Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations: an alternative to fed-batch cultures

Overflow metabolism is an undesirable characteristic of aerobic cultures of Escherichia coli. It results from elevated glucose consumption rates that cause a high substrate conversion to acetate, severely affecting cell physiology and bioprocess performance. Such phenomenon typically occurs in batch cultures under high glucose concentration. Fed-batch culture, where glucose uptake rate is controlled by external addition of glucose, is the classical bioprocessing alternative to prevent overflow metabolism. Despite its wide-spread use, fed-batch mode presents drawbacks that could be overcome by simpler batch cultures at high initial glucose concentration, only if overflow metabolism is effectively prevented. In this study, an E. coli strain (VH32) lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) with a modified glucose transport system was cultured at glucose concentrations of up to 100 g/L in batch mode, while expressing the recombinant green fluorescence protein (GFP). At the highest glucose concentration tested, acetate accumulated to a maximum of 13.6 g/L for the parental strain (W3110), whereas a maximum concentration of only 2 g/L was observed for VH32. Consequently, high cell and GFP concentrations of 52 and 8.2 g/L, respectively, were achieved in VH32 cultures at 100 g/L of glucose. In contrast, maximum biomass and GFP in W3110 cultures only reached 65 and 48%, respectively, of the values attained by the engineered strain. A comparison of this culture strategy against traditional fed-batch culture of W3110 is presented. This study shows that high cell and recombinant protein concentrations are attainable in simple batch cultures by circumventing overflow metabolism through metabolic engineering. This represents a novel and valuable alternative to classical bioprocessing approaches.     

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.     

Enhanced production of plasmid DNA by engineered Escherichia coli strains

Escherichia coli strains VH33 (PTS? GalP? strain displaying a strongly reduced overflow metabolism) and VH34 (additionally lacking the pyruvate kinase A) were evaluated for the production of a plasmid DNA (pDNA) vaccine. The parent (W3110) and mutant strains were cultured using 10 g of glucose/L. While the specific growth rates of the three strains were similar, they presented differences in the accumulation of acetate. W3110 accumulated up to 4 g/L of acetate, VH33 produced 1.4 g/L, and VH34 only 0.78 g/L. VH33 and VH34 produced 76% and 300% more pDNA than W3110. Moreover, VH34 demanded 33% less oxygen than VH33 and W3110, which can be advantageous for large-scale applications.     

Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

ABSTRACT: BACKGROUND: In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS- pykA- and PTS- pykF -). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. RESULTS: When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. CONCLUSIONS: Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS- pykA-) and VH35 (PTS- pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.     

Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions

Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed-acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate-formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale-down system, to simulate gradients occurring in large-scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by-product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9-12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by-products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large-scale cultures.     

Glucose and acetate influences on the behavior of the recombinant strainEscherichia coli HB 101 (GAPDH)

Summary This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) byE. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L^–1 of biomass and 6 g L^–1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L^–1, and in fed-batch experiments for glucose availability of 10 g h^–1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.     

Elevated Fis expression enhances recombinant protein production in Escherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.     

Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109

Two Escherichia coli strains, widely used for the production of various recombinant proteins, were compared for their pre-induction growth and acetate accumulation patterns. The strains studied were E. coli BL21 (lambdaDE3), transformed with a plasmid encoding Pseudomonas exotoxin A, and an E. coli K12 derived strain, JM109, carrying a plasmid encoding maltose-binding protein fused with HIV protease. Cultures were grown in controlled bench-top fermentors to the optimal pre-induction density in both high glucose batch and low glucose fed batch strategies. The results showed the superiority of E. coli BL21 (lambdaDE3) as a host for a recombinant protein expression system. For example, JM109 responds differently to high glucose concentration and to low glucose concentration. Its acetate concentration was as high as 10 g/L in a batch mode and 5 g/L in a fed batch mode. In comparison, strain BL21 (lambdaDE3) reached 2 g/L acetate when grown in batch mode and not more than 1 g/L acetate when grown in a fed batch mode. E. coli BL21 (lambdaDE3), most likely, possesses an acetate self-control mechanism which makes it possible to grow to the desired pre-induction density in a high glucose medium using simple batch propagation techniques. Such a technique is cost effective, reproducible, and easy to scale up. (c) 1996 John Wiley & Sons, Inc.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme II^Glc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme II^Glc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme II^Glc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y _Glc values. Assuming equal Y _ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme II^Glc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease     

Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli

The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM.     

Engineering E. coli for improved microaerobic pDNA production

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA⁻, which, together with W3110recA⁻vgb⁺, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

     

Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation

Escherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.     

理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌

利用代谢工程手段理性改造野生大肠杆菌的莽草酸(Shikimic acid,SA)合成途径及相关代谢节点,以构建高产莽草酸的工程菌株.根据细胞代谢网络分析,利用Red-Xer重组系统连续删除了野生型大肠杆菌CICIMB0013的莽草酸激酶基因(aroL、aroK),葡萄糖磷酸转移酶系统(PTS)的关键组分EIICBglc的编码基因(ptsG)以及奎宁酸/莽草酸脱氢酶基因(ydiB)并系统评价了基因删除对细胞的生长、葡萄糖代谢和莽草酸积累的影响.aroL、aroK的删除阻断了莽草酸进一步转化成为莽草酸-3-磷酸,初步提高莽草酸的累积.删除ptsG基因使大肠杆菌PTS系统部分缺失,细胞通过GalP-glk(半乳糖透性酶-葡萄糖激酶)途径,利用ATP将葡萄糖磷酸化后进入细胞.利用该途径运输葡萄糖能够减少PEP的消耗,使得更多的碳代谢流进入莽草酸合成途径,从而显著提高了莽草酸的产量.在此基础上删除ydiB基因,阻止了莽草酸合成的前体物质3-脱氢奎宁酸转化为副产物奎宁酸(Quinic acid,QA),进一步提高了莽草酸的累积.初步发酵显示4个基因缺失的大肠杆菌代谢工程菌生产莽草酸的能力比原始菌提高了90多倍.