metabolic flux
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Author(s):  
Renjun Mao ◽  
Zhenqing Bai ◽  
Jiawen Wu ◽  
Ruilian Han ◽  
Xuemin Zhang ◽  
...  

Senna obtusifolia is a famous medicinal plant that is widely used in Asian countries. Its seed plays an important role in the treatment of many diseases because it contains various anthraquinones and flavonoids. Our previous studies have indicated that three space environment-induced S. obtusifolia lines (SP-lines) i.e., QC10, QC29, and QC46, have higher seed yield and aurantio-obtusin (AO) content. However, the underlying mechanism of higher AO content in SP-lines is still unknown. Herein, transcriptome sequencing and HPLC were employed to analyze the differences between SP-lines and ground control (GC3) and elucidate the regulatory mechanisms of AO accumulation in SP-lines. The results show that 4002 differentially expressed genes (DEGs) were identified in SP-lines versus (vs.) GC3. DEGs in the QC10 vs. GC3, QC29 vs. GC3, and QC46 vs. GC3 comparisons were classified into 28, 36, and 81 GO terms and involved in 63, 74, and 107 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene expression analysis revealed that DEGs involved in anthraquinone pathways were significantly elevated in QC10 and QC46. Integrating the results of GO annotation, KEGG enrichment, and gene expression analysis, we propose that the elevated genes such as DAHPS, DHQS, and MenB enhance the metabolic flux in the anthraquinone pathway and promote AO content in QC10 and QC46. Taken together, this study elucidated the mechanism of AO content in SP-lines and provides valuable genetic information for S. obtusifolia. In addition, to the best of our knowledge, this study presents the first transcriptome analysis of environment-induced medicinal plants and paves the way to select elite S. obtusifolia varieties in the future.


2022 ◽  
Author(s):  
Paul Dijkstra ◽  
Weichao Wu ◽  
Michaela Dippold ◽  
Egbert Schwartz ◽  
Bruce Hungate ◽  
...  

Abstract Biochemistry is an essential yet often undervalued aspect of soil ecology, especially in soil C cycling. We assume based on tradition, intuition or hope that the complexity of biochemistry is confined to the microscopic world, and can be ignored when dealing with whole soil systems. This opinion paper draws attention to patterns caused by basic biochemical processes that permeate the world of ecosystem processes. From these patterns, we can estimate activities of the biochemical reactions of the central C metabolic network and gain insights into the ecophysiology of microbial biosynthesis and growth and maintenance energy requirements; important components of Carbon Use Efficiency (CUE).The biochemical pathways used to metabolize glucose vary from soil to soil, with mostly glycolysis in some soils, and pentose phosphate or Entner-Doudoroff pathways in others. However, notwithstanding this metabolic diversity, glucose use efficiency is high and thus substrate use for maintenance energy and overflow respiration is low in these three soils. These results contradict current dogma based on four decades of research in soil ecology. We identify three main shortcomings in our current understanding of substrate use efficiency: 1) in numeric and conceptual models, we lack appreciation of the strategies that microbes employ to quickly reduce energy needs in response to starvation; 2) production of exudates and microbial turnover affect whole-soil CUE more than variation in maintenance energy demand; and 3) whether tracer experiments can be used to measure the long-term substrate use efficiency of soil microbial communities depends critically on the ability of non-growing cells to take up tracer substrates, how biosynthesis responds to these substrates, as well as on how cellular activities scale to the community level.To move the field of soil ecology forward, future research must consider the details of microbial ecophysiology and develop new tools that enable direct measurement of microbial functioning in intact soils. We submit that 13C metabolic flux analysis is one of those new tools.


2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiao Bu ◽  
Jing‑Yuan Lin ◽  
Chang‑Qing Duan ◽  
Mattheos A. G. Koffas ◽  
Guo‑Liang Yan

Abstract Background The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve β-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. Results The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of β-carotene in S. cerevisiae. However, due to the competition for precursor between β-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on β-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in β-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to β-carotene pathway and achieved additional 31.7% increase in β-carotene content without adversely affecting cell growth. By inducing an extra constitutive β-carotene synthesis pathway for further conversion precursor farnesol to β-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of β-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. Conclusions This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae. Graphical Abstract


2021 ◽  
Vol 2021 ◽  
pp. 1-22
Author(s):  
Pengfei Yang ◽  
Xiangxia Luo ◽  
Jin Li ◽  
Tianyi Zhang ◽  
Xiaoling Gao ◽  
...  

Glutamine metabolism provides energy to tumor cells and also produces reactive oxygen species (ROS). Excessive accumulation of ROS can damage mitochondria and eventually lead to cell death. xCT (SLC7A11) is responsible for the synthesis of glutathione in order to neutralize ROS. In addition, mitophagy can remove damaged mitochondria to keep the cell alive. Ionizing radiation kills tumor cells by causing the accumulation of ROS, which subsequently induces nuclear DNA damage. With this in mind, we explored the mechanism of intracellular ROS accumulation induced by ionizing radiation and hypothesized new methods to enhance the effect of radiotherapy. We used MCF-7 breast cancer cells and HCT116 colorectal cancer cells in our study. The above-mentioned cells were irradiated with different doses of X-rays or carbon ions. Clone formation assays were used to detect cell proliferation, enzyme-linked immunosorbent assay (ELISA) detected ATP, and glutathione (GSH) production, while the expression of proteins was detected by Western blot and quantitative real-time PCR analysis. The production of ROS was detected by flow cytometry, and immunofluorescence was used to track mitophagy-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays in order to further explore the protein expression found in tumors with the use of immunohistochemistry. Ionizing radiation increased the protein expressions of ASCT2, GLS, and GLUD in order to upregulate the glutamine metabolic flux in tumor cells. This caused an increase in ATP secretion. Meanwhile, ionizing radiation inhibited the expression of the xCT (SLC7A11) protein and reduced the generation of glutathione, leading to excessive accumulation of intracellular ROS. The mitophagy inhibitor, or knockdown Parkin gene, is able to enhance the ionizing radiation-induced ROS production and increase nucleus DNA damage. This combined treatment can significantly improve the killing effect of radiation on tumor cells. We concluded that ionizing radiation could upregulate the glutamine metabolic flux and enhance ROS accumulation in mitochondria. Ionizing radiation also decreased the SLC7A11 expression, resulting in reduced GSH generation. Therefore, inhibition of mitophagy can increase ionizing radiation-induced cell death.


Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 28
Author(s):  
Gerardo Della Sala ◽  
Consiglia Pacelli ◽  
Francesca Agriesti ◽  
Ilaria Laurenzana ◽  
Francesco Tucci ◽  
...  

Defining the metabolic phenotypes of cancer-initiating cells or cancer stem cells and of their differentiated counterparts might provide fundamental knowledge for improving or developing more effective therapies. In this context we extensively characterized the metabolic profiles of two osteosarcoma-derived cell lines, the 3AB-OS cancer stem cells and the parental MG-63 cells. To this aim Seahorse methodology-based metabolic flux analysis under a variety of conditions complemented with real time monitoring of cell growth by impedentiometric technique and confocal imaging were carried out. The results attained by selective substrate deprivation or metabolic pathway inhibition clearly show reliance of 3AB-OS on glycolysis and of MG-63 on glutamine oxidation. Treatment of the osteosarcoma cell lines with cisplatin resulted in additive inhibitory effects in MG-63 cells depleted of glutamine whereas it antagonized under selective withdrawal of glucose in 3AB-OS cells thereby manifesting a paradoxical pro-survival, cell-cycle arrest in S phase and antioxidant outcome. All together the results of this study highlight that the efficacy of specific metabolite starvation combined with chemotherapeutic drugs depends on the cancer compartment and suggest cautions in using it as a generalizable curative strategy.


2021 ◽  
Author(s):  
Jeffrey J Czajka ◽  
Deepanwita Banerjee ◽  
Thomas T Eng ◽  
Javier Menasalvas ◽  
Chunsheng Yan ◽  
...  

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ning Zhang ◽  
Limin Song ◽  
Yang Xu ◽  
Xueyuan Pei ◽  
Ben F Luisi ◽  
...  

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalysed by FoSir5, that support conidial germination in F. oxysporum.


2021 ◽  
Vol 8 ◽  
Author(s):  
Sophie Landon ◽  
Oliver Chalkley ◽  
Gus Breese ◽  
Claire Grierson ◽  
Lucia Marucci

Whole-cell modelling is a newly expanding field that has many applications in lab experiment design and predictive drug testing. Although whole-cell model output contains a wealth of information, it is complex and high dimensional and thus hard to interpret. Here, we present an analysis pipeline that combines machine learning, dimensionality reduction, and network analysis to interpret and visualise metabolic reaction fluxes from a set of single gene knockouts simulated in the Mycoplasma genitalium whole-cell model. We found that the reaction behaviours show trends that correlate with phenotypic classes of the simulation output, highlighting particular cellular subsystems that malfunction after gene knockouts. From a graphical representation of the metabolic network, we saw that there is a set of reactions that can be used as markers of a phenotypic class, showing their importance within the network. Our analysis pipeline can support the understanding of the complexity of in silico cells without detailed knowledge of the constituent parts, which can help to understand the effects of gene knockouts and, as whole-cell models become more widely built and used, aid genome design.


2021 ◽  
Author(s):  
Rosemary Yu ◽  
Egor Vorontsov ◽  
Carina Sihlbom ◽  
Jens Nielsen

Metabolic flux can be regulated by a variety of different mechanisms, but the organization of these mechanisms within the metabolic network has remained unknown. Here we test the hypothesis that flux control mechanisms are not distributed randomly in the metabolic network, but rather organized according to pathway. Combining proteomics, phosphoproteomics, and metabolic modeling, we report the largest collection of flux-enzyme-phosphoenzyme relationships to date in Saccharomyces cerevisiae. In support of the hypothesis, we show that (i) amino acid metabolic pathways are predominantly regulated by enzyme abundance stemming from transcriptional regulation; (ii) upper glycolysis and associated pathways, by inactivating enzyme phosphorylation; (iii) lower glycolysis and associated pathways, by activating enzyme phosphorylation; and (iv) glycolipid/glycophospholipid pathways, by a combination of enzyme phosphorylation and metabolic compartmentalization. We delineate the evolutionary history for the observed organization of flux control mechanisms in yeast central metabolic pathways, furthering our understanding of the regulation of metabolism and its evolution.


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