Ammonium sulfate supplementation enhances erythromycin biosynthesis by augmenting intracellular metabolism and precursor supply in Saccharopolyspora erythraea.

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.

Characterization of crude oil degrading bacterial communities and their impact on biofilm formation.

In the present study, produced water sample collected from the Indian crude oil reservoir is used to enrich the bacterial communities. The impact of these enriched bacterial communities on the biodegradation of crude oil, biofilm formation, and biocorrosion process are elucidated. A crude oil degradation study is carried out with the minimal salt medium and 94% of crude oil was utilized by enriched bacterial communities. During the crude oil degradation many enzymes including alkane hydroxylase, alcohol dehydrogenase, and lipase are playing a key role in the biodegradation processes. The role of enriched bacterial biofilm on biocorrosion reactions are monitored by weight loss studies and electrochemical analysis. Weight loss study revealed that the biotic system has vigorous corrosion attacks compared to the abiotic system. Both AC-Impedance and Tafel analysis confirmed that the nature of the corrosion reaction take place in the biotic system. Very less charge transfer resistance and higher corrosion current are observed in the biotic system than in the abiotic system. Scanning electron microscope confirms that the dense biofilm formation favoured the pitting type of corrosion. X-ray diffraction analysis confirms that the metal oxides formed in the corrosion systems (biotic). From the metagenomic analysis of the V3-V4 region revealed that presence of diverse bacterial communities in the biofilm, and most of them are uncultured/unknown. Among the known genus, Bacillus, Halomonas, etc are dominant in the enriched bacterial biofilm sample. From this study, we conclude that the uncultured bacterial strains are found to be playing a key role in the pitting type of corrosion and they can utilize crude oil hydrocarbons, which make them succeeded in extreme oil reservoir environments.

Enhanced biodegradation of hydrophobic organic pollutants by the bacterial consortium: Impact of enzymes and biosurfactants.

Hydrocarbons and their derivative compounds are recalcitrant in nature and causing adverse impacts to the environment and are classified as important pollutants. Removal of these pollutants from the atmosphere is a challenging process. Hydrophobic organic pollutants (HOPs) including crude oil, diesel, dotriacontane (C32), and tetracontane (C40) are subjected to the biodegradation study by using a bacterial consortium consist of Bacillus subtilis, Pseudomonas stutzeri, and Acinetobacter baumannii. The impact of pH and temperature on the biodegradation process was monitored. During the HOPs biodegradation, the impact of hydrocarbon-degrading extracellular enzymes such as alcohol dehydrogenase, alkane hydroxylase, and lipase was examined, and found average activity about 47.2, 44.3, and 51.8 µmol/mg-1, respectively. Additionally, other enzymes such as catechol 1,2 dioxygenase and catechol 2,3 dioxygenase were found as 118 and 112 µmol/mg-1 Enzyme as an average range in all the HOPs degradation, respectively. Also, the impact of the extracellular polymeric substance and proteins were elucidated during the biodegradation of HOPs with the average range of 116.90, 54.98 mg/L-1 respectively. The impact of biosurfactants on the degradation of different types of HOPs is elucidated. Very slight changes in the pH were also noticed during the biodegradation study. Biodegradation efficiency was calculated as 90, 84, 76, and 72% for crude oil, diesel, C32, and C40, respectively. Changes in the major functional groups (CH, C-O-C, CO, =CH2, CH2, CH3) were confirmed by FTIR analysis and intermediated metabolites were identified by GCMS analysis. The surface-active molecules along with the enzymes played a crucial role in the biodegradation process.

Artesunate inhibits osteoclastogenesis through the miR-503/RANK axis.

Osteoporosis is a metabolic bone disease that is characterized by decreased bone density and strength due to excessive loss of bone protein and mineral content, which can be induced by increased osteoclast activity. Developing agents targeting osteoclast activation is considered to be the most effective method to reverse bone destruction and alleviate the pain caused by osteoporosis. MTT assay was conducted to detect the cell viability after artesunate treatment of RAW264.7 cells. TRACP staining and pit formation assays were performed to examine the TRACP-positive cells and pit-forming activity of osteoclasts. qRT-PCR and Western blot analysis were performed to assess the mRNA and protein expression levels of the osteoclastogenesis-related genes NFATc1, TRAP, and cathepsin k. The protein levels of RANK, p-Akt, p-p38, and p-ERK were examined by Western blotting. Luciferase reporter assay was conducted to determine whether miR-503 targeted RANK directly. Artesunate inhibited TRACP-positive cells and the pit-forming activity of osteoclasts. However, artesunate increased the expression of miR-503. Artesunate suppressed osteoclastogenesis-related gene expression and RANKL-induced activation of MAPKs and the AKT pathway. In addition, miR-503 inhibited RANK expression by directly targeting RANK during osteoclast differentiation. Artesunate inhibited osteoclastogenesis and osteoclast functions in vitro by regulating the miR-503/RANK axis and suppressing the MAPK and AKT pathways, which resulted in decreased expression of osteoclastogenesis-related markers.

Occurrence and distribution of neonicotinoid insecticides in surface water and sediment of the Guangzhou section of the Pearl River, South China.

Little information is available about the occurrence of neonicotinoid insecticides in surface water and sediment of the metropolitan regions around the rivers in China. Here we investigate the residual level of neonicotinoids in the Guangzhou section of the Pearl River. At least one or two neonicotinoids was detected in each surface water and sediment, and the total amount of neonicotinoids (∑5neonics) in surface water ranged from 92.6 to 321 ng/L with a geometric mean (GM) of 174 ng/L. Imidacloprid, thiamethoxam and acetamiprid were three frequently detected neonicotinoids (100%) from surface water. As for the sediment, total concentration was varied between 0.40 and 2.59 ng/g dw with a GM of 1.12 ng/g dw, and acetamiprid and thiacloprid were the common sediment neonicotinoids. Western and Front river-route of the Guangzhou section of the Pearl River suffered a higher neonicotinoids contamination than the Rear river-route, resulting from more effluents of WWTPs receiving, and intensive commercial and human activities. Level of residual neonicotinoids in surface water was significantly correlated with the water quality (p < 0.01), especially items of pH, DO and ORP, and nitrogen and phosphorus contaminants. Compared with reports about residual neonicotinoids in water and sediment previously, the metropolitan regions of the Guangzhou could be confronted with a moderate contamination and showed serious ecological threats (even heavier than the Pearl Rivers). Our results will provide valuable data for understanding of neonicotinoids contamination in the Pearl River Delta and be helpful for further assessing environmental risk of neonicotinoids.

Impacts of high ß-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via (13)C metabolic flux analysis.

The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high ß-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of ß-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.

Quantitative metabolic flux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil.

A metabolic network was constructed for the Acremonium chrysogenum cultivation fed with soybean oil. Metabolic flux analysis indicated that the shift from exponential growth to rapid cephalosporin C (CPC) formation was accompanied by 1.63- and 5-fold carbon flux enlargement in TCA cycle and glyoxylate by-pass, respectively. The flux via pentose phosphate pathway branch was little affected during the rapid CPC formation period; the contributory explanation was that 35.6% of NADPH was consumed in the dissimilation of fatty acids. Estimation of NADPH, ATP generation, and consumption demonstrated that, with soybean oil as carbon source in rapid CPC formation phase, the NADPH consumed in fatty acid catabolism was fourfold greater than that used in the CPC biosynthesis-relevant part; simultaneously, more than 90% energy spent was not directly related to the CPC formation. Therefore, the improvement of CPC production yield through optimization of the NADPH, ATP generation, and consumption was put forward.

Synthesis and herbicidal activity of isoindoline-1,3-dione substituted benzoxazinone derivatives containing a carboxylic ester group.

A carboxylic ester group was introduced to three series of isoindolinedione substituted benzoxazinone derivatives. Some of these analogues exhibited good herbicidal activities, and the injury symptoms against weeds included leaf cupping, crinkling, bronzing, and necrosis, typical of protox inhibitor herbicides. Structurally, they were classified as Chemical Group A (4-carboxylic ester group-6-isoindolinyl-benzoxazinones), B (4-carboxylic ester group-7-isoindolinyl-benzoxazinones), and C (4-carboxylic ester group-6- tetrahydroisoindolinyl-benzoxazinones). All of the tested compounds were structurally confirmed by (1)H NMR, IR, mass spectroscopy, and elemental analysis. Preliminary bioassay data of these three classes of compounds showed that, in general, the order of the herbicidal effectiveness is C > A > B. Several of the lead compounds, for example, C10 (methyl 2-(6-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-7-fluoro-2-methyl-3-oxo-2H-benzo[b][1,4] oxazin-4(3H)-yl) propano-ate), C12 (ethyl 2-(6-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-7-fluoro-2- methyl-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl) propanoate), and C13 (ethyl 2-(6-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-7-fluoro-2-methyl-3-oxo-2H-benzo-[b][1,4]oxazin-4(3H)-yl) butanoate), exhibited greater than 80% control at 75 g a.i./ha in both pre- and postemergence treatments against dicotyledonous weeds, such as Abutilon theophrasti Medic, Chenopodium album L., and Amaranthus ascendens L., and monocotyledon weeds, such as Digitaria sanguinalis L., Echinochloa crus-galli L., and Setaria viridis L. On the basis of advanced screening tests and crop selectivity, compounds C10, C12, and C13 are safer to crops than flumioxazin. Compounds C10, C12, and C13 are potent to develop as pre-emergent herbicides used in peanut, soybean, maize, and cotton fields.