Metabolic engineering for the development of microbial production strains, such as carotenoid overproducing bacteria, has a long history in industrial biotechnology. In contrast to classical strain development that mostly relies on the generation and screening of mutant libraries, rational strain development relies on the identification of a genetic target that has to be engineered in order to overcome metabolic bottlenecks facilitating the production of the desired valuable compounds. In this work, two synthetic biology approaches, namely, a CRISPRi-library and a genetically encoded biosensor, are demonstrated as tools for metabolic engineering purposes with a focus on carotenoid biosynthesis in C. glutamicum. The methods presented here gave insights into carotenoid biosynthesis and facilitated development of new metabolic engineering strategies. The use of a genetically encoded biosensor, the screening of a CRISPRi-library, and their combination can be transferred to study a wide range of organisms and target compounds.

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (AβPP) processing and regulates clusterin protein levels. AβPP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sAβPPα) presence in AβPP-wt cells and Aβ expression in AβPP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 μM) or simvastatin (50 μM) as well as cholesterol chelator methyl-β-cyclodextrin (0.2 mM) diminished cell viability (p < 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p < 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in AβPP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.

To evaluate the fluctuation of secondary metabolites in Arabian lilac during a year, aerial parts of the plant were harvested in the middle of each month. The essential oils content from fresh and dried plant materials was analyzed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometer (GC-MS), individually. Phytochemical contents, along with antiradical scavenging potential of the related methanol extracts were separately assessed. The spring and autumn samples (fresh and dried) yielded more essential oil than the other samples. Forty-one compounds were identified totally in the oils and the major constituents characterized were β-caryophyllene, sabinene, and caryophyllene oxide. The extracts obtained from winter and summer plants possessed the highest total phenolics. The maximum amount of total flavonoid content was measured in winter (December and January), whereas the minimum one was observed in spring (March). The summer and winter samples showed the highest and lowest content of flavones and flavanols, respectively, whereas the anthocyanin content was higher in winter than in summer. Moreover, antiradical activity of the extracts in summer and winter samples was higher than in other seasons. Overall, this study can provide useful information regarding the best harvest period of Arabian lilac to yield the desired compounds for application in phytopharmaceutical and food industries.

The N-methylanthraniloylisoprenoid diphosphate derivatives 1 and 2 bind to RabGGTase II and are enzymatically transferred to Rab 7 (the Rab proteins are small G-proteins that control events of docking and fusion of intracellular vesicles). The fluorescent Rab 7 proteins thus obtained may become important tools for further biological studies on vesicular trafficking in cells.

This in vitro study analyzed the influence of geranylgeraniol (GGOH) on human oral keratinocytes (HOK) after exposure to bisphosphonates. HOK were incubated with four different bisphosphonates (clodronate, ibandronate, pamidronate, zoledronate) in two experimental set-ups: with and without GGOH. MTT and PrestoBlue assays were used to analyze HOK cell viability. HOK migration ability was examined with Boyden and Scratch assays, and Tunel and ToxiLight assays were used to detect the HOK apoptosis rate. No significant differences between the experimental set-ups, with and without GGOH, could be found for clodronate (p each >0.3). For the nitrogen-containing bisphosphonates, negative effects could be shown in the experimental set-ups without GGOH in all assays. In the GGOH experimental set-ups, the levels of HOK cell viability were significantly increased (MTT: p each ≤0.001; PrestoBlue: p each ≤0.012). The HOK migration ability was also greater (Boyden: p each <0.001; Scratch: p each ≤0.015). Regarding the apoptosis rate, reduced numbers of apoptotic HOK in the Tunel assay (p each <0.001) and decreased adenylate kinase release in the ToxiLight assay (p each ≤0.002) were observed. GGOH reversed the negative effects of bisphosphonates on HOK. These findings provide evidence that GGOH could be a promising treatment option for BP-ONJ.