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121.
This work aimed to identify the key operational factors that significantly affect succinate production by the high succinate producing Escherichia coli strain SBS550MG (pHL413), which bears mutations inactivating genes adhE ldhA iclR ackpta::Cm(R) and overexpresses the pyruvate carboxylase from Lactococcus lactis. The considered factors included glucose concentration, cell density, CO(2) concentration in the gas stream, pH, and temperature. The results showed that high glucose concentrations inhibited succinate production and that there is a compromise between the total succinate productivity and succinate specific productivity, where the total productivity increased with the increase in cell density and the specific productivity decreased with cell density, probably due to mass transfer limitation. On the other hand, a CO(2) concentration of 100% in the gas stream showed the highest specific succinate productivity, probably by favoring pyruvate carboxylation, increasing the OAA pool that later is converted into succinate. A full factorial design of experiments was applied to analyze the pH and temperature effects on succinate production in batch bioreactors, where succinate yield was not significantly affected by either temperature (37 to 43°C) or pH (6.5 to 7.5). Additionally, the temperature effect on succinate productivity and titer was not significant, in the range tested. On the other hand, a pH of 6.5 showed very low productivity, whereas pH values of 7.0 and 7.5 resulted in significantly higher specific productivities and higher titers. The increase on pH value from 7.0 to 7.5 did not show significant improvement. Then, pH 7.0 should be chosen because it involves a lower cost in base addition. 相似文献
122.
Bashir Sajo Mienda 《Journal of biomolecular structure & dynamics》2013,31(14):3680-3686
Genome-scale metabolic model (GEM) of Escherichia coli has been published with applications in predicting metabolic engineering capabilities on different carbon sources and directing biological discovery. The use of glycerol as an alternative carbon source is economically viable in biorefinery. The use of GEM for predicting metabolic gene deletion of lactate dehydrogenase (ldhA) for increasing succinate production in Escherichia coli from glycerol carbon source remained largely unexplored. Here, I hypothesized that metabolic gene knockout of ldhA in E. coli from glycerol could increase succinate production. A proof-of-principle strain was constructed and designated as E. coli BMS5 (ΔldhA), by predicting increased succinate production in E. coli GEM and confirmed the predicted outcomes using wet cell experiments. The mutant GEM (ΔldhA) predicted 11% increase in succinate production from glycerol compared to its wild-type model (iAF1260), and the E. coli BMS5 (ΔldhA) showed 1.05 g/l and its corresponding wild-type produced .05 g/l (23-fold increase). The proof-of-principle strain constructed in this study confirmed the aforementioned hypothesis and further elucidated the fact that E. coli GEM can prospectively and effectively predict metabolic engineering interventions using glycerol as substrate and could serve as platform for new strain design strategies and biological discovery. 相似文献
123.
Bashir Sajo Mienda Mohd Shahir Shamsir 《Journal of biomolecular structure & dynamics》2013,31(11):2380-2389
Systems metabolic engineering and in silico analyses are necessary to study gene knockout candidate for enhanced succinic acid production by Escherichia coli. Metabolically engineered E. coli has been reported to produce succinate from glucose and glycerol. However, investigation on in silico deletion of ptsG/b1101 gene in E. coli from glycerol using minimization of metabolic adjustment algorithm with the OptFlux software platform has not yet been elucidated. Herein we report what is to our knowledge the first direct predicted increase in succinate production following in silico deletion of the ptsG gene in E. coli GEM from glycerol with the OptFlux software platform. The result indicates that the deletion of this gene in E. coli GEM predicts increased succinate production that is 20% higher than the wild-type control model. Hence, the mutant model maintained a growth rate that is 77% of the wild-type parent model. It was established that knocking out of the ptsG/b1101 gene in E. coli using glucose as substrate enhanced succinate production, but the exact mechanism of this effect is still obscure. This study informs other studies that the deletion of ptsG/b1101 gene in E. coli GEM predicted increased succinate production, enabling a model-driven experimental inquiry and/or novel biological discovery on the underground metabolic role of this gene in E. coli central metabolism in relation to increasing succinate production when glycerol is the substrate. 相似文献
124.
125.
菌株CICIM B0013-030 (B0013,ack-pta,pps,pflB) 可积累D-乳酸作为主要发酵产物,然而副产物琥珀酸和乙酸的含量分别高达乳酸的11.9%和7.1%。为构建副产物含量低的产D-乳酸重组大肠杆菌菌株,本研究删除了菌株B0013-030的琥珀酸 (frdA) 和乙酸 (tdcDE) 合成途径,并考察了重组菌株在摇瓶和发酵罐中经两阶段发酵 (好氧生长菌体和厌氧发酵产酸) 利用葡萄糖发酵D-乳酸的性能。结果表明,分别构建含有frdA::difGm和tdcDE::difGm突变盒的重组质粒,并利用Red重组系统将突变盒整合于染色体上的目的基因,再利用Xer重组系统去除抗生素抗性基因,依次获得了重组菌株B0013-040B (B0013-030,frdA) 和B0013-050B (B0013-040B,tdcDE)。摇瓶发酵结果表明,frdA基因的删除使得菌株B0013-040B副产物琥珀酸的含量降低了80.8%;在7 L发酵罐中进行乳酸发酵,菌株B0013-040B的D-乳酸产量达114.5 g/L,光学纯度大于99.9%,但仍积累1.0 g/L琥珀酸和5.4 g/L乙酸。进一步删除了tdcD和tdcE基因的菌株B0013-050B,在7 L发酵罐中生产111.9 g/L D-乳酸,乙酸和琥珀酸的合成量分别降低为0.4 g/L,其他副产物含量也维持较低水平,表明该菌株具有较优良的D-乳酸发酵性能。 相似文献
126.
The allosteric enzyme aspartate transcarbamoylase (ATCase) exists in two conformational states. The enzyme, in the absence of substrates is primarily in the low-activity T state, is converted to the high-activity R state upon substrate binding, and remains in the R state until substrates are exhausted. These conformational changes have made it difficult to obtain structural data on R-state active-site complexes. Here we report the R-state structure of ATCase with the substrate Asp and the substrate analog phosphonoactamide (PAM) bound. This R-state structure represents the stage in the catalytic mechanism immediately before the formation of the covalent bond between the nitrogen of the amino group of Asp and the carbonyl carbon of carbamoyl phosphate. The binding mode of the PAM is similar to the binding mode of the phosphonate moiety of N-(phosphonoacetyl)-l-aspartate (PALA), the carboxylates of Asp interact with the same residues that interact with the carboxylates of PALA, although the position and orientations are shifted. The amino group of Asp is 2.9 A away from the carbonyl oxygen of PAM, positioned correctly for the nucleophilic attack. Arg105 and Leu267 in the catalytic chain interact with PAM and Asp and help to position the substrates correctly for catalysis. This structure fills a key gap in the structural determination of each of the steps in the catalytic cycle. By combining these data with previously determined structures we can now visualize the allosteric transition through detailed atomic motions that underlie the molecular mechanism. 相似文献
127.
Overlooked until recently, mitochondrial protein phosphorylation is now emerging as a key post-translational mechanism in the regulation of mitochondrial functions. In particular, tyrosine phosphorylation represents a promising field to discover new mechanisms of bioenergetic regulation. Tyrosine kinases belonging to the Src kinase family have been observed in mitochondrial compartments, however their substrates are almost unknown. Here, we provide evidence that the flavoprotein of succinate dehydrogenase and aconitase are "in vitro" substrates of Fgr tyrosine kinase. Fgr phosphorylates flavoprotein of succinate dehydrogenase at Y535 and Y596 and aconitase at Y71, Y544 and Y665. The significance of these findings is discussed. 相似文献
128.
Prospects for a bio-based succinate industry 总被引:4,自引:4,他引:0
Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based
bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated
research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming
cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with
little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described
the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise
for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial
species are described, and the advantages and disadvantages of each bacterial system are discussed. 相似文献
129.
Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K+]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aβ1–42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed. 相似文献
130.
We have shown that mitochondrial c-Src regulates reactive oxygen species (ROS) production by phosphorylating the succinate dehydrogenase A of respiratory complex II (CxII). To elucidate the molecular mechanisms underlying ROS production regulated by c-Src in the CxII, we investigated the CxII protein complex derived from cells treated with Src family kinase inhibitor PP2. We identified flotillin-1 as a c-Src target that prevents ROS production from CxII. Phosphorylation-site analysis suggests Tyr56 and Tyr149 on flotillin-1 as sites for phosphorylation by c-Src. A comparison of cells expressing flotillin-1 and its phosphorylation defective mutants confirms the requirement for flotillin-1 phosphorylation for its interaction with CxII and subsequent reduction in ROS production. Our findings suggest a critical role of flotillin-1 in ROS production mediated by c-Src. 相似文献