Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture

The recombinant Escherichia coli B strain KO11, containing chromosomally-integrated genes for ethanol production, was developed for use in lignocellulose-to-ethanol bioconversion processes but suffers from instability in continuous culture and a low ethanol tolerance compared to yeast. Here we report the ability cell immobilization to improve its phenotypic stability and ethanol tolerance during continuous culture on a 50 g/L xylose feed. Experiments conducted in a vertical tubular fermentor operated as a liquid-fluidized bed with the cells immobilized on porous glass microspheres were compared to control experiments in the same reactor operated as a chemostat without the support particles. Without cell immobilization the ethanol yield fell sharply following start-up, declining to 60% of theoretical after only 8-9 days of continuous fermentation. While immobilizing the cells did not prevent this decline, it delayed its onset and slowed its rate. With immobilization, a stable high ethanol yield (>85%) was maintained for at least 10 days, thereafter declining slowly, but remaining above 70% even after up to 40 days of fermentation. The ethanol tolerance of E. coli KO11 cells was substantially increased by immobilization on the glass microspheres. In ethanol tolerance tests, immobilized cells released from the microspheres had survival rates 2.3- to 15-fold higher than those of free cells isolated from the same broth. Immobilization is concluded to be an effective means of increasing ethanol tolerance in E. coli KO11. While immobilization was only partially effective in combating its phenotypic instability, further improvements can be expected following optimization of the immobilization conditions.     

Fermentation of sweet whey by ethanologenic Escherichia coli

Whey, an abundant byproduct of the dairy industry, contains large amounts of protein and lactose which could be used for fuel ethanol production. We have investigated a new organism as a candidate for such fermentations: recombinant Escherichia coli containing the genes encoding the ethanol pathway from Zymomonas mobilis. The highest level of ethanol achieved, 68 g/L, was produced after 108 hours in Luria broth containing 140 g lactose/L. Fermentations of lower lactose concentrations were completed more rapidly with approximately 88% of theoretical yields. Reconstituted sweet whey (60 g lactose/L)was fermented more slowly than lactose in Luria broth requiring 144 hours to produce 26 g ethanol/L. Supplementing sweet whey with a trace metal mix and ammonium sulfate reduced the required fermentation time to 72 hours and increased final ethanol concentration (28 g ethanol/L). By adding proteinases during fermentation, the requirement for ammonia was completely eliminated, and the rate of fermentation further improved (30 g ethanol/L after 48 hours). This latter incresed in rate of ethanol production and ethanol yield are presumed to result from incorporation of amino acids released by hydrolysis of whey proteins. The fermentation of sweet whey by ethanologenic E. coil reduced the nonvolatile residue by approximately 70%. This should reduce biological oxygen demand and reduce the cost of waste treatment. Whey supplemented with trace metals and small amounts of proteinase may represent an economically attractive feedstock for the production of ethanol and other useful chemicals.     

Kinetics of ethanol production by recombinant Escherichia coli KO11

The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xyclose fermentation than vice versa. The fermentation of condensate from steampretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate. The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. (c) 1995 John Wiley & Sons, Inc.     

Comparative stability of ethanol production by Escherichia coliKO11 in batch and chemostat culture

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded. Received 13 January 1999/ Accepted in revised form 23 April 1999     

Effects of process errors on the production of ethanol by Escherichia coli KO11

Escherichia coli KO11 was previously constructed for the production of ethanol from both hexose and pentose sugars in hemicellulose hydrolysates by inserting the Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). This biocatalyst appears relatively resistant to potential process errors during fermentation. Antibiotics were not required to maintain the maximum catabolic activity of KO11 even after deliberate contamination with up to 10% soil. Fermentations exposed to extremes of temperature (2 h at 5°C or 50°C) or pH (2 h at pH 3 or pH 10) recovered after re-adjustment to optimal fermentation conditions (35°C, pH6) although longer times were required for completion in most cases. Ethanol yields were not altered by exposure to extremes in temperature but were reduced by exposure to extremes in pH. Re-inoculation with 5% (by volume) from control fermentors reduced this delay after exposure to pH extremes. Received 24 July 1997/ Accepted in revised form 16 April 1998     

An integrated metabolic modeling approach to describe the energy efficiency of Escherichia coli fermentations under oxygen-limited conditions: Cellular energetics, carbon flux, and acetate production

An integrated metabolic model for the production of acetate by growing Escherichia coli on glucose under aerobic conditions is presented. The model is based on parameters which are easily determined by experiments. Forming the basis for this integrated metabolic model are the 12 principal precursor metabolites for biosynthetic pathways, the Embden-Meyerhof-Parnas pathway, the pentose phosphate cycle, the tricarboxylic acid cycle and the anapleurotic reactions, the Crabtree effect, the Pasteur effect, and the details of bacterial respiration. The result can be used to explain phenomena often observed in industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low oxygen concentration, a high specific growth rate, or a combination of these conditions. (c) 1993 John Wiley & Sons, Inc.     

Ethanol production from dilute acid hydrolysate of rice hulls using genetically engineered Escherichia coli

Approximately 30% of rice hulls, which represent an abundant residue with little commercial value, was solubilized with 0.4 M H2SO4 acid to produce a syrup containing over 100 g monomer sugar/l. Toxins generated during hydrolysis were mitigated with Ca(OH)2. Treated hydrolysate plus additional nutrients was fermented with Escherichia coli KO11 to produce over 46 g ethanol/L in 72 h (92% of theoretical yield). © Rapid Science Ltd. 1998     

The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli

AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC) and enteropathogenic E. coli (EPEC). METHODS AND RESULTS: A total of 100 strains of E. coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR. Fifty-four per cent produced an amplicon with the R3 primer set. Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25. However, 46% of enteroaggregative E. coli and 50% of enteropathogenic E. coli were also shown to have an R3 LPS-core structure. CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.     

Culture conditions' impact on succinate production by a high succinate producing Escherichia coli strain

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.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth

Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations. (c) 1994 John Wiley & Sons, Inc.     

Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

In the genome‐engineering era, it is increasingly important that researchers have access to a common set of platform strains that can serve as debugged production chassis and the basis for applying new metabolic engineering strategies for modeling and characterizing flux, engineering complex traits, and optimizing overall performance. Here, we describe such a platform strain of E. coli engineered for ethanol production. Starting with a fully characterized host strain (BW25113), we site‐specifically integrated the genes required for homoethanol production under the control of a strong inducible promoter into the genome and deleted the genes encoding four enzymes from competing pathways. This strain is capable of producing >30 g/L of ethanol in minimal media with <2 g/L produced of any fermentative byproduct. Using this platform strain, we tested previously identified ethanol tolerance genes and found that while tolerance was improved under certain conditions, any effect on ethanol production or tolerance was lost when grown under production conditions. Thus, our findings reinforce the need for a metabolic engineering “commons” that could provide a set of platform strains for use in more sophisticated genome‐engineering strategies. Towards this end, we have made this production strain available to the scientific community. Biotechnol. Bioeng. 2013; 110: 1520–1526. © 2013 Wiley Periodicals, Inc.     

Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli

To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.     

重组大肠杆菌的分批补料培养方法

在重组大肠杆菌的培养过程中,存在着菌体的高浓度与外源蛋白的高表达这一矛盾,使得重组菌的比生长速率通常远远低于宿主菌,限制了基因工程菌由实验室规模向工业化规模的转变。要实现重组大肠杆菌的高密度培养,最常用和最有效的方法就是分批补料流加培养。     

大肠杆菌murB和birA基因的转录通读现象

在对应用限制性显示 PCR技术构建的大肠杆菌 poly(A)化mRNA的cDNA文库鉴定中 ,发现了一克隆的基因片段同时跨越了murB和birA基因的部分序列 ,提示这 2个基因存在转录通读现象 ,设计引物 ,利用逆转录后的产物为模板 ,进行了验证。结果表明murB和birA基因存在转录通读现象。     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

大肠杆菌梭曼水解酶的纯化和性质

大肠杆菌梭曼水解酶的纯化和性质邵煌,刘昌玲,肖美珍,孙曼霁（北京军事医学科学院毒物药物研究所,北京１００８５０）梭曼属Ｇ类神经性有机磷毒剂．自然界发现多种细菌中均存在梭曼水解酶（Ｓｏｍａｎａｓｅ）活性［１－３］．研究细菌梭曼水解酶,寻求生物解毒的方法...     

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.