Metabolomics approach used for understanding temperature-related pectinase activity in Bacillus licheniformis DY2.

Pectinases are enzymes that catalyze pectin degradation. There is a global demand for pectinases because of their wide utility and catalytic efficiency. Optimization of the fermentation process to increase the pectolytic enzyme activity is generally practiced to lower process costs, but whether temperature influences the metabolome, enhancing pectinase activity, is not known. Here, we developed a metabolomics approach to explore it. The activity of P-DY2 pectinase produced by Bacillus licheniformis DY2 was higher in cells grown at 30°C than those grown at 37°C. Differential metabolome analysis revealed fluctuating tricarboxylic acid (TCA) cycle at 30°C. Consistently, the transcripts of TCA cycle genes and activities of pyruvate dehydrogenase and α-Ketoglutaric dehydrogenase were lower at 30°C than 37°C. Furthermore, inhibition of pyruvate dehydrogenase and succinate dehydrogenase enhanced the activity of P-DY2, supporting the conclusion that the inactivated pyruvate metabolism and TCA cycle were required for pectinase activity, and that P-DY2 was TCA cycle-independent. Collectively, these findings indicated that fermentation temperature affected P-DY2 activity by metabolic modulation, with an inactivated TCA cycle as a characteristic feature of high P-DY2 activity. More importantly, the present study highlights an approach of promoting pectinase activity through metabolic modulation by using metabolic pathway inhibitors.

Gas Chromatography/Mass Spectrometry Metabolomics of Urine and Serum from Nonhuman Primates Exposed to Ionizing Radiation: Impacts on the Tricarboxylic Acid Cycle and Protein Metabolism.

Ionizing radiation (IR) directly damages cells and tissues or indirectly damages them through reactive free radicals that may lead to longer term adverse sequelae such as cancers, persistent inflammation, or possible death. Potential exposures include nuclear reactor accidents, improper disposal of equipment containing radioactive materials or medical errors, and terrorist attacks. Metabolomics (comprehensive analysis of compounds <1 kDa) by mass spectrometry (MS) has been proposed as a tool for high-throughput biodosimetry and rapid assessment of exposed dose and triage needed. While multiple studies have been dedicated to radiation biomarker discovery, many have utilized liquid chromatography (LC) MS platforms that may not detect particular compounds (e.g., small carboxylic acids or isomers) that complementary analytical tools, such as gas chromatography (GC) time-of-flight (TOF) MS, are ideal for. The current study uses global GC-TOF-MS metabolomics to complement previous LC-MS analyses on nonhuman primate biofluids (urine and serum) 7 days after exposure to 2, 4, 6, 7, and 10 Gy IR. Multivariate data analysis was used to visualize differences between control and IR exposed groups. Univariate analysis was used to determine a combined 26 biomarkers in urine and serum that significantly changed after exposure to IR. We found several metabolites involved in tricarboxylic acid cycle function, amino acid metabolism, and host microbiota that were not previously detected by global and targeted LC-MS studies.

Urinary metabolic signatures and early triage of acute radiation exposure in rat model.

After a large-scale radiological accident, early-response biomarkers to assess radiation exposure over a broad dose range are not only the basis of rapid radiation triage, but are also the key to the rational use of limited medical resources and to the improvement of treatment efficiency. Because of its high throughput, rapid assays and minimally invasive sample collection, metabolomics has been applied to research into radiation exposure biomarkers in recent years. Due to the complexity of radiobiological effects, most of the potential biomarkers are both dose-dependent and time-dependent. In reality, it is very difficult to find a single biomarker that is both sensitive and specific in a given radiation exposure scenario. Therefore, a multi-parameters approach for radiation exposure assessment is more realistic in real nuclear accidents. In this study, untargeted metabolomic profiling based on gas chromatography-mass spectrometry (GC-MS) and targeted amino acid profiling based on LC-MS/MS were combined to investigate early urinary metabolite responses within 48 h post-exposure in a rat model. A few of the key early-response metabolites for radiation exposure were identified, which revealed the most relevant metabolic pathways. Furthermore, a panel of potential urinary biomarkers was selected through a multi-criteria approach and applied to early triage following irradiation. Our study suggests that it is feasible to use a multi-parameters approach to triage radiation damage, and the urinary excretion levels of the relevant metabolites provide insights into radiation damage and repair.

Variations of metabolites and proteome in Lonicera japonica Thunb. buds and flowers under UV radiation.

Lonicera japonica Thunb., also known as Jin Yin Hua and Japanese honeysuckle, is used as a herbal medicine in Asian countries. Its flowers have been used in folk medicine in the clinic and in making food or healthy beverages for over 1500years in China. To investigate the molecular processes involved in L. japonica development from buds to flowers exposed to UV radiation, a comparative proteomics analysis was performed. Fifty-four proteins were identified as differentially expressed, including 42 that had increased expression and 12 that had decreased expression. The levels of the proteins related to glycolysis, TCA/organic acid transformation, major carbohydrate metabolism, oxidative pentose phosphate, stress, secondary metabolism, hormone, and mitochondrial electron transport were increased during flower opening process after exposure to UV radiation. Six metabolites in L. japonica buds and flowers were identified and relatively quantified using LC-MS/MS. The antioxidant activity was performed using a 1,1-diphenyl-2-picrylhydrazyl assay, which revealed that L. japonica buds had more activity than the UV irradiated flowers. This suggests that UV-B radiation induces production of endogenous ethylene in L. japonica buds, thus facilitating blossoming of the buds and activating the antioxidant system. Additionally, the higher metabolite contents and antioxidant properties of L. japonica buds indicate that the L. japonica bud stage may be a more optimal time to harvest than the flower stage when using for medicinal properties.

In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid "cycle" in illuminated leaves.

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, (13)C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA "cycle" does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.