Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli

Microbial cells possess numerous sensing/regulator systems in order to respond rapidly to environmental changes. Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. A group of global regulators, which include the one component Fnr protein and the two-component Arc system, coordinate the adaptive responses. To quantitate the contribution of Arc and FNR-dependent regulation under microaerobic conditions, the gene expression pattern of the electron transfer chain genes and the TCA cycle genes in wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that the TCA cycle genes, icd, gltA, sucC, and sdhC are repressed by ArcA while Fnr has a minor or no effect on the expression of these genes under microaerobic conditions. The expression levels of the electron transfer chain genes, nuoA, ndh, and ubiE, were not significantly affected by either ArcA or Fnr regulation proteins, while a lower expression of cydA (up to 9-fold lower) and a higher expression of cyoA (up to 31-fold higher) were observed in cultures of the arcA mutant strain compared to those of the wild type. Since significantly higher NADH/NAD+ ratios were previously observed in cultures of the arcA mutant strain compared to the wild type it seems that the cytochrome o oxidase (the product of cyoABCDE) cannot efficiently support aerobic respiration when the cells are grown under microaerobic conditions.     

Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism

The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. To analyse how Saccharomyces cerevisiae (S. cerevisiae) metabolism during growth on glucose might be altered when intracellular NADH pool is decreased, we expressed noxE encoding a water-forming NADH oxidase from Lactococcus lactis (L. lactis) in the S. cerevisiae strain V5. During batch fermentation under controlled microaeration conditions, expression of the NADH oxidase under the control of a yeast promoter lead to large decreases in the intracellular NADH concentration (five-fold) and NADH/NAD+ ratio (six-fold). This increased NADH consumption caused a large redistribution of metabolic fluxes. The ethanol, glycerol, succinate and hydroxyglutarate yields were significantly reduced as a result of the lower NADH availability, whereas the formation of more oxidized metabolites, acetaldehyde, acetate and acetoin was favoured. The biomass yield was low and consumption of glucose, at concentration above 10%, was impaired. The metabolic redistribution in cells expressing the NADH oxidase was a consequence of the maintenance of a redox balance and of the management of acetaldehyde formation, which accumulated at toxic levels early in the process.     

Increased recombinant protein production in Escherichia coli strains with overexpressed water-forming NADH oxidase and a deleted ArcA regulatory protein

Glycolytic flux is increased and acetate production is reduced in Escherichia coli by the expression of heterologous NADH oxidase (NOX) from Streptococcus pneumoniae coupled with the deletion of the arcA gene, which encodes the ArcA regulatory protein. In this study, we examined the overproduction of a model recombinant protein in strains of E. coli expressing NOX with or without an arcA mutation. The presence of NOX or the absence of ArcA reduced acetate by about 50% and increased beta-galactosidase production by 10-20%. The presence of NOX in the arcA strain eliminated acetate production entirely in batch fermentations and resulted in a 120% increase in beta-galactosidase production.     

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 μmol per min per g cells (dry weight), the absence of limitations by O₂ and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.     

Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source

ArcA is a global regulator that switches on the expression of fermentation genes and represses the aerobic pathways when Escherichia coli enters low oxygen growth conditions. The metabolic profile of E. coli CT1062 (DeltaarcA)and CT1061 (arcA2) grown in microaerobiosis with glycerol as carbon source were determined and compared with E. coli K1060, the arcA+ parent strain. Both arcA mutants achieved higher biomass yields than the wild-type strain. The production of acetate, formate, lactate, pyruvate, succinate and ethanol were determined in the supernatants of cultures grown on glycerol under microaerobic conditions for 48 h. The yield of extracellular metabolites on glycerol showed lower acid and higher ethanol values for the mutants. The ethanol/acetate ratio was 0.87 for the parent strain, 2.01 for CT1062, and 12.51 for CT1061. Accordingly, the NADH/NAD+ ratios were 0.18, 0.63, and 0.97, respectively. The extracellular succinate yield followed a different pattern, with yield values of 0.164 for K1060, 0.442 for CT1062 and 0.214 for CT1061. The dissimilarities observed can be attributed to the different effects exerted by the deletion and point mutations in a global regulator.     

过量表达烟酸单核苷酸腺苷酰转移酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因(ldhA)和丙酮酸-甲酸裂解酶的编码基因(pflB)的发酵生产丁二酸的潜力菌株。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。nadD为催化NAD(H)合成途径中烟酸单核苷酸(NaMN)生成烟酸腺嘌呤二核苷酸(NaAD)的烟酸单核苷酸腺苷酰转移酶(Nicotinic acid mononucleotide adenylyltransferase,NAMNAT)的编码基因,通过过量表达nadD基因能够提高NAD(H)总量与维持合适的NADH/NAD+比例。文中构建了重组菌E.coli NZN111/pTrc99a-nadD,在厌氧摇瓶发酵过程中通过添加终浓度为1.0 mmol/L的IPTG诱导表达,重组菌E.coli NZN111/pTrc99a-nadD中NAD+和NADH的浓度分别比宿主菌E.coli NZN111提高了3.21倍和1.67倍,NAD(H)总量提高了2.63倍,NADH/NAD+从0.64降低为0.41,使重组菌株恢复了厌氧条件下生长和代谢葡萄糖的能力。重组菌与对照菌相比,72 h内可以消耗14.0 g/L的葡萄糖产6.23 g/L的丁二酸,丁二酸产量增加了19倍。     

Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase

We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.     

Alterations of glucose metabolism in Escherichia coli mutants defective in respiratory-chain enzymes

The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo? oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted L-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

Improvement of pyruvate production based on regulation of intracellular redox state in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The commercial demand for pyruvate has been expanding. However, some challenges need to be overcome in the microbial production of pyruvate, such as low glucose consumption caused by excessive accumulation of NADH. In this study, weakening or block of the TCA cycle, overexpression of foreign NADH oxidase, and carbon sources with different oxidation state was attempted to decrease NADH accumulation in engineered strain YP211. Results showed that blocking or weakening TCA cycle could not lower the intracellular redox state in strain YP211.Overexpressing NADH oxidase from Lactococcus lactis significantly decreased the intracellular NADH content and increased the consumption rate of glucose. However, the yield of pyruvate did not increase significantly. Compared with glucose as carbon source, sodium gluconate with a higher oxidation state resulted in a significant decrease of NADH/NAD⁺, and the concentration and yield of pyruvate increased by 62 and 6%, respectively. In the fed-batch fermentation, the yield of pyruvate increased to 0.78 g/g gluconate, and the concentration of pyruvate reached 78.8 g/L. It was suggested that sodium gluconate was a more ideal carbon source for strain YP211, which could effectively decrease NADH content and improve the pyruvate production.     

Specific growth inhibition by acetate of an Escherichia coli strain expressing Era-dE,a dominant negative Era mutant

Escherichia coli Era is a GTP binding protein and essential for cell growth. We have previously reported that an Era mutant, designated Era-dE, causes a dominant negative effect on the growth and the loss of the ability to utilize TCA cycle metabolites as carbon source when overproduced. To investigate the role of Era, the gene expression in the cells overproducing Era-dE was examined by DNA microarray analysis. The expression of lipA and nadAB, which are involved in lipoic acid synthesis and NAD synthesis, respectively, was found to be reduced in the cells overproducing Era-dE. Lipoic acid and NAD are essential cofactors for the activities of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and glycine cleavage enzyme complex. The expression of numerous genes involved in dissimilatory carbon metabolism and carbon source transport was increased. This set of genes partially overlaps with the set of genes controlled by cAMP-CAP in E coli. Moreover, the growth defect of Era-dE overproduction was specifically enhanced by acetate but not by TCA cycle metabolites both in rich and synthetic media. Intracellular serine pool in Era-dE overproducing cells was found to be increased significantly compared to that of the cells overproducing wild-type Era. It was further found that even the wild-type E. coli cells not overproducing Era-dE became sensitive to acetate in the presence of serine in a medium. We propose that when Era-dE is overproduced, carbon fluxes to the TCA cycle and to C1 units become impaired, resulting in a higher cellular serine concentration. We demonstrated that such cells with a high serine concentration became sensitive to acetate, however the reason for this acetate sensitivity is not known at the present.     

异源表达NADH选择性氧化酶提高光滑球拟酵母酵解速率及丙酮酸生产强度

摘要：【目的】为进一步提高光滑球拟酵母(Torulopsis glabrata)葡萄糖代谢速率及丙酮酸生产强度。【方法】将源于荚膜胞浆菌(Histoplasma capsulatum)的编码选择性氧化酶的AOX1基因过量表达于T. glabrata中,获得了一株线粒体内NADH氧化途径发生改变且胞内总NADH 氧化酶活性提高1.8倍的重组菌株AOX。【结果】与出发菌株CON比较,细胞浓度以及发酵周期降低了20.3%和10.7%,而平均比葡萄糖消耗速率和丙酮酸合成速率分别提高了34.7%和54.1%。其原因     

过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111厌氧发酵的主要产物为丁二酸,是发酵生产丁二酸的潜力菌株。但是由于敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸甲酸裂解酶的编码基因 (pflB),导致辅酶NADH/NAD+不平衡,厌氧条件下不能利用葡萄糖生长代谢。构建烟酸转磷酸核糖激酶的重组菌Escherichia coli NZN111/pTrc99a-pncB,在厌氧摇瓶发酵过程中通过添加0.5 mmol/L的烟酸、0.3 mmol/L的IPTG诱导后重组菌的烟酸转磷酸核糖激酶 (Nicotinic acid phosphor     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions

Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.     

Characterization of the Arc two-component signal transduction system of the capnophilic rumen bacterium Mannheimia succiniciproducens

The ArcB/A two-component signal transduction system of Escherichia coli modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the putative arcA and arcB genes of Mannheimia succiniciproducens MBEL55E, a capnophilic (CO2-loving) rumen bacterium, encode functional proteins that specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro, and the arcA and arcB genes of M. succiniciproducens confer toluidine blue resistance to E. coli arcA and arcB mutants. However, neither the quinone analogs (ubiquinone 0 and menadione) nor the cytosolic effectors (d-lactate, acetate, and pyruvate) affect the net phosphorylation of M. succiniciproducens ArcB. Our results indicate that different types of signaling molecules and distinct modes of kinase regulation are used by the ArcB proteins of E. coli and M. succiniciproducens.