Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

Towards catalyst compartimentation in combined chemo- and biocatalytic processes: Immobilization of alcohol dehydrogenases for the diastereoselective reduction of a β-hydroxy ketone obtained from an organocatalytic aldol reaction

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis

The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities.     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因,我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析,发现酵母在对数生长中期的表达谱非常稳定,而一旦进入对数生长后期.则出现明显的代谢重构现象.许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调;而许多涉及酵母转座和DNA重组的基因则表达下调;一些中心代谢途径也发生了代谢重构.包括:琥珀酸和α-酮戊二酸生成途径基因的一致上调,都与氨基酸合成和代谢相关基因表达的结果相吻合.结果表明:由于氨基酸合成的需求量增加,进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环,导致酒精的生产速率降低.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. Redirection of metabolic fluxes

The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.     

Engineering of Corynebacterium glutamicum for high-level γ-aminobutyric acid production from glycerol by dynamic metabolic control

Synthetic biology seeks to reprogram microbial cells for efficient production of value-added compounds from low-cost renewable substrates. A great challenge of chemicals biosynthesis is the competition between cell metabolism and target product synthesis for limited cellular resource. Dynamic regulation provides an effective strategy for fine-tuning metabolic flux to maximize chemicals production. In this work, we created a tunable growth phase-dependent autonomous bifunctional genetic switch (GABS) by coupling growth phase responsive promoters and degrons to dynamically redirect the carbon flux for metabolic state switching from cell growth mode to production mode, and achieved high-level GABA production from low-value glycerol in Corynebacterium glutamicum. A ribosome binding sites (RBS)-library-based pathway optimization strategy was firstly developed to reconstruct and optimize the glycerol utilization pathway in C. glutamicum, and the resulting strain CgGly2 displayed excellent glycerol utilization ability. Then, the initial GABA-producing strain was constructed by deleting the GABA degradation pathway and introducing an exogenous GABA synthetic pathway, which led to 5.26 g/L of GABA production from glycerol. In order to resolve the conflicts of carbon flux between cell growth and GABA production, we used the GABS to reconstruct the GABA synthetic metabolic network, in which the competitive modules of GABA biosynthesis, including the tricarboxylic acid (TCA) cycle module and the arginine biosynthesis module, were dynamically down-regulated while the synthetic modules were dynamically up-regulated after sufficient biomass accumulation. Finally, the resulting strain G7-1 accumulated 45.6 g/L of GABA with a yield of 0.4 g/g glycerol, which was the highest titer of GABA ever reported from low-value glycerol. Therefore, these results provide a promising technology to dynamically balance the metabolic flux for the efficient production of other high value-added chemicals from a low-value substrate in C. glutamicum.     

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

Effects of Varying Nitrogen Sources on Amino Acid Synthesis Costs in Arabidopsis thaliana under Different Light and Carbon-Source Conditions

Plants as sessile organisms cannot escape their environment and have to adapt to any changes in the availability of sunlight and nutrients. The quantification of synthesis costs of metabolites, in terms of consumed energy, is a prerequisite to understand trade-offs arising from energetic limitations. Here, we examine the energy consumption of amino acid synthesis in Arabidopsis thaliana. To quantify these costs in terms of the energy equivalent ATP, we introduce an improved cost measure based on flux balance analysis and apply it to three state-of-the-art metabolic reconstructions to ensure robust results. We present the first systematic in silico analysis of the effect of nitrogen supply (nitrate/ammonium) on individual amino acid synthesis costs as well as of the effect of photoautotrophic and heterotrophic growth conditions, integrating day/night-specific regulation. Our results identify nitrogen supply as a key determinant of amino acid costs, in agreement with experimental evidence. In addition, the association of the determined costs with experimentally observed growth patterns suggests that metabolite synthesis costs are involved in shaping regulation of plant growth. Finally, we find that simultaneous uptake of both nitrogen sources can lead to efficient utilization of energy source, which may be the result of evolutionary optimization.     

Entamoeba histolytica: observations on metabolism based on the genome sequence

The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E. histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.     

植物对金属纳米材料胁迫响应的蛋白质组学研究进展

土壤已逐渐成为金属纳米材料在环境中的主要沉积库,植物作为生态系统中的重要组成部分,在纳米材料的归趋中起着十分重要的作用。研究表明,金属纳米材料会通过改变植物蛋白质的表达影响植物的生长发育。本文阐述了多种典型金属纳米材料胁迫引起的植物蛋白质组差异表达,总结了金属纳米材料胁迫下纳米材料理化性质、植物特征和胁迫条件等因素对植物蛋白质组响应的影响,综述了植物蛋白质组对金属纳米材料胁迫的响应机制,主要涉及能量合成与代谢响应、氨基酸合成与信息传导响应、氧化应激和胁迫防御响应,并对今后的研究方向进行了展望。     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.     

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, β‐oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP‐ribosylation by sirtuins and poly(ADP‐ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging‐associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age‐associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging‐related declines in NAD levels. We also demonstrated that Nmnat3‐overexpressing (Nmnat3 Tg) mice were protected against diet‐induced and aging‐associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Impact of balanced substrate flux on the metabolic process employing fuzzy logic during the cultivation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var.<Emphasis Type="Italic"> Galleriae</Emphasis>

Summary The insecticidal crystal protein (ICP) synthesized at the onset of sporulation by Bacillus thuringiensis var. galleriae (Btg) is lethal against specific pests. Attempts were made to enhance the synthesis of biomass and ICP by Btg employing process optimization strategies. The process optimization was carried out with residual glucose concentration control in a bench scale bioreactor. A fuzzy logic-based feedback control system for maintaining the residual glucose concentration at a constant level during cultivation was developed in LabVIEW. This control system indicated the possibilities in providing a balanced substrate flux during cultivation. The identified optimum level of 2.72 g/l in residual glucose concentration was maintained by fed-batch cultivation with glucose and yeast extract fed at equal concentration with the above control system. High cell density of 16.0 g/l with specific growth rate of 0.69 h^-1 was obtained during the cultivation. The balanced flux of substrate during cultivation has resulted in the enhanced synthesis of biomass and ICP. This optimized process could be commercially exploited by comparing the fluxes of basal compounds in different media sources used in fermentation.     

Characterization of NADPH–cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera

A complete cDNA encoding the NADPH–cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4 kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis–Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC₅₀ = 1.69 μM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC₅₀ value ranged from 15 to 90 μM) was also observed.     

Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.