Succinate production from sucrose by metabolic engineered Escherichia coli strains under aerobic conditions

Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.     

Purification and phosphorylation of the Arc regulatory components of Escherichia coli.

In Escherichia coli, a two-component signal transduction system, consisting of the transmembrane sensor protein ArcB and its cognate cytoplasmic regulatory protein ArcA, controls the expression of genes encoding enzymes involved in aerobic respiration. ArcB belongs to a subclass of sensors that have not only a conserved histidine-containing transmitter domain but also a conserved aspartate-containing receiver domain of the regulator family. 'ArcB (a genetically truncated ArcB missing the two transmembrane segments on the N-terminal end) and ArcA were purified from overproducing cells. Autophosphorylation of 'ArcB was revealed when the protein was incubated with [gamma-32P]ATP but not with [alpha-32P]ATP or [gamma-32P]GTP. When ArcA was incubated in the presence of 'ArcB and [gamma-32P]ATP, ArcA acquired radioactivity at the expense of the phosphorylated protein 'ArcB-32P. When a limited amount of 'ArcB was incubated with excess ArcA and [gamma-32P]ATP, ArcA-32P increased linearly with time. Under such conditions, for a given time period the amount of ArcA phosphorylated was proportional to the concentration of 'ArcB. Thus, 'ArcB acted as a kinase for ArcA. Chemical stabilities of the phosphorylated proteins suggested that 'ArcB-32P contained both a histidyl phosphate and an aspartyl phosphate(s) and that ArcA-32P contained only an aspartyl phosphate(s).     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Structural similarity of YbeD protein from Escherichia coli to allosteric regulatory domains

Lipoic acid is an essential prosthetic group in several metabolic pathways. The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon. YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region. Here, we report the nuclear magnetic resonance structure of YbeD from E. coli. The structure includes a beta alpha beta beta alpha beta fold with two alpha-helices on one side of a four-strand antiparallel beta-sheet. The beta 2-beta 3 loop shows the highest sequence conservation and is likely functionally important. The beta-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions. YbeD shows striking structural homology to the regulatory domain from d-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system.     

Quantification and analysis of metabolic characteristics of aerobic succinate-producing Escherichia coli under different aeration conditions

The production of succinate by engineered Escherichia coli strains has been widely investigated. In this study, quantitative comparison of metabolic fluxes was carried out for the wild-type E. coli strain and a quintuple mutant strain QZ1111 that was designed for the production of succinate aerobically by knocking out five genes (ptsG, poxB, pta, sdhA, iclR) of the wild-type E. coli MG1655. Metabolic flux distributions of both strains were quantified by ¹³C-labeling experiments, together with the determination of physiological parameters and the expression level of key genes. The experimental results indicated that under the same aeration condition the fraction of oxaloacetate molecules originating from phosphoenolpyruvate was increased in E. coli QZ1111 compared to that in the wild-type E. coli MG1655. The glyoxylate shunt was likely activated in E. coli QZ1111 only under high aeration condition but repressed in other conditions, indicating that the deletion of the iclR gene could not completely remove the repression of the glyoxylate shunt with limited oxygen supply. Our results also suggested further genetic manipulation strategies to enhance the production yield of succinate.     

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at Y_P/S  =  0.36 ± 0.029 g_Pyruvate/g_Glucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.     

Very slow growth of Escherichia coli.

A recycling fermentor (a chemostat with 100% biomass feedback) was used to study glucose-limited behavior of Escherichia coli B. The expectation from mass transfer analysis that growth would asymptotically approach a limit mass determined by the glucose provision rate (GPR) and the culture's maintenance requirement was not met. Instead, growth proceeded at progressively lower rates through three distinct phases. After the fermentor was seeded, but before glucose became limiting, growth followed the usual, exponential path (phase 1). About 12 h postseeding, residual glucose in the fermentor fell below 1 microgram . ml-1 and the growth rate (dx/dt) became constant and a linear function of GPR (phase 2). The specific growth rate, mu, therefore fell continuously throughout the phase. Biomass yield and glucose assimilation (13%) were near the level for exponential growth, however, and independent of GPR over a broad range. At a critical specific growth rate (0.04 h-1 for this strain), phase 2 ended abruptly and phase 3 commenced. In phase 3, the growth rate was again constant, although lower than in phase 2, so that mu continued to fall, but growth rates and yields were praboloid functions of GPR. They were never zero, however, at any positive value of GPR. By inference, the fraction of metabolic energy used for maintenance functions is constant for a given GPR, although different for phases 2 and 3, and independent of biomass. In both phases 2 and 3, orcinol, diphenylamine, and Lowry reactive materials were secreted at near-constant rates such that over 50% as much biosynthetic mass was secreted as was retained by the cells.     

Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.     

Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase

Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17,000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6). This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts. After r2 and c3 subunits were separated, -SH groups of r2 were titrated with p-(hydroxymercuri)benzenesulfonate (PMPS) at pH 7.0. The concomitant release of Zn2+ (2 equiv/r dimer) was quantitated with 4-(2-pyridylazo)resorcinol (PAR) and was a linear function of PMPS added until 8 mercaptide bonds per r2 were formed. Breakage of 1 of 4 Zn2(+)-sulfur bonds in a Zn2+ binding cluster therefore makes the other three bonds more labile. From stopped-flow measurements, the PMPS-promoted Zn2+ release from r2 or mercaptide bond formation with 10- to 20-fold excess PMPS/r2-SH at pH 7.0 was first order with an Arrhenius activation energy Ea = 10 kcal/mol and a half-time t 1/2 = 9 +/- 2 ms at 20 degrees C without inhibitory anions present. The rate of mercurial-promoted Zn2+ release from r2 is at least 77 times faster than that from intact c6r6 [Hunt, J.B., Neece, S.H., Schachman, H.K., and Ginsburg, A. (1984) J. Biol. Chem. 259, 14793]; this indicates that Zn2+ binding clusters are more accessible to attack by PMPS than are those in ATCase. The addition of a 25-fold excess of the multidentate fluorescent chelator quin-2 to r2 gave a rate of Zn2+ dissociation that was 1/210th of that observed with excess mercurial. Furthermore, the Zn(PAR)1 complex was identified as the active species in the transfer of Zn2+ from Zn(PAR)2 to aporegulatory subunits, with kappa = (8 +/- 3) x 10(5) M-1 s-1 at pH 7.0 and 15 degrees C for this second-order association reaction. Although kinetic results are dependent on the mechanisms involved, an affinity constant K'A = (1.3 +/- 0.6) x 10(12) M-1 for Zn2+ binding to r dimer at pH 7.0 and 20 degrees C in the absence and presence of 100 mM KCl could be determined spectrally by rapid equilibration with the high-affinity, sensitive metalloindicators indo-1 and quin-2. This K'A value is based on the assumptions that Zn2+ binding sites in r2 are equivalent (noninteracting) and that apo-r2 does not dissociate; if apo-r2 dissociates, K'A approximately 10(14) M-1. Within experimental error, the K'A value was independent of [indo-1]/[r2] ratios from 36 to 3 with 0.3-8 microM r2.(ABSTRACT TRUNCATED AT 400 WORDS)     

重组大肠杆菌生产L-色氨酸发酵条件优化

为了提高重组大肠杆菌FB讲／pSV－04发酵生产L－色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L．色氨酸的影响。在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53．4g／L,比优化前提高了141．6％。经30L发酵罐初步放大,L－色氨酸的产量达53．6g／L,发酵结果稳定,具有工业化应用前景。     

Redox studies of subunit interactivity in aerobic ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.     

Redox control of fast ligand dissociation from Escherichia coli cytochrome bd

Bacterial bd-type quinol oxidases, such as cytochrome bd from Escherichia coli, contain three hemes, but no copper. In contrast to heme-copper oxidases and similarly to globins, single electron-reduced cytochrome bd forms stable complexes with O(2), NO and CO at ferrous heme d. Kinetics of ligand dissociation from heme d(2+) in the single electron- and fully-reduced cytochrome bd from E. coli has been investigated by rapid mixing spectrophotometry at 20 degrees C. Data show that (i) O(2) dissociates at 78 s(-1), (ii) NO and CO dissociation is fast as compared to heme-copper oxidases and (iii) dissociation in the single electron-reduced state is hindered as compared to the fully-reduced enzyme. Presumably, rapid ligand dissociation requires reduced heme b(595). As NO, an inhibitor of respiratory oxidases, is involved in the immune response against microbial infection, the rapid dissociation of NO from cytochrome bd may have important bearings on the patho-physiology of enterobacteria.     

Differential sensitivities of the growth of Escherichia coli to acrylate under aerobic and anaerobic conditions and its effect on product formation

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.     

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.