Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers

The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose-inducible promoters P(tac) and P(trc) by the natural inducer lactose and the synthetic inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of P(tac) and P(trc). In E. coli strains carrying a functional LacY, induction of P(trc) and P(tac) with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY(+) strains.     

Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring

A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized.     

乳糖诱导的重组人血管内皮抑素(rhEndostatin)蛋白的表达

研究用乳糖替代IPTG作为诱导剂进行重组蛋白的表达,观察乳糖对乳糖操纵子调控的基因工程菌发酵及重组血管内皮抑素表达的影响,从而选取最佳诱导表达条件。以重组人血管内皮抑素表达工程菌pETrhEN/BL21(DE3)作为研究对象,分别用IPTG和乳糖作为诱导剂,在摇瓶中进行表达实验。并对重组蛋白质表达量进行分析。然后在5 L发酵罐中进行验证。在摇瓶培养条件下,乳糖浓度大于0.5 g/L即可以诱导目的蛋白的表达。乳糖浓度1 g/L时诱导目的蛋白表达量与1 mmol/L的IPTG相当,当乳糖浓度为10 g/L,目的蛋白表达量达到最大。在发酵罐培养条件下,补料4 h后葡萄糖浓度基本耗尽,此时开始加入乳糖。诱导后1 h,即有重组蛋白表达,在诱导后4 h达到高峰(占菌体可溶性蛋白的56%),与此同时,诱导后5 h菌体浓度也达到最高值。在以乳糖操纵子为调控手段的工程菌表达系统中,可以使用乳糖作为诱导剂,诱导应在葡萄糖消耗完后进行。     

An inducible β-lactamase in a strain ofEscherichia coli

The production of beta-lactamase (penicillin/cephalosporin beta-lactam amidohydrolase, E.C.3.5.2.6) was found to be inducible in a clinically isolated strain of Escherichia coli. This is the first report of an inducible beta-lactamase in E. coli. The optimal concentration of inducer was 400 mug/ml of ml of benzylpenicillin, or 800 mug/ml of 6-aminopenicillanic acid. About fiftyfold induction was achieved. Maximum induction took ninety minutes from the time of adding the inducer. Induction was abolished by the presence of chloramphenicol(10 mug/ml). The enzyme has a molecular wieght of 23,000, and is inhibited by rho-chloromercuribenzoate and by iodine. It is active against a wide range of substrates, including cephaloridine and cloxacillin.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Functionally important residues of the Tet repressor inducing peptide TIP determined by a complete mutational analysis

Tet repressor (TetR) is widely used to control gene expression in pro- and eukaryotes. The mechanism of induction by its natural inducer tetracycline is well characterized. A 16-mer oligopeptide, called TIP, fused to thioredoxin A (TrxA) of Escherichia coli is an artificial inducer of TetR. We analyzed the sequence requirements of TIP by directed and random single amino acid substitutions and identified residues important for TetR induction. An alanine scanning analysis of the first twelve residues showed that all except the ones at position eleven and twelve are important for induction. A randomization of residues at positions one to twelve of TIP revealed the properties of each residue necessary for induction. These further insights into the specificity of TIP-TetR interaction are discussed in the light of the X-ray structure of the [TetR-TIP] complex. The last four residues of TIP contribute indirectly to TetR induction by increasing the steady-state level of the fusion protein. TIP mutants fused N-terminally or C-terminally to TrxA in E. coli induce with the same efficiency indicating identical binding and induction mechanisms, and the lack of contribution from TrxA.     

乳糖诱导甜蛋白Monellin在大肠杆菌中的表达

根据已报道的单链Monellin甜蛋白的氨基酸序列,按大肠杆菌基因偏爱密码子,设计和人工合成了单链monellin基因。将单链monellin基因克隆到大肠杆菌表达载体pET-28a中,构建了重组表达载体pET28a-mon,转化大肠杆菌BL21(DE3),得到表达Monellin的大肠杆菌工程菌株。借助SDS-PAGE分析方法,研究了乳糖代替IPTG诱导大肠杆菌表达甜蛋白Monellin。通过对乳糖作为诱导剂表达条件进行优化,Monellin的表达量可占细胞总蛋白的33.09%,与IPTG诱导表达量接近。本研究结果为乳糖作为诱导剂应用于重组大肠杆菌生产甜蛋白Monellin提供了参考依据。     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Dynamics of induced CAT expression in E. coli

The dynamics of chemically induced chloramphenicolaceyl-transferase (CAT) expression are determined in batch cultures of Escherichia coli DH5alphaF'IQ [pKK262-1]. This article is directed towards understanding the coupling of induced cloned-protein synthesis and reduced cell growth which are balanced in the optimal system. Experimental results indicate that the best inducer (IPTG) concentration is near 1.0 mM when added during midexponential growth. Lower concentrations cause only weak induction, whereas higher concentrations cause sufficiently strong induction that cell growth is suppressed. Induction at the onset of the stationary phase results in high expression but is accompanied by stimulated protease activity. Also, cell mass yield is adversely affected by enhanced protein synthesis. A structured metabolic model is shown to predict the responses of instantaneous growth rate and productivity which result from protein overexpression. This model can be employed to predict alternative reactor strategies and operating conditions necessary for the design of efficient bioprocess.     

Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates.

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.     

Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport

Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols. Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92:601-608. 1966.-The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization ("glucose effect") was studied in Escherichia coli. Experiments on the uptake into the cell of labeled inducers (d-galactose-C(14) and d-fucose-H(3)) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport inhibition model was provided by the finding that glucose did not substantially inhibit induction of the gal enzymes when glucose and galactose were produced intracellularly by beta-galactosidase hydrolysis of lactose, even if excess glucose was added. The inducer uptake experiments also showed that d-galactose and d-fucose probably enter the cell via different transport systems, although uptake of both compounds was inhibited by glucose.     

Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to d-threo chloramphenicol which lacks a C(3) hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.     

By IL-1 signaling,monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide

Epithelia react to microbial pathogens by mounting a defensive response that includes the production of antimicrobial peptides. In this study, we show that, in human epidermal cultures, Escherichia coli LPS was a very weak direct inducer of human beta-defensin (HBD)-2 mRNA and peptide, but the induction was greatly amplified when monocyte-derived cells (MoDeC) acted as intermediaries between LPS and the epidermis. IL-1R antagonist largely reversed the effect of MoDeC on epidermal HBD-2, indicating that, from among the many products of MoDeC, IL-1 was the dominant inducer of HBD-2 synthesis. In normal fresh human skin, which contains Langerhans cells and other myeloid cell types, in addition to keratinocytes, LPS also induced HBD-2 in an IL-1-dependent manner. In DNA microarray expression studies, HBD-2 was one of the most abundant mRNAs induced in epidermis by LPS-treated MoDeC, and its induction was reversed by IL-1Ra. Thus, epidermal response to LPS is potently amplified by MoDeC through IL-1-mediated signaling, leading to a selective increase in the synthesis of the antimicrobial peptide HBD-2. This pattern of responses establishes a key role for both IL-1 and HBD-2 in the host defense reaction of the epidermis.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

Penicillinamidohydrolase inEscherichia coli

Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) in Escherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme in Escherichia coli are substantially influenced by cyclic 3',5'-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylacetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolase by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a "catabolically neutral" source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.     

Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction.

The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined. The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase. H2S production by the E. coli host was, as in S. typhimurium, suppressed by nitrate or glucose in the growth medium. The presence of plasmid-borne phs genes in a S. typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate. These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate.