The pharmacological effects of Oren-gedoku-to (OGT), a Japanese-Chinese traditional herbal medicinal mixture on lipid biosynthesis were investigated in cultured human hepatocyte HepG2 cells. The addition of OGT (0.5 and 4.2 mg/ml), which had no effect on cell proliferation and cellular protein content, caused a marked decrease in the cellular cholesterol content, particularly cholesteryl ester content following 24 h incubation. The incorporation of 14C-oleate into cellular cholesteryl ester fraction was also reduced remarkably during incubation for 6 and 24 h. The effects of OGT, its components and its main active chemicals on acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity were studied in vitro to explore the mechanism by which OGT inhibits cholesteryl ester formation. The data confirmed that OGT, in a dose-dependent manner, and its components (Scutellaria baicalensis, Coptis japonica, Gardenia jasminoides and Phellodendron amurense) remarkably inhibit ACAT activity. Among the main active chemicals of OGT, baicalein, a kind of flavonoid, decreased ACAT activity in a dose-dependent fashion from the level of 10(-6)M. These results strongly suggest that OGT reduces the cholesteryl ester formation in human hepatocytes by inhibiting ACAT, and that baicalein may, in part, be responsible for ACAT inhibition.
2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.
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Styrene is an important industrial chemical. Although several studies have reported microbial styrene production, the amount of styrene produced in batch cultures can be increased. In this study, styrene was produced using genetically engineered Escherichia coli. First, we evaluated five types of phenylalanine ammonia lyases (PALs) from Arabidopsis thaliana (AtPAL) and Brachypodium distachyon (BdPAL) for their ability to produce trans-cinnamic acid (Cin), a styrene precursor. AtPAL2-expressing E. coli produced approximately 700 mg/L of Cin and we found that BdPALs could convert Cin into styrene. To assess styrene production, we constructed an E. coli strain that co-expressed AtPAL2 and ferulic acid decarboxylase from Saccharomyces cerevisiae. After a biphasic culture with oleyl alcohol, styrene production and yield from glucose were 3.1 g/L and 26.7% (mol/mol), respectively, which, to the best of our knowledge, are the highest values obtained in batch cultivation. Thus, this strain can be applied to the large–scale industrial production of styrene.
In previous studies, titanium peroxide nanoparticles (PAA-TiOx NPs) with surfaces functionalized using polyacrylic acid (PAA) and hydrogen peroxide (H2O2) demonstrated a synergistic effect when combined with X-ray irradiation. The combination generated H2O2 and reactive oxygen species (ROS) that enhanced the irradiation efficacy. In the present study, we examined the relationship between catalase and PAA-TiOx NPs sensitization to X-ray radiation because catalase is the primary antioxidant enzyme that converts H2O2 to water and oxygen. Catalase-knockout PANC-1 (dCAT) cells were generated using the CRISPR/Cas9 system, which was confirmed by the suppression of catalase expression in mRNA and protein levels that resulted in an 81.7% decrease in catalase activity compared with levels in wild-type cells. Catalase deficiency was found to increase the production of ROS, particularly in hypoxia. Also, the combination of PAA-TiOx NPs and X-ray 5 Gy resulted in a 7-fold decrease in the survival fraction (SF; p < 0.01) of dCAT cells compared with rates documented in wild-type cells. Interestingly, the combination treatment with X-ray 3 Gy in dCAT cells resulted in an SF similar to that observed in wild-type cells treated with the same combination but at a higher radiation dose (5 Gy). These results suggest that a strategy of catalase inhibition could be used to establish an advanced combination treatment of PAA-TiOx NPs and X-ray irradiation for pancreatic cancer cells.
Abstract The demand for the essential commodity chemical 1,2‐propanediol (1,2‐PDO) is on the rise, as its microbial production has emerged as a promising method for a sustainable chemical supply. However, the reliance of 1,2‐PDO production in Escherichia coli on anaerobic conditions, as enhancing cell growth to augment precursor availability remains a substantial challenge. This study presents glucose‐based aerobic production of 1,2‐PDO, with xylose utilization facilitating cell growth. An engineered strain was constructed capable of exclusively producing 1,2‐PDO from glucose while utilizing xylose to support cell growth. This was accomplished by deleting the gloA , eno , eda , sdaA , sdaB , and tdcG genes for 1,2‐PDO production from glucose and introducing the Weimberg pathway for cell growth using xylose. Enhanced 1,2‐PDO production was achieved via yagF overexpression and disruption of the ghrA gene involved in the 1,2‐PDO‐competing pathway. The resultant strain, PD72, produced 2.48 ± 0.15 g L −1 1,2‐PDO with a 0.27 ± 0.02 g g −1 ‐glucose yield after 72 h cultivation. Overall, this study demonstrates aerobic 1,2‐PDO synthesis through the isolation of the 1,2‐PDO synthetic pathway from the tricarboxylic acid cycle.
