Trehalose-6-phosphate synthase influences polysaccharide synthesis and cell wall components in Ganoderma lucidum.

Trehalose-6-phosphate synthase (TPS) is a key enzyme that participates in trehalose metabolism, which can synthesize trehalose in a two-step pathway with trehalose phosphatase, but its role in fungi is rarely studied, especially in large basidiomycetes. In this study, the tps gene of Ganoderma lucidum was cloned and named as gltps. And gltps-silenced strains were constructed by RNA interference. In this study, it is found that the extracellular polysaccharide content increased 1.6-2-fold, but there was no significant change on intracellular polysaccharide content in gltps-silenced strains compared with the wild-type (WT) strain. Furthermore, the cell wall compositions of the gltps-silenced strains were also altered, which showed that the chitin and ß-1,3-glucan contents were significantly decreased. Compared with WT, the concentration of chitin decreased by 20%-50% and that of ß-1, 3-glucan decreased by 15%-30%. The study found that the cells of gltps-silenced strains were more sensitive to cell wall stress, which might be due to changes in the compounds and structure of the cell wall. These results showed that gltps had an important effect on carbohydrate metabolism of G. lucidum cells.

Spermidine Regulates Mitochondrial Function by Enhancing eIF5A Hypusination and Contributes to Reactive Oxygen Species Production and Ganoderic Acid Biosynthesis in Ganoderma lucidum.

Spermidine, a kind of polycation and one important member of the polyamine family, is essential for survival in many kinds of organisms and participates in the regulation of cell growth and metabolism. To explore the mechanism by which spermidine regulates ganoderic acid (GA) biosynthesis in Ganoderma lucidum, the effects of spermidine on GA and reactive oxygen species (ROS) contents were examined. Our data suggested that spermidine promoted the production of mitochondrial ROS and positively regulated GA biosynthesis. Further research revealed that spermidine promoted the translation of mitochondrial complexes I and II and subsequently influenced their activity. With a reduction in eukaryotic translation initiation factor 5A (eIF5A) hypusination by over 50% in spermidine synthase gene (spds) knockdown strains, the activities of mitochondrial complexes I and II were reduced by nearly 60% and 80%, respectively, and the protein contents were reduced by over 50%, suggesting that the effect of spermidine on mitochondrial complexes I and II was mediated through its influence on eIF5A hypusination. Furthermore, after knocking down eIF5A, the deoxyhypusine synthase gene (dhs), and the deoxyhypusine hydroxylase gene (dohh), the mitochondrial ROS level was reduced by nearly 50%, and the GA content was reduced by over 40%, suggesting that eIF5A hypusination contributed to mitochondrial ROS production and GA biosynthesis. In summary, spermidine maintains mitochondrial ROS homeostasis by regulating the translation and subsequent activity of complexes I and II via eIF5A hypusination and promotes GA biosynthesis via mitochondrial ROS signaling. The present findings provide new insight into the spermidine-mediated biosynthesis of secondary metabolites. IMPORTANCE Spermidine is necessary for organism survival and is involved in the regulation of various biological processes. However, the specific mechanisms underlying the various physiological functions of spermidine are poorly understood, especially in microorganisms. In this study, we found that spermidine hypusinates eIF5A to promote the production of mitochondrial ROS and subsequently regulate secondary metabolism in microorganisms. Our study provides a better understanding of the mechanism by which spermidine regulates mitochondrial function and provides new insight into the spermidine-mediated biosynthesis of secondary metabolites.

Mitochondrial pyruvate carrier regulates the lignocellulosic decomposition rate through metabolism in Ganoderma lucidum.

The activity of mitochondrial pyruvate carrier (MPC) can be modulated to regulate intracellular metabolism under different culture conditions. In Ganoderma lucidum, the role of MPC in regulating carbon sources remains unknown. By knocking down MPC genes (MPC1 and MPC2), this research found that the loss of MPC increased the growth rate of G. lucidum by ~30% in a medium with wood chips as a carbon source. Then cellulase and laccase activities were tested. Endoglucanase and laccase activity increased by ~50% and ~35%, respectively, in MPC knockdown mutants compared with that in the wild type strain. Finally, the expression levels of genes related to glycolysis were assayed, and the transcription levels of these enzymes were found to be increased by ~250% compared with the wild type strain. In conclusion, the regulation of intracellular metabolism by MPC provides a new way to improve the use of nondominant carbon sources such as lignocellulose.

A quantitative determination of fluorochloridone in rat plasma by UPLC-MS/MS method: application to a pharmacokinetic study.

A precise, high-throughput and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of fluorochloridone (FLC) in rat plasma. The extraction of analytes from plasma samples was carried out by protein precipitation procedure using acetonitrile prior to UPLC-MS/MS analysis. Verapamil was proved as a proper internal standard (IS) among many candidates. The chromatographic separation based on UPLC was well optimized. Multiple reaction monitoring in positive electrospray ionization was used with the optimized MS transitions at: m/z 312.0 → 292.0 for FLC and m/z 456.4 → 165.2 for IS. This method was well validated with good linear response (r(2) > 0.998) observed over the investigated range of 3-3000 ng/mL and with satisfactory stability. This method was also characterized with adequate intra- and inter-day precision and accuracy (within 12%) in the quality control samples, and with high selectivity and less matrix effect observed. Total running time was only 1.5 min. This method has been successfully applied to a pilot FLC pharmacokinetic study after oral administration. Copyright © 2016 John Wiley & Sons, Ltd.

Ninety day toxicity and toxicokinetics of fluorochloridone after oral administration in rats.

The ninety day toxicity and toxicokinetics of fluorochloridone (FLC) were accessed in Wistar rats. Animals were gavaged with FLC at doses of 31.25 mg/kg, 125 mg/kg and 500 mg/kg for ninety days, followed by thirty days for recovery. On the 1st, 60th, 75th and 90th days of the dosing phase, plasma of ten animals of all groups treated with FLC was collected for toxicokinetic analysis of FLC by an UPLC-MS/MS method. Numerous changes in body weight, hematology, serum chemistry, and organ weight ratios were observed by the 45th and 90th dosing day. Most changes in groups treated with FLC were absent on the last recovery day. Testis and epididymis lesions were consistently seen in histopathological observations on the 45th, 90th dosing day and the last recovery day. Repeated administration of FLC increased the level of testosterone in serum in male rats on the 90th dosing day. FLC plasma concentrations could be detected in all animal drug-treated groups during the dosing phase, and a dose proportional relationship was seen between FLC dose and AUC or Cmax. This study will support future studies on the mechanism of FLC-induced toxicity.