重组大肠杆菌的高密度发酵和甘油生产条件的初步研究

在摇瓶中进行重组大肠杆菌菌株BL21高密度发酵条件的研究,考察了葡萄糖浓度、盐离子浓度、温度、接种量、发酵时间等对该菌株生产甘油的影响。初步确定底物浓度为2.5%,盐离子浓度0.2%,温度为37℃,接种量为2%,经24h的摇瓶发酵,甘油产量最高达6.8g/L。在30L发酵罐实验中、按初步确定的优化条件发酵26h,甘油产量可达46.67g/L,是LB/葡萄糖培养基中甘油产量的2.06倍。     

Metabolic engineering and transhydrogenase effects on NADPH availability in escherichia coli

The synthesis of several industrially useful compounds are cofactor‐dependent, requiring reducing equivalents like NADPH in enzymatic reactions leading up to the synthesis of high‐value compounds like polymers, chiral alcohols, and antibiotics. However, NADPH is costly and has limited intracellular availability. This study focuses on the study of the effect of the two transhydrogenase enzymes of Escherichia coli, PntAB and UdhA (SthA) on reducing equivalents‐dependent biosynthesis. The production of (S)‐2‐chloropropionate from 2‐chloroacrylate is used as a model system for monitoring NADPH availability because 2‐haloacrylate reductase, the enzyme catalyzing the one‐step conversion to (S)‐2‐chloropropionate in the synthesis pathway, requires NADPH as a cofactor. Results suggest that the presence of UdhA increases product yield and NADPH availability while the presence of PntAB has the opposite effect. A maximum product yield of 1.4 mol product/mol glucose was achieved aerobically in a pnt‐deletion strain with udhA overexpression, a 150% improvement over the wild‐type control strain. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1124–1130, 2013     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Cloning of the histidine transport genes from salmonella typhimurium and characterization of an analogous transport system in escherichia coli

The genes for the well-characterized high-affinity histidine transport system of S typhimurium have been cloned in λgt4. Genetic and physiological analyses of the analogous transport system of E coli were undertaken in order that available λ vectors, recombinant DNA techniques, and a genetic selection for transport function might be used to isolate the Salmonella genes. The presence of the transport genes on a 12.4 Kb cloned DNA fragment has been confirmed (1) genetically, by complementation studies; (2) physiologically, by the rates of histidine uptake by bacteria containing this DNA; and (3) by demonstrating that the cloned DNA codes for the previously identified transport proteins J and P. The isolated fragment carries the entire transport operon, the argT gene and the ubiX locus, but neither the purF gene nor the ack/pta loci.     

基于辅因子调控对大肠杆菌两阶段发酵产丁二酸的影响

考察了E.coli NZN111及其重组菌株E.coli NZN111/pTrc99a-pncB发酵生产丁二酸的性能。E.coli NZN111两阶段发酵丁二酸的同时,会造成丙酮酸的大量积累。研究发现:通过过量表达烟酸转磷酸核糖激酶,两阶段发酵重组菌株E.coli NZN111/pTrc99a-pncB,减少丙酮酸的积累且无副产物乙酸生成,提高丁二酸的产量,丁二酸得率和耗糖速率分别提高了139%和20%。     

Automatic containment sampling of recombinant escherichia coli fermentations

A containment sampling system for shake flasks and fermentors has been developed from a blood collection system used in hospitals. The core of the system is a collection vial with a vacuum inside. When a needle connected to the fermentation fluid penetrates a rubber seal on the vial, a sample is withdrawn. The system has been developed in two versions, a manual method for shake flasks, and an automated version for fermentors including cool storage of samples. The sampling system offers the same safety for fermentation containment as the original system offers safety for patients and hospital staff. (c) 1992 John Wiley & Sons, Inc.     

Predictive model for reduction of Escherichia coli during acetic acid decontamination of chicken skin

AIMS: The response surface methodology was used to evaluate the effect of operating variables (acetic acid concentration, spraying time and temperature) on the reduction of Escherichia coli populations on poultry breast skin in a laboratory showering process, as well as to identify the best conditions that are required to develop this operation. METHODS AND RESULTS: Skin samples were inoculated with a 24-h E. coli culture and afterwards treated according to experimental design under selected acetic acid concentration, spraying time, and solution temperature. The E. coli reduction model was significantly affected by the acetic acid concentration and spraying time (P < or = 0.05 and < or =0.01), while temperature did not show a significant effect (P > 0.05). CONCLUSION: The predictive model obtained was validated through additional confirmatory experiments and showed to be adequate, and it could be used as an approach to optimize the acetic acid spray washes during poultry carcasses processing. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of acetic acid washes in the processing of poultry does not have the capability of eliminating E. coli populations from carcasses. However, significant reductions in the initial load could be achieved.     

Influence of strain and medium composition on filtration of escherichia coli suspensions

Cross-flow filtration of Escherichia coli strains was examined at the laboratory and pilot scales using Romicon 500,000 molecular-weight-cutoff hollow fiber membranes. Both the series resistance and macrosolute polarization models were employed to compare performances. Total dissolved solids content above 90 g/L and viscosity above 1.1 x 10(-3) pac s of cell-free culture media were found to decrease average filtration fluxes by over 60% both in the absence and presence of cells. Broth filtration with culture media of dissolved solids levels below 80 g/L were influenced to a greater extent by harvest cell density. The collodial nature of the complex nutrient responsible for the total solids increase affected prediction of filtration performance. Differences in strain filterability were observed with JM109 preferred over DH5 in high solids-containing media and RR1 preferred over JM109 in low dissolved solids-containing media. Their research demonstrates the importance of cell strain and media selection in the performance of early downstream processing steps. (c) 1994 John Wiley & Sons, Inc.     

Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。     

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。     

共表达烟酸转磷酸核糖激酶和丙酮酸羧化酶对大肠杆菌NZN111产丁二酸的影响

构建了共表达烟酸转磷酸核糖激酶(NAPRTase)和丙酮酸羧化酶(PYC)的重组质粒pTrc99a-pncB-pyc,并考察了重组菌E.coli NZN111/pTrc99a-pncB-pyc生产丁二酸的能力。结果表明:重组菌NZN111/pTrc99a-pncB-pyc的NAPRTase和PYC的比酶活达到最高,分别为20.75和1.04 U/mg,同时,辅酶NADH、NAD+及NAD(H)总量达到最高。厌氧摇瓶发酵结果:48 h能够消耗17.5 g/L的葡萄糖生成14.08 g/L的丁二酸,而丙酮酸的产量大幅度降低,仅为0.11 g/L。本研究为基因工程菌大肠杆菌厌氧条件下发酵生产丁二酸提供了一定的基础。     

Efficient export of prefolded,disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high‐value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide‐containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild‐type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide‐bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:281–290, 2014     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Effect of ColV plasmids on the hydrophobicity of Escherichia coli

Abstract The hydrophobicity of E. coli strains carrying or lacking the colicin V ( ColV ) plasmids, ColV , I-K94 or ColV -K30 was assayed. ColV ⁺ derivatives of strain 1829, produced by conjugation or transformation, were more hydrophobic than either the original 1829 parental strain or a Col ^- derivative formed by curing 1829 ColV -K30 of its plasmid by an SDS/high temperature growth technique. Transfer of ColV into other E. coli strains also led to increased hydrophobicity. This effect of ColV plasmids was observed for organisms grown at 37°C; ColV ⁺ and ColV^- strains did not differ in hydrophobicity of grown at 21°C. This finding and other studies suggest that sex pili may be involved in the increased hydrophobicity.     

Effect of modified glucose uptake using genetic engineering techniques on high-level recombinant protein production in escherichia coli dense cultures

Reduction of acetate excretion using a modified cellular glucose uptake rate was examined. An Escherichia coli strain bearing a mutationin ptsG, a gene encoding enzyme II in glucose phosphotransferase system (PTS), was constructed and characterized. The growth rate of the mutant strain was slower than its parent in glucose defined medium, butwas not affected in complex medium. Experimental results using this mutant strain showed a significant improvement in culture performance in simple batch cultivations due to reduced acetate excretion through the modified glucose uptake. Both biomass and recombinant protein productivity were increased by more than 50% with the ptsG mutant when compared to the parent strain. Recombinant protein productivity by the newly constructed strain at a level of more than 1.6 g/L was attained consistently in a simple batch bioreactor. (c) 1994 John Wiley & Sons, Inc.     

过表达氨基葡萄糖脱氨酶对大肠杆菌氨基葡萄糖合成及中心碳代谢的影响

考察过表达氨基葡萄糖脱氨酶对氨基葡萄糖合成及大肠杆菌（Escherichia coli）中心碳代谢的影响。实验结果表明：过表达氨基葡萄糖脱氨酶使得在36 g/L葡萄糖,pH为9.0的发酵条件下,发酵24 h后,重组菌发酵液中氨基葡萄糖、丙酮酸和乙酸的量分别是对照菌Rosetta的2.1、1.48和1.74倍;而乳酸的量为2.53 g/L,对照菌Rosetta发酵液中的乳酸含量未检测到,重组菌发酵液中柠檬酸及α-酮戊二酸的含量分别是Rosetta的2.99和2.73倍。