farnesyl-pyrophosphate and geranylgeraniol

Combinatorial design is an effective strategy to acquire the optimal solution in complex systems. In this study, the combined effects of pathway combination, promoters' strength fine-tuning, copy numbers and integration locus variations caused by δ-integration were explored in Saccharomyces cerevisiae using geranylgeraniol (GGOH) production as an example. Two GGOH biosynthetic pathway branches were constructed. In branch 1, GGOH was converted from isopentenyl pyrophosphate (IPP) and farnesyl diphosphate (FPP). In branch 2, GGOH was derived directly from IPP and dimethylallyl pyrophosphate (DMAPP). Regulated by 10 combinations of 11 diverse promoters, a fusion gene BTS1-ERG20, a heterologous geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius (GGPPSsa) and an endogenous N-terminal truncated gene 3-hydroxyl-3-methylglutaryl-CoA reductase isoenzyme 1 (tHMGR), were incorporated into yeast by δ-integration, leading to a series of GGOH producing strains with yields ranging from 18.45 mg/L to 161.82 mg/L. The yield was further increased to 437.52 mg/L by optimizing the fermentation medium. Consequently, the GGOH yield reached 1315.44 mg/L in a 5-L fermenter under carbon restriction strategy. Our study not only opens large opportunities for downstream diterpenes overproductions, but also demonstrates that pathway optimization based on combinatorial design is a promising strategy to engineer microbes for overproducing natural products with complex structure.

Topics: Bacterial Proteins; Biosynthetic Pathways; Diterpenes; Farnesyltranstransferase; Hemiterpenes; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; Metabolic Engineering; Organophosphorus Compounds; Polyisoprenyl Phosphates; Promoter Regions, Genetic; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sesquiterpenes

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Topics: Adult; Apoptosis; Bone Density Conservation Agents; Bone Morphogenetic Protein 2; Cell Culture Techniques; Cell Survival; Cells, Cultured; Diphosphonates; Diterpenes; Epiregulin; Farnesol; Female; Fibroblasts; Gene Expression Regulation; Gingiva; Humans; Imidazoles; Interferon-alpha; Mevalonic Acid; Microscopy, Electron, Transmission; Middle Aged; Neovascularization, Physiologic; Polyisoprenyl Phosphates; rhoB GTP-Binding Protein; Sesquiterpenes; Signal Transduction; Vascular Endothelial Growth Factor A; Zoledronic Acid

Simvastatin (SV), a competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and a widely prescribed treatment for hypercholesterolemia, exerts numerous positive pleiotropic effects that are thought to occur independent of its cholesterol-lowering properties. In previously published work, we have shown that chronic SV treatment rescues cognitive function in a transgenic mouse model of Alzheimer's disease, and enhances learning and memory in non-transgenic mice without affecting total brain cholesterol and amyloid-beta levels. More recently, we demonstrated the ability of SV to enhance long-term potentiation (LTP) in the CA1 region of the hippocampus in slices from wild-type C57BL/6 mice via a mechanism dependent upon phosphatidylinositol 3-kinase (PI3-K)/Akt activation during LTP induction. The present study was conducted to better understand the molecular mechanisms underlying SV-induced enhancement of LTP. Specifically, it was found that inhibiting production of isoprenoid intermediates in the biosynthetic pathway for cholesterol triggers the downstream events leading to enhanced LTP. Interestingly, two major isoprenoid intermediates exhibit differential effects. Replenishment of farnesyl pyrophosphate, but not geranylgeranyl pyrophosphate, abolished the LTP-enhancing ability of SV. In parallel to this finding, inhibiting farnesylation, but not geranylgeranylation, replicated the enhancement of LTP caused by SV. Finally, inhibiting farnesylation promotes the activation of Akt during the induction phase. Together, these results suggest that SV enhances LTP in CA1 by modulating isoprenylation-dependent molecular pathways downstream of farnesyl transferase. These findings will aid in the identification of novel therapeutic targets that modulate synaptic and cognitive function.

Topics: Alkyl and Aryl Transferases; Animals; Blotting, Western; CA1 Region, Hippocampal; Diterpenes; Electrophysiological Phenomena; Farnesol; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Long-Term Potentiation; Male; Mevalonic Acid; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Polyisoprenyl Phosphates; Prenylation; Proto-Oncogene Proteins c-akt; Sesquiterpenes; Signal Transduction; Simvastatin

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Topics: Actin Cytoskeleton; Adenocarcinoma; Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Division; Cell Size; Diterpenes; Drug Interactions; Enzyme Activation; Farnesol; G1 Phase; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Transgenic; Polyisoprenyl Phosphates; Prostatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); rac GTP-Binding Proteins; rhoA GTP-Binding Protein; Sesquiterpenes; Tumor Cells, Cultured