Investigation of the Metabolic Flux Analysis Method for Overdetermined System

With the xanthan synthesis in Xanthomoaas campestris as an example, two methods for metabolic flux analysis of overdetermined system, the experimental data error minimization method and the equation error minimization method, are compared from their mathematical basis, rationality of the results and the easiness of computation. The results show that the experimental data error minimization method is appropriate in metabolic flux analysis of overdetermined system.

Engineering and finetuning expression of SerC for balanced metabolic flux in vitamin B6 production

Vitamin B6 plays a crucial role in cellular metabolism and stress response,making it an essential component for growth in all known organisms.However,achieving efficient biosynthesis of vitamin B6 faces the challenge of maintaining a balanced distribution of metabolic flux between growth and production.In this study,our focus is on addressing this challenge through the engineering of phosphoserine aminotransferase(SerC)to resolve its redundancy and promiscuity.The enzyme SerC was semi-designed and screened based on sequences and predicted kcat values,respectively.Mutants and heterologous proteins showing potential were then fine-tuned to optimize the production of vitamin B6.The resulting strain enhances the production of vitamin B6,indicating that different fluxes are distributed to the biosynthesis pathway of serine and vitamin B6.This study presents a promising strategy to address the challenge posed by multifunctional enzymes,with significant implications for enhancing biochemical production through engineering processes.

Metabolic flux analysis on arachidonic acid fermentation

The analysis of flux distributions in metabolic networks has become an important approach for understand-ing the fermentation characteristics of the process.A model of metabolic flux analysis of arachidonic acid(AA)synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source.During the expo-nential,decelerating and stationary phase,carbon fluxes to AA were 3.28%,8.80%and 6.97%,respectively,with sufficient N-source broth based on the flux of glucose uptake,and those were increased to 3.95%,19.21%and 39.29%,respectively,by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05% NaNO_(3) at 96 h.Eventually AA yield was increased from 1.3 to 3.5 g·L^(−1).These results suggest a way to improve AA fermentation,that is,fermentation with limited N-source broth and adding low concentration N-source during the stationary phase.

Metabolic flux simulation of microbial systems based on optimal planning algorithms

The genomic scale metabolic networks of the microorganisms can be constructed based on their genome se-quences,functional annotations,and biochemical reactions,reflecting almost all of the metabolic functions.Mathematical simulations of metabolic fluxes could make these functions be visualized,thereby providing guidance for rational engineering design and experimental operations.This review summarized recently devel-oped flux simulation algorithms of microbial systems.For the single microbial systems,the optimal planning algorithm has low complexity because there is no interaction between microorganisms,and it can quickly simulate the stable metabolic states through the pseudo-steady hypothesis.Besides,the experimental conditions of single microbial systems are easier to reach or close to the optimal states of simulation,compared with pol-ymicrobial systems.The polymicrobial culture systems could outcompete the single microbial systems as they could relieve metabolic pressure through metabolic division,resource exchange,and complex substrate co-utilization.Besides,they provide varieties of intracellular production environments,which render them the po-tential to achieve efficient bioproduct synthesis.However,due to the quasi-steady hypothesis that restricts the simulation of the dynamic processes of microbial interactions and the algorithm complexity,there are few re-searches on simulation algorithms of polymicrobial metabolic fluxes.Therefore,this review also analyzed and combed the microbial interactions based on the commonly used hypothesis of maximizing growth rates,and studied the strategies of coupling interactions with optimal planning simulations for metabolism.Finally,this review provided new insights into the genomic scale metabolic flux simulations of polymicrobial systems.

Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide

There is an urgent need to elucidate the pathogenesis of myocardial ischemia(MI)and potential drug treatments.Here,the anti-MI mechanism and material basis of Ginkgo biloba L.extract(GBE)were studied from the perspective of energy metabolism flux regulation.Metabolic flux analysis(MFA)was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol(ISO)-induced ischemia-like cardiomyocytes.It showed that[U-13 C]glucose derived m+2 isotopologues from the upstream tricarboxylic acid(TCA)cycle metabolites were markedly accumulated in ISO-injured cardiomyocytes,but the opposite was seen for the downstream metabolites,while their total cellular concentrations were increased.This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources.A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction.It showed that bilobalide protected against impaired mitochondrial aerobic respiration.MFA also showed that bilobalide significantly modulated the TCA cycle flux,reduced abnormal metabolite accumulation,and balanced the demand of different carbon sources.Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells.Bilobalide’s efficacy was verified by in vivo experiments in rats.This is the first report to show that bilobalide,the active ingredient of GBE,protects against MI by rescuing impaired TCA cycle flux.This provides a new mechanism and potential drug treatment for MI.It also shows the potential of MFA/Seahorse combination as a powerful strategy for pharmacological research on herbal medicine.

Analysis of Data on Xanthan Fermentation in Stationary Phase Using Black Box and Metabolic Network Models

The xanthan fermentation data in the stationary phase was analyzed using the black box and the metabolic network models. The data consistency is checked through the elemental balance in the black box model. In the metabolic network model, the metabolic flux distribution in the cell is calculated using the metabolic flux analysis method, then the maintenance coefficients is calculated.

Effects of Propionate on Streptolydigin Production and Carbon Flux Distribution in Streptomyces lydicus AS 4.2501

To achieve higher antibiotic streptolydigin productivity through metabolic regulation, propionate was fed during the fermentation of Streptomyces lydicus AS 4.2501. The effects of propionate feeding on streptolydigin production and intracellular fluxes were investigated. The highest streptolydigin production yield of 95.10mg·L-1 was obtained when 2mmol·L-1 of sodium propionate was added at 60h of cultivation into shake-flask culture. This yield is 23.06% higher when compared to that of a batch culture without propionate supplementation. It was also found that when propionate was added, much more organic acids were excreted. Metabolic flux analysis was per-formed and it demonstrated that the carbon fluxes of the pentose phosphate pathway and the anaplerotic reaction were significantly increased after propionate feeding. The carbon flux from pyruvate to acetyl-CoA was determined to be 24.7, which was 12.27% higher than that in the batch culture. This study indicated that the glu-cose-6-phosphate and pyruvate nodes were potential bottlenecks for increasing streptolydigin productivity. Potential targets and strategies that could be manipulated through genetic and process engineering to increase the production of streptolydigin were also suggested.

Simulation of Flux Distribution in Central Metabolism of Saccharo-myces cerevisiae by Hybridized Genetic Algorithm

A scheme of investigating the intracellular metabolic fluxes in central metabolism of Saccharomyces cerevisiae based on isotope model and tracer experiment was developed. The metabolic model applied in this study includes the Embden-Meyerhof-Parnas pathway,the pentose phosphate pathway,the tricarboxylic acid cycle,CO2 anaplerotic reactions,ethanol and acetate formation,and pathways involved in amino acid synthesis. The approach of hybridized genetic algorithm combined with the sequential simplex technique was used to optimize a quadratic error function without the requirement of the information on the partial derivatives. The impact of some key pa-rameters on the algorithm was studied. This approach was proved to be rapid and numerically stable in the analysis of the central metabolism of S.cerevisiae.

Optimization of the bioconversion of glycerol to ethanol using Escherichia coli by implementing a bi-level programming framework for proposing gene transcription control strategies based on genetic algorithms

In silico approaches for metabolites optimization have been derived from the flood of sequenced and annotated genomes. However, there exist still numerous degrees of freedom in terms of optimization algorithm approaches that can be exploited in order to enhance yield of processes which are based on biological reactions. Here, we propose an evolutionary approach aiming to suggest different mutant for augmenting ethanol yield using glycerol as substrate in Escherichia coli. We found that this algorithm, even though is far from providing the global optimum, is able to uncover genes that a global optimizer would be incapable of. By over-expressing accB, eno, dapE, and accA mutants in ethanol production was augmented up to 2 fold compared to its counterpart E. coli BW25113.

Metabolic reprogramming in tumors:Contributions of the tumor microenvironment

The genetic alterations associated with cell transformation are in large measure expressed in the metabolic phenotype as cancer cells proliferate and change their local environment,and prepare for metastasis.Qualitatively,the fundamental biochemistry of cancer cells is generally the same as in the untransformed cells,but the cancer cells produce a local environment,the TME,that is hostile to the stromal cells,and compete for nutrients.In order to proliferate,cells need sufficient nutrients,either those that cannot be made by the cells themselves,or must be made from simpler precursors.However,in solid tumors,the nutrient supply is often limiting given the potential for rapid proliferation,and the poor quality of the vasculature.Thus,cancer cells may employ a variety of strategies to obtain nutrients for survival,growth and metastasis.Although much has been learned using established cell lines in standard culture conditions,it is becoming clear from in vivo metabolic studies that this can also be misleading,and which nutrients are used for energy production versus building blocks for synthesis of macromolecules can vary greatly from tumor to tumor,and even within the same tumor.Here we review the operation of metabolic networks,and how recent understanding of nutrient supply in the TME and utilization are being revealed using stable isotope tracers in vivo as well as in vitro.

Inhibition of xanthine oxidase alleviated pancreatic necrosis via HIF-1α-regulated LDHA and NLRP3 signaling pathway in acute pancreatitis

Acute pancreatitis(AP)is a potentially fatal condition with no targeted treatment options.Although inhibiting xanthine oxidase(XO)in the treatment of AP has been studied in several experimental models and clinical trials,whether XO is a target of AP and what its the main mechanism of action is remains unclear.Here,we aimed to re-evaluate whether XO is a target aggravating AP other than merely generating reactive oxygen species that trigger AP.We first revealed that XO expression and enzyme activity were significantly elevated in the serum and pancreas of necrotizing AP models.We also found that allopurinol and febuxostat,as purine-like and non-purine XO inhibitors,respectively,exhibited protective effects against pancreatic acinar cell death in vitro and pancreatic damage in vivo at different doses and treatment time points.Moreover,we observed that conditional Xdh overexpression aggravated pancreatic necrosis and severity.Further mechanism analysis showed that XO inhibition restored the hypoxia-inducible factor 1-alpha(HIF-1α)-regulated lactate dehydrogenase A(LDHA)and NOD-like receptor family pyrin domain containing 3(NLRP3)signaling pathways and reduced the enrichment of^(13)C_(6)-glucose to^(13)C_(3)-lactate.Lastly,we observed that clinical circulatory XO activity was significantly elevated in severe cases and correlated with C-reactive protein levels,while pancreatic XO and urate were also increased in severe AP patients.These results together indicated that proper inhibition of XO might be a promising therapeutic strategy for alleviating pancreatic necrosis and preventing progression of severe AP by downregulating HIF-1α-mediated LDHA and NLRP3 signaling pathways.

Integrating Urban Metabolism With Landscape Disciplines by Applying Technomass

Urban metabolism provides a robust framework for analyzing urban development and its impacts.However,several conceptual and operational shortcomings have constrained the application of urban metabolism in understanding the overall urban processes,limiting the transfer of its potential benefits to design and planning.This article systematically analysed the rationale of the current urban metabolism models,focusing on four prevailing shortcomings from a transdisciplinary perspective:1)utilizing an isolated state approach,which treats urban systems as isolated from other ecosystems;2)ignoring internal processes within urban systems,known as the black box paradox;3)employing a linear material approach that focuses on the path of single materials;and 4)overlooking the material productivity of urban systems,where energy and materials entering the system are used to reproduce the urban material structure and generate goods and tradable products.While these issues have been identified individually in existing scientific literature,there is a lack of holistic solutions.This article proposes an enhanced urban metabolism analytical approachDthe ecosystem approach applying atechnomassoDto address these shortcomings and provide practical solutions in landscape architecture and planning disciplines for sustainable urban development.

Contribution of phenylpropanoid metabolism to plant development and plant–environment interactions

Phenylpropanoid metabolism is one of the most important metabolisms in plants, yielding more than 8,000 metabolites contributing to plant development and plant-environment interplay.Phenylpropanoid metabolism materialized during the evolution of early freshwater algae that were initiating terrestrialization and land plants have evolved multiple branches of this pathway, which give rise to metabolites including lignin, flavonoids, lignans, phenylpropanoid esters, hydroxycinnamic acid amides, and sporopollenin.Recent studies have revealed that many factors participate in the regulation of phenylpropanoid metabolism, and modulate phenylpropanoid homeostasis when plants undergo successive developmental processes and are subjected to stressful environments. In this review, we summarize recent progress on elucidating the contribution of phenylpropanoid metabolism to the coordination of plant development and plant–environment interaction, and metabolic flux redirection among diverse metabolic routes. In addition, our review focuses on the regulation of phenylpropanoid metabolism at the transcriptional, post-transcriptional, post-translational,and epigenetic levels, and in response to phytohormones and biotic and abiotic stresses.

Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana

Anthocyanins, a group of L-phenylalanine （Phe）-derived flavonoids, have been demonstrated to play impor- tant roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases （ADTs）, the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adtl adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression ofADT2, hyperaccumulate Phe and produce elevated level of antho- cyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sus- taining anthocyanin biosynthesis.