Defense Mechanism of Phosphorothioated DNA under Peroxynitrite-Mediated Oxidative Stress.

DNA phosphorothioation (PT) exists in many pathogenic bacteria; however, the mechanism of PT-DNA resistance to the immune response is unclear. In this work, we meticulously investigated the peroxynitrite (PN) tolerance using PT-bioengineered E. coli strains. The in vivo experiment confirms that the S+ strain survives better than the S- strain under moderately oxidative stress. The LCMS, IC, and GCMS experiments demonstrated that phosphorothioate partially converted to phosphate, and the byproduct included sulfate and elemental sulfur. When O,O-diethyl thiophosphate ester (DETP) was used, the reaction rate k1 was determined to be 4.3 ± 0.5 M-1 s-1 in the first-order for both phosphorothioate and peroxynitrite at 35 °C and pH of 8.0. The IC50 values of phosphorothioate dinucleotides are dramatically increased by 400-700-fold compared to DETP. The SH/OH Yin-Yang mechanism rationalizes the in situ DNA self-defense against PN-mediated oxidative stress at the extra bioenergetic cost of DNA modification.

Alterations of the Brain Proteome and Gut Microbiota in d-Galactose-Induced Brain-Aging Mice with Krill Oil Supplementation.

Brain aging is commonly associated with neurodegenerative disorders, but the ameliorative effect of krill oil and the underlying mechanism remain unclear. In this study, the components of krill oil were measured, and the antiaging effects of krill oil were investigated in mice with d-galactose (d-gal)-induced brain aging via proteomics and gut microbiota analysis. Krill oil treatment decreased the expression of truncated dopamine- and cAMP-regulated phosphoproteins and proteins involved in the calcium signaling pathway. In addition, the concentrations of dopamine were increased in the serum (p < 0.05) and brain (p > 0.05) due to the enhanced expressions of tyrosine-3-monooxygenase and aromatic l-amino acid decarboxylase. Moreover, krill oil alleviated gut microbiota dysbiosis, decreased the abundance of bacteria that consume the precursor tyrosine, and increased the abundance of Lactobacillus spp. and short-chain fatty acid producers. This study revealed the beneficial effect of krill oil against d-gal-induced brain aging and clarified the underlying mechanism through proteomics and gut microbiota analysis.

Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis.

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert L-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.

A quantitative analytical method for valienone and its application in the evaluation of valienone production by a breakthrough microbial process.

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L-1 H3PO4 at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C18 (5 µm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 µg·mL-1. The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

Over-expression of UDP-glucose pyrophosphorylase increases validamycin A but decreases validoxylamine A production in Streptomyces hygroscopicus var. jinggangensis 5008.

During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.