Production of l-phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110

The market of l-phenylalanine has been stimulated by the great demand for the low-calorie sweetener aspartame. In this paper, the effects of pivotal genes on l-phenylalanine production were evaluated by metabolic engineering of wild type Escherichia coli. The bifunctional PheA protein contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by l-phenylalanine). The catalytic domain of PheA was overexpressed to increase l-phenylalanine production. It was firstly indicated that this domain could enhance the metabolic influx to overproduce l-phenylalanine and improve the survival ability under m-Fluoro-dl-phenylalanine stress. Furthermore, the fermentation performance of aroG feedback inhibition resistant mutants was firstly compared, aroG29 and aroG15 increased the l-phenylalanine concentration by 5-fold. After that the expression of aroK and ydiB was also elevated, and the l-phenylalanine yield on cell (0.79 g/g) and maximum l-phenylalanine productivity (0.073 g/L/h) were subsequently doubled. Meanwhile, the l-phenylalanine yield on glucose increased from 0.124 g/g to 0.153 g/g. It was found that genes ydiB and aroK could elevate the l-phenylalanine yield and productivity and shorten the lag phase.     

Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose

Efficient microbial cell factory for the production of homoserine from glucose has been developed by iterative and rational engineering of Escherichia coli W3110. The whole pathway from glucose to homoserine was divided into three groups, namely, glucose transport and glycolysis (‘up-stream’), TCA and glyoxylate cycles (‘mid-stream’), and homoserine module (conversion of aspartate to homoserine and its secretion; ‘down-stream’), and the carbon flux in each group as well as between the groups were accelerated and balanced. Altogether, ∼18 genes were modified for active and consistent production of homoserine during both the actively-growing and non-growing stages of cultivation. Finally, fed-batch, two-stage bioreactor experiments, separating the growth from the production stage, were conducted for 61 h, which gave the high titer of 110.8 g/L, yield of 0.64 g/g glucose and volumetric productivity of 1.82 g/L/h, with an insignificant amount of acetate (<0.5 g/L) as the only noticeable byproduct. The metabolic engineering strategy employed in this study should be applicable for the biosynthesis of other amino acids or chemicals derived from aspartic acid.     

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.     

Oxygen availability and the growth of Escherichia coli W3110: A problem exacerbated by scale-up

By examining the effects of oxygen availability on the batch growth of Escherichia coli on glucose the present study seeks to determine the responses of growing cells to varying degrees of oxygen excess, limitation or starvation as might be experienced in an industrial-scale bioreactor. It was found that as the degree of oxygen limitation increases so too does the byproduct acetate production in addition to a concomitant decrease in the substrate based biomass yield coefficient and maximum specific growth rate. Similar, although not as severe, responses to excess oxygen growth conditions were also observed. This result supports the concept of oxygen being potentially toxic to growing organisms, resulting in, at the very least, a degree of inhibition.     

Reduction of aerobic acetate production by Escherichia coli.

Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.     

Proteomic analysis of extracellular proteins from Escherichia coli W3110

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.     

Beta-lactamase production by Escherichia coli under different growth conditions

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.     

Purification and characterization of an 'actomyosin' complex from Escherichia coli W3110

Abstract An 'actomyosin' complex was purified from Escherichia coli W3110 using selective precipitation. The complex contains three major components of 19.5, 18.5 and 17 kDa. The 19.5- and 17-kDa proteins were purified by electroelution, peptide mapped and N-terminally sequenced. The structural gene for the 17-kDa protein was found to have been previously identified in an operon containing several other genes including the essential lpxA, lpxB and dnaE . The possible function of the 17-kDa protein and the other 'actomyosin' components is discussed.     

Oxygen availability and growth of Escherichia coli W3110: Dynamic responses to limitation and starvation

It is well known that oxygen availability can have a profound effect upon the growth of an Escherichia coli culture with respect to acetate excretion or lower product yield. The current investigation seeks to determine the dynamic responses of steady state continuous cultures to abrupt changes in the oxygen supply. This should yield information regarding the behaviour of such cells as they circulate through areas of low and high oxygen availability in an industrial-scale bioreactor. It was found that a decoupling of catabolism and anabolism occurred following sudden switches both from and to oxygen limitation. It also appeared that the imposed growth rate had an effect on the speed of recovery of the system following any such changes.     

利用同源重组的方法提高大肠杆菌W3110天冬氨酸的积累

大肠杆菌中存在3种天冬氨酸激酶,分别为LysC,MetL,ThrA,使天冬氨酸磷酸化后分别进入Lys、Met、Thr的合成途径.因此大肠杆菌菌体中无法积累大量天冬氨酸. 以大肠杆菌W3110为出发菌株,利用Red同源重组系统分别构建了LysC、ThrA和MetL单基因缺陷株和LysC-ThrA和LysC-MetL双基因缺陷株. 采用高效液相色谱法测定L-天冬氨酸积累量. 发现除MetL单基因突变株外,其余突变株均积累了比野生型更多的L-天冬氨酸,这为代谢工程改造菌株并通过发酵法生产天冬氨酸奠定了基础.     

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.     

Kinetic models for the growth of Escherichia coli with mixtures of sugars under carbon-limited conditions

In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the micromax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low.     

Bioenergetic aspects of aerobic glucose metabolism of Escherichia coli K-12 under varying specific growth rates and glucose concentrations.

An attempt was made to find a bioenergetical explanation for the differential effect of specific growth rate and glucose concentration on glucose metabolism of Escherichia coli K-12 with the help of 2,4-dinitrophenol (DNP). The effect of DNP on biomass occurred only at high glucose concentrations. The presence of this uncoupler strongly stimulated glucose uptake rates and oxygen uptake rates, but repressed severly Yg values. Increase in glucose concentration, however, sharply decreased QO2. The amount of oxygen required for maintenance was not affected by DNP, but Yomax values were much lower in the presence of DNP. The results are discussed and it is suggested that aerobic fermentation is caused by a severe reduction of site 1 of the respiratory chain region, whereas biomass formation is affected by repression of the terminal cytochrome a2. In comparing the effect of glucose on biomass formation at similar Qglucose levels aerobic and anaerobic fermentation, repression occurred in both cases at glucose concentrations of 0.3% and above. Although the analyses of 15 enzymes established the metabolic differences, the repression of growth was common to both fermentation types.     

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.