Characterization of sexual maturity-associated N6-methyladenosine in boar testes.

BACKGROUND: The health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated. RESULTS: Using the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. CONCLUSION: The results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Screening of Selenium/Glutathione-Enriched Candida utilis and Its Anti-inflammatory and Antioxidant Activities in Mice.

This study aimed to screen a mutant of Candida utilis SE-172 with high selenite tolerance and glutathione (GSH) biosynthesis capability via 60Co γ-radiation mutagenesis to prepare selenium (Se)-enriched yeast. The maximal intracellular contents of GSH and organic Se of 22.94 mg/g and 1308.1 µg/g were obtained, respectively, under a batch culture of SE-172. The physiological mechanism underlying increased GSH and organic Se contents in Se/GSH-enriched C. utilis SE-172 was revealed based on assaying activities of γ-glutamylcysteine synthase (γ-GCS) involved in GSH biosynthesis and selenophosphate synthase (SPS) related to organic Se bioconversion, and by determining intracellular ATP and NADH contents and ATP/ADP and NADH/NAD+ ratios associated with energy supply and regeneration. Moreover, the effect of this selenized yeast on anti-inflammatory and antioxidant activities in mice with colitis was investigated. The supplementation of Se/GSH-enriched yeast decreased the dextran sodium sulfate-induced damage to colon tissues, reduced the expression of pro-inflammatory factors [interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α)] in serum, increased the antioxidant-related enzyme [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)] activities, and decreased the malondialdehyde content in colon. The Se/GSH-enriched C. utilis SE-172 showed potent anti-inflammatory and antioxidant activities in mice with colitis.

MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans.

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for ß-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

Metabolic Engineering of Escherichia coli for Production of α-Santalene, a Precursor of Sandalwood Oil.

α-Santalene belongs to a class of natural compounds with many physiological functions and medical applications. Advances in metabolic engineering enable non-native hosts (e.g., Escherichia coli) to produce α-santalene, the precursor of sandalwood oil. However, imbalances in enzymatic activity often result in a metabolic burden on hosts and repress the synthetic capacity of the desired product. In this work, we manipulated ribosome binding sites (RBSs) to optimize an α-santalene synthetic operon in E. coli, and the best engineered E. coli NA-IS3D strain could produce α-santalene at a titer of 412 mg·L-1. Concerning the observation of the inverse correlation between indole synthesis and α-santalene production, this study speculated that indole-associated amino acid metabolism would be competitive to the synthesis of α-santalene rather than indole toxicity itself. The deletion of tnaA could lead to a 1.5-fold increase in α-santalene production to a titer of 599 mg·L-1 in E. coli tnaA- NA-IS3D. Our results suggested that the optimization of RBS sets of the synthetic module and attenuation of the competitive pathway are promising approaches for improving the production of terpenoids including α-santalene.

Regulatory molecule cAMP changes cell fitness of the engineered Escherichia coli for terpenoids production.

Terpenoids are a class of natural compounds with many important functions and applications. They are synthesized from a long synthetic pathway of isoprenyl unit coupling with the myriads of terpene synthases. Owing to the catalytic divergence of terpenoids synthesis, microbial production of terpenoids is compromised to the complexity of pathway engineering and suffers from the metabolic engineering burden. In this work, the adaptive Escherichia coli HP variant exhibited a general cell fitness in terpenoid synthesis. Especially, it could yield taxadiene of 193.2 mg/L in a test tube culture, which is a five-fold increase over the production in the wild type E. coli DH5α. Mutational analyses indicated that IS10 insertion in adenylate cyclase CyaA (CyaAHP) resulted in lowering intracellular cyclic AMP (cAMP), which could regulate its receptor protein CRP to rewire cell metabolism and contributed to the improved cell fitness. Our results suggested a way to manipulate cell fitness for terpenoids production and other products.

A gas chromatography-flame ionization detection method for direct and rapid determination of small molecule volatile organic compounds in aqueous phase.

It is still difficult to directly detect low content of volatile organic compounds (VOCs) in water samples by gas chromatography (GC) because when water is the only solvent, it would result in the instability and poor repeatability of peak retention time and peak shape. The adverse effects of water on direct GC analysis of VOCs cannot be significantly reduced or eliminated by simply changing the detection condition of GC. However, it was found that the addition of methanol in samples to a certain final proportion, such as 50 or 75% (v/v), could greatly reduce or eliminate the adverse effects of water. By using 75% (v/v) methanol as a solvent, the standard curves of ethanol, acetic acid, acetone, and isopropanol with correlation coefficient (R 2) over 0.99 were successfully plotted by gas chromatography-flame ionization detection (GC-FID) in a certain concentration range, respectively. The results showed that the retention time and peak shape stability of ethanol, acetic acid, acetone, and isopropanol in aqueous solution were greatly improved by the addition of methanol to final concertation of 75% (v/v). To verify the practical application potential of this method, the method was applied to the detection of components in isopropanol fermentation wastewater. The results showed that the method has well applicability and reliability. The key points in the application of this method were also summarized. This GC analysis method would have a wider and better application prospect in the detection of water-soluble organic matters.

Knockout of acetoacetate degradation pathway gene atoDA enhances the toxicity tolerance of Escherichia coli to isopropanol and acetone.

Isopropanol and acetone are important chemical products and potential high-quality new fuels. Both of them are metabolites of isopropanol synthesis pathway, but they are toxic to most bacteria. In this study, toxicity tolerance of Escherichia coli strains was evaluated by detecting their growth rates under different concentrations of isopropanol and acetone. It was showed that isopropanol was more toxic to E. coli than acetone, and the native strain MG1655 had better tolerance over DH5α to either acetone or isopropanol of 300 mM. Key genes of ethanol synthesis pathway, acetic acid metabolism pathway, and acetoacetic acid degradation pathway, including adhE, ackA-pta, and atoDA, were knocked out in MG1655 to form mutants MGΔadhE, MGΔackA-pta, and MGΔatoDA. The tolerance performances of the mutants to isopropanol and acetone were determined under various concentrations including 300 mM, 500 mM, and 700 mM, respectively. The mutant MGΔatoDA exhibited excellent tolerance to both acetone and isopropanol of 500 mM, and MGΔackA-pta could tolerate acetone at 500 mM rather than isopropanol, while the deletion of adhE in MGΔadhE resulted in a severe cell growth defection. Although isopropanol and acetone at 700 mM caused severe growth inhibition on each strain, cell growth could be restored to varying degrees with the prolongation of culture time. This phenomenon was suggested to be related to the volatilization of isopropanol and acetone based on volatilization tests. It was envisioned that MG1655 was a suitable host strain for isopropanol metabolic engineering research, and the acetoacetic acid degradation pathway gene atoDA, was probably the key optimizing point for isopropanol production.

Transcriptome analysis reveals the mechanism underlying improved glutathione biosynthesis and secretion in Candida utilis during selenium enrichment.

The effect of sodium selenite on batch culture of Candida utilis CCTCC M 209298 was investigated. Cell growth was inhibited while glutathione biosynthesis and secretion were improved during selenium enrichment. To reveal the mechanism underlying the decrease in biomass and the increase in glutathione, both metabolic flux analysis of key intermediates involved in glutathione metabolic pathway and transcriptome analysis of C. utilis by RNA-seq were carried out for selenized cells and the control without selenium enrichment. Results indicated that sodium selenite decreased carbon fluxes towards biomass but increased fluxes towards amino acids for the biosynthesis of glutathione and related amino acids. Selenium enrichment down-regulated a large number of genes involved in cell components and the cell cycle, resulting in decreased biomass as well as increased cell permeability. Moreover, several genes associated with transportation, binding, and mitochondrial and ribosomal functions for energy metabolism and protein synthesis were up-regulated in the presence of sodium selenite. All of these results disclosed the physiological response of C. utilis to sodium selenite.

MiR-222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene.

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA-222 (miR-222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR-222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR-222 functions as an anti-apoptotic factor in pGCs. MiR-222 mimics in pGCs result in the upregulation of the anti-apoptotic BCL-2 gene, down-regulation of the proapoptotic caspase-3 gene, and inhibition of apoptosis. MiR-222 inhibitors reduced BCL-2 and had no significant effect on caspase-3. MiR-222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1-siRNA reduced the proapoptotic BAX gene. MiR-222 can directly target the 3'-untranslated region of the THBS1 gene. MiR-222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR-222 inhibitor. Transfection of THBS1-siRNA resulted in the inhibition of the miR-222 inhibitor, which suggests that miR-222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR-222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.

Disruption of por1 gene in Candida utilis improves co-production of S-adenosylmethionine and glutathione.

The por1 gene encoding one of the mitochondrial porin channels in C. utilis CCTCC M 209298 was disrupted using a homologous recombination method. The co-production of S-adenosylmethionine (SAM) and glutathione (GSH) in the mutant C. utilis Δpor1 increased by 34.9% and 25.1%, respectively, during batch and fed-batch fermentation, relative to the parental strain. The average oxygen consumption rate, activities of key enzymes involved in SAM and GSH biosynthesis, levels of intracellular cofactors such as NADH and ATP, and carbon fluxes of key metabolites were compared between the parental strain and the Δpor1 mutant. The disruption of por1 gene increased the rate of mitochondrial respiration, increased the activities of both methionine adenosyltransferase and γ-glutamylcysteine synthetase, and enhanced the supply of energy and substrates for SAM and GSH biosynthesis, all of which favored the overproduction of SAM and GSH in the Δpor1 mutant.

Improved S-adenosylmethionine and glutathione biosynthesis by heterologous expression of an ATP6 gene in Candida utilis.

ATP is indispensable to the biosynthesis of both S-adenosylmethionine (SAM) and glutathione (GSH) in yeast cells. To improve ATP supply for overproduction of SAM and GSH in Candida utilis CCTCC M 209298, an exogenous ATP6 gene from Arabidopsis thaliana was expressed in the parental strain to construct the mutant C. utilis ATP6 by genomic integration. The maximal production of SAM and GSH in the mutant increased by 46.6 and 28.7%, respectively, when compared with those obtained in the parental strain. The mechanism underlying improved SAM and GSH biosynthesis by exogenous ATP6 gene expression revealed that the mutant had higher activities of key enzymes involved in SAM and GSH biosynthesis as well as energy metabolism. Increased NADH availability and F0 F1 -ATPase activity subsequently resulted in improved ATP regeneration and intracellular ATP supply for SAM and GSH overproduction. The present study not only developed an effective method for improving SAM and GSH biosynthesis by energy metabolism regulation, but also offered a novel approach for efficient production of similar energy-consuming products in eukaryotic cells.