Farnesyl Diphosphate (FPP) Toxicity in E. coli, M9 medium
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ABSTRACT: This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in LB and fed 10 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in LB and fed 10 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 2.5, 5, 7, and 29.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 6, 10, 14, and 26.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:Simvastatin has been widely used for treatment of hypercholesterolemia due to its ability to inhibit HMG-CoA reductase, the rate limiting enzyme of de novo cholesterol synthesis via mevalonate pathway. Its inhibitory action causes also depletion of pathway intermediates, farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), which are inevitable for proper targeting of small GTPases (e.g. Ras proteins) to their site of action. We profiled by array the gene expression of MIA PaCa-2 cells treated with simvastatin, FPP, GGPP and their combinations. The inhibitory effect of statins on GFP-K-Ras protein trafficking were partially prevented by addition of the mevalonate pathway intermediates. We conclude that the anticancer effect of simvastatin is to a large extent mediated through isoprenoid intermediates of the mevalonate pathway.
Project description:Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into sixteen, seventeen and eighteen carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing sixteen carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel sixteen carbon terpenes by GC-QTOF. Based on the available FPP analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.
Project description:In this experiment we performed the transcriptional profiling of the wild type yeast Saccharomyces cerevisiae upon treatment with lovastatin or zaragozic acid. These drugs are known to exert a repressing effect on the sterol branch of the isoprenoid pathway, but their action differs at the level of FPP biosynthesis. While lovastatin decreases FPP availability because it is an inhibitor of HMGR, zaragozic acid increases this level because it inhibits squalene synthase. In this work, we were interested especially in genes, whose expression would be oppositely regulated in the presence of lovastatin or zaragozic acid, since these genes could be affected by variation in FPP level and thus related somehow to the isoprenoid pathway.
Project description:Glyphosate (GLY) is an effective antimetabolite that acts against the shikimate pathway 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, However, little is known about the genome-scale transcriptional responses of bacteria after glyphosate shock. To investigate further the mechanisms by which E. coli response to a glyphosate shock, a DNA-based microarray was used for transcriptional analysis of E. coli exposed to 200 mM glyphosate.
Project description:Glyphosate (GLY) is an effective antimetabolite that acts against the shikimate pathway 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, However, little is known about the genome-scale transcriptional responses of bacteria after glyphosate shock. To investigate further the mechanisms by which E. coli response to a glyphosate shock, a DNA-based microarray was used for transcriptional analysis of E. coli exposed to 200 mM glyphosate. RNA extracted from cells of E. coli K-12 JM109 cells after 4 h of growth to OD600 achieve 0.4, or cells after 200 mM glyphosate shock for 1 h when their OD600 achieved 0.4.
Project description:RNA Sequencing of claudin-low triple negative breast cancer cells T11 treated with FPP-1746-Omomyc, Omomyc, FPP-1746 and vehicle control (PBS) to determine differentially regulated genes induced by FPP-1746-Omomyc
Project description:E. coli TG1 with pBS(Kan)Synhox can produce more hydrogen than TG1/pBS(Kan). To reveal the difference of metabolic activity (gene expression) between these strains in producing hydrogen, the differential gene expression analyses were performed. All samples cultured in complex medium with fructose containg 5 mM IPTG. Keywords: hydrogen production