Relationship between β-Carotene Accumulation and Geranylgeranyl Pyrophosphate Synthase in Different Species of Dunaliella

To study the relationship between β-carotene synthesis and geranylgeranyl pyrophosphate synthase (GGPS) activity, 15 species of Dunaliella were used to determine the changes in photosynthetic pigment contents, chlorophyll fluorescence parameters, β-carotene content, and GGPS activity. By observing the morphology and size of 15 species of Dunaliella, D8 has the largest individual algal cell and D9 has the smallest individual. Growth was relatively slow during days one through seven. After about eight days, the cells entered the logarithmic growth period and grew rapidly to a high density. After about 45 days, they entered a mature period, and growth slowed down. The contents of chlorophyll, carotenoids, and β-carotene increased during growth. D1 has the highest accumulation of β-carotene, and GGPS enzyme activity has a positive linear relationship with the β-carotene synthesis content. Phylogenetic analysis showed that the GGPS proteins of the 15 species were highly homologous, and the GGPS protein was not part of the membrane.

Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension

Background: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. Methods: We developed a sensitive HPLC technique to quantify dietary GGPP and conducted proteomics, qRT-PCR screening and western blot to determine signaling cascades, gene expression, protein-protein interaction and protein membrane trafficking in wild type and transgenic rats. Results: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells (HPASMCs) treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2/Rab10 signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and HPASMCs upregulated the expression of Ca 2+ -sensing receptor (CaSR) and hypoxia-induced mitogenic factor/FIZZ1 (HIMF), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10-knockdown almost abrogated hypoxia-promoted CaSR membrane-trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed with a high GGPP diet, blood GGPP levels were increased, and this abolished statin-lowering effects on plasma GGPP and hypoxia-enhanced RhoA activity of blood monocytes that were both also rescued by garlic extracts. Conclusions: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of i) RhoA-ROCK2 cascade activation and ii) Rab10-faciliated CaSR membrane trafficking with iii) subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin together with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9

Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.

Ggps1 deficiency in the uterus results in dystocia by disrupting uterine contraction

Dystocia is a serious problem for pregnant women, and it increases the cesarean section rate. Although uterine dysfunction has an unknown etiology, it is responsible for cesarean delivery and clinical dystocia, resulting in neonatal morbidity and mortality; thus, there is an urgent need for novel therapeutic agents. Previous studies indicated that statins, which inhibit the mevalonate pathway of cholesterol synthesis, can reduce the incidence of preterm birth, but the safety of statins for pregnant women has not been thoroughly evaluated. Therefore, to unambiguously examine the function of the mevalonate pathway in pregnancy and delivery, we employed a genetic approach by using myometrial cell-specific deletion of geranylgeranyl pyrophosphate synthase (Ggps1) mice. We found that Ggps1 deficiency in myometrial cells caused impaired uterine contractions, resulting in disrupted embryonic placing and dystocia. Studies of the underlying mechanism suggested that Ggps1 is required for uterine contractions to ensure successful parturition by regulating RhoA prenylation to activate the RhoA/Rock2/p-MLC pathway. Our work indicates that perturbing the mevalonate pathway might result in problems during delivery for pregnant females, but modifying protein prenylation with supplementary farnesyl pyrophosphate or geranylgeranyl pyrophosphate might be a strategy to avoid side effects.

Corynebacterium glutamicum CrtR and Its Orthologs in Actinobacteria: Conserved Function and Application as Genetically Encoded Biosensor for Detection of Geranylgeranyl Pyrophosphate

Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.