不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析

[目的]探索L-苏氨酸生物合成机理及影响因素.[方法]建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布.[结果]5%溶氧条件下,25.5%碳架进入HMP途径,74.5%碳架进入糖酵解途径,获得33.9%质量转化率;20%溶氧条件下,58.08%碳架进入HMP途径,41.92%碳架进入糖酵解途径,获得46.5%质量转化率;[结论]与理想代谢流(88.23%质量转化率)相比,应从菌种改造和发酵控制方面通过改变6-磷酸葡萄糖异构酶借以增加HMP途径代谢流量,通过增加磷酸烯醇式丙酮酸羧化反应代谢流提高天冬氨酸族合成代谢流,减少TCA循环代谢流量,从而达到减少副产物生成,增加L-苏氨酸生物合成的目的.     

Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: flux variability analysis in relation to ¹³C metabolic flux analysis

Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large‐scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior ¹³C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH₄⁺ assimilation. Analysis of the light‐dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO₂ efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the ¹³C flux studies, ribulose‐1,5‐bisphosphate carboxylase activity is predicted to account fully for the light‐dependent changes in carbon balance.     

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h⁻¹. The ¹³C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO₂ production rate and the biomass concentration decreased, where the ¹³C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.     

Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements

The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by (13)C-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the ppc mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway significantly decreased in the ppc mutant.     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis

Carbon flow in Bacillus subtilis through the pentose phosphate (PP) pathway was modulated by overexpression of glucose-6-phosphate dehydrogenase (G6PDH) under the control of the inducible Pxyl promoter in B. subtilis PY. Alteration of carbon flow into the PP pathway will affect the availability of ribulose-5-phosphate (Ru5P) and the riboflavin yield. Overexpression of G6PDH resulted in the glucose consumption rate increasing slightly, while the specific growth rate was unchanged. An improvement by 25% ± 2 of the riboflavin production was obtained. Compared to by-products formation in flask culture, low acid production (acetate and pyruvate) and more acetoin were observed. Metabolic analysis, together with carbon flux redistribution, indicated that the PP pathway fluxes are increased in response to overexpression of G6PDH. Moreover, increased flux of the PP pathway is associated with an increased intracellular pool of Ru5P, which is a precursor for riboflavin biosynthesis. The high concentrations of Ru5P could explain the increased riboflavin production.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several “proof of principle” studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by ¹³C metabolic flux analysis (¹³C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report ¹³C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of ¹²C and ¹³C from unlabeled CO₂ and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism’s preferred mode under nitrogen-fixing conditions. The ¹³C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism’s distinct metabolic features under nitrogen-fixing and -non-fixing conditions.     

葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响*

麦芽糖和葡萄糖对粪产碱杆菌发酵合成凝胶多糖有着显著的影响,为了详细分析两种底物对凝胶多糖合成的影响机制,利用恒化培养实验及稳态碳平衡代谢分析,研究发现在稀释速率为0.1h^-1时,利用麦芽糖和葡萄糖为碳源底物的条件下粪产碱杆菌的微观代谢途径通量有较大的差异。以麦芽糖为底物时凝胶多糖的摩尔得率为53.8%,比葡萄糖为碳源时的摩尔得率(36.9%)高出了45.8%以上。同时以麦芽糖为碳源时HMP途径的绝对代谢通量比葡萄糖时的通量提升了40%以上。这条途径通量的增加,提升了NADPH还原力供给速率,促进了依赖于还原力NADPH的凝胶多糖合成途径通量,提升了碳源底物向产物的摩尔转化速率。而且代谢流分析结果显示ED途径通量和能量提供也是影响粪产碱杆菌凝胶多糖合成效率的关键因素。麦芽糖作为碳源底物过程中维持的较低的残留葡萄糖浓度解除了高葡萄糖浓度条件下对凝胶多糖合成的抑制,能够实现更高通量的ATP能量提供效率,更加促进了凝胶多糖合成通量。     

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.     

Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13C- and 31P-NMR spectroscopy

The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on ¹³C-labelled glucose, fructose and xylose in defined continuous cultures. The ¹³C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The ³¹P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate. Received: 7 January 1999 / Accepted: 22 March 1999     

Quantification of metabolism in Saccharomyces cerevisiae under hyperosmotic conditions using elementary mode analysis

Yeast metabolism under hyperosmotic stress conditions was quantified using elementary mode analysis to obtain insights into the metabolic status of the cell. The fluxes of elementary modes were determined as solutions to a linear program that used the stoichiometry of the elementary modes as constraints. The analysis demonstrated that distinctly different sets of elementary modes operate under normal and hyperosmotic conditions. During the adaptation phase, elementary modes that only produce glycerol are active, while elementary modes that yield biomass, ethanol, and glycerol become active after the adaptive phase. The flux distribution in the metabolic network, calculated using the fluxes in the elementary modes, was employed to obtain the flux ratio at key nodes. At the glucose 6-phosphate (G6P) node, 25% of the carbon influx was diverted towards the pentose phosphate pathway under normal growth conditions, while only 0.3% of the carbon flux was diverted towards the pentose phosphate pathway during growth at 1?M NaCl, indicating that cell growth is arrested under hyperosmotic conditions. Further, objective functions were used in the linear program to obtain optimal solution spaces corresponding to the different accumulation rates. The analysis demonstrated that while biomass formation was optimal under normal growth conditions, glycerol synthesis was closer to optimal during adaptation to osmotic shock.     

Use of the flux model of amino acid metabolism of Escherichia coli

A program implementing a flux model of Escherichia coli metabolism was used to analyze the effects of the addition of amino acids (tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, histidine, lysine, threonine, cysteine, methionine, arginine, proline) to minimal medium or media lacking nitrogen, carbon, or both. The overall response of the metabolic system to the addition of various amino acids to the minimal medium is similar. Glycolysis and the synthesis of pyruvate with its subsequent degradation to acetate via acetyl-CoA become more efficient, whereas the fluxes through the pentose phosphate pathway and the TCA cycle decrease. If amino acids are used as the sole source of carbon, nitrogen, or both, the changes in the flux distribution are determined mainly by the carbon limitation. The phosphoenolpyruvate to glucose-6-phosphate flux increases; the flux through the pentose phosphate path is directed towards ribulose-5-phosphate. Other changes are determined by the compounds that are the primary products of catabolism of the added amino acid.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.     

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

Metabolic flux analysis of<Emphasis Type="Italic"> pykF</Emphasis> gene knockout<Emphasis Type="Italic"> Escherichia coli</Emphasis> based on <Superscript>13</Superscript>C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

Metabolic flux analysis based on ¹³C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF^– mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF^– mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.     

基于代谢流量分布信息理解大肠杆菌中异柠檬酸裂解酶调节因子的代谢调控作用

基因的表达受不同的转录调节因子调节。大肠杆菌中的异柠檬酸裂解酶调节因子(IclR)能够抑制编码乙醛酸支路酶的aceBAK操纵子的表达。本研究基于代谢物的13C同位体物质分布来定量解析代谢反应,主要研究了iclR基因在大肠杆菌生理和代谢中的作用。大肠杆菌iclR基因缺失突变株的生长速率、糖耗速率和乙酸的产量相对于原始菌株都有所降低,但菌体得率略有增加。通过代谢途径的流量比率分析发现基因缺失株的乙醛酸支路得到了激活,33%的异柠檬酸流经了乙醛酸支路;戊糖磷酸途径的流量变小,使得CO2的生成量减少。同时,乙醛酸支路激活,但草酰乙酸形成磷酸烯醇式丙酮酸的流量基本不变,说明磷酸烯醇式丙酮酸-乙醛酸循环没有激活,没有过多的碳原子在磷酸烯醇式丙酮酸羧化激酶反应中以CO2形式排出,从而确保了菌体得率。葡萄糖利用速率的降低、乙酰辅酶A的代谢效率提高等使得iclR基因敲除菌的乙酸分泌较原始菌株有所降低。