced cell lysates were used both singly and in combination along with the trans-supply of hexadecanoic acid and 9-hexadecenoic acid, followed by product profiling by GC–MS. Formation of 9,10-dihydroxyhexadecanoic acid was successfully achieved when combination of induced cell lysates of recombinant E. coli containing FAD, EH, and EPOX were incubated with 9-hexadecenoic acid. Conclusions The in vitro production of 9,10-dihydroxyhexadecanoic acid synthesis using three fatty acid modification genes from different sources has been successfully demonstrated. The strategy adopted can be used for the production of similar compounds

Crucial role of cytosolic tryparedoxin peroxidase in Leishmania donovani survival, drug response and virulence

Leishmania donovani, the causative agent of visceral leishmaniasis, uses a cascade of enzymes that include cytosolic tryparedoxin peroxidase (cTXNPx) for detoxification of peroxides, an event pivotal for survival of digenic parasites living in two disparate biological environments. In this study, we observed an increase in promastigote cTXNPx levels after exposure to H2O2 and this group did not show any cell death; however, exposure to a combination of H2O2 and nitric oxide resulted in significant reduction of cTXNPx levels accompanied by high cell death. The protective relationship between higher levels of cTXNPx and survival was further substantiated by the improved ability of L. donovani promastigotes overexpressing cTXNPx to withstand exposure to H2O2 and nitric oxide combination as compared with vector transfectants. In addition, cTXNPx transfectants demonstrated increased virulence, causing higher parasite burden in macrophages as compared with vector transfectants. Interestingly, the cTXNPx transfectants as promastigotes or amastigotes were resistant to clearance by the anti-leishmanial drug antimony, suggesting a cTXNPx link to drug response. Mechanistically, cTXNPx overexpression was protective against changes in Ca2+ homeostasis but not against mitochondrial hyperpolarization brought about by exposure to H2O2 and nitric oxide. Therefore, this study provides a link between cTXNPx expression to survival, virulence and drug response in L. donovani

MOESM3 of In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli

Additional file 3: Figure S1. Gas-chromatogram of pure 9,10-Dihydroxyhexadecanoic acid (A) and the mass spectrum of the same (B)

MOESM2 of In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli

Additional file 2: Figure S1. 12% SDS PAGE showing induction of three genes at 16 °C and 37 °C using E. coli strain BL21(DE3)-Gold. Figure S2. 12% SDS PAGE showing induction of FAD at 42 °C using E. coli strain BL21(DE3)CodonPlus-RIL, BL21(DE3)-Gold and BL21(DE3)-pLysS

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Not AvailableButea monosperma is the most important host tree for the rangeeni strain of lac insect, Kerria lacca, exploited commercially in India for lac cultivation. Six flower colour variants of B. monosperma viz., scarlet, yellow, golden yellow, chrome yellow, white and mustard colour were collected from different regions of Jharkhand. Inter Simple Sequence Repeat (ISSR) marker system was employed to investigate the genetic structure and diversity among these flower colour variants. Out of 45 ISSR primers, 12 were found to be informative generating 127 scorable bands. A maximum of 19 and a minimum of 7 scorable bands per primer were obtained. The resolving power of the primers ranged between 2 and 11.3. Average polymorphic band percentage for all the primers was 85%. The marker index ranged from 2.9 to 15.85. The estimated Jaccard similarity coefficient ranged from 0.35 to 0.52. The clustering analysis using Jaccard’s coefficient showed 2 clusters; first - yellow and golden yellow and second - mustard, scarlet, chrome yellow and white flower colour variants.Not Availabl