Modulation of gene expression from the arabinose-inducible araBAD promoter

The arabinose-inducible P_BAD promoter suffers from all-or-none gene expression in which cells harboring the natively controlled arabinose transport gene (araE) are either induced or uninduced, the relative fraction of which is controlled by the concentration of arabinose. The population-averaged variation in expression from P_BAD as a function of inducer concentration is proportional to the percentage of cells that are fully induced (vs. uninduced) rather than the level of expression in individual cells. Because of its undesirable effects on the expression of heterologous genes, the all-or-none phenomenon was eliminated in Escherichia coli by expression of araE from arabinose-independent (either the Lactococcus lactis constitutive or IPTG-inducible lac) promoters. In these arabinose-transport engineered cells, variation in P_BAD expression with arabinose concentration was a result of variation of the expression level in individual cells with all cells in the population having approximately the same induction level. Journal of Industrial Microbiology & Biotechnology (2002) 29, 34–37 doi:10.1038/sj.jim.7000259 Received 20 November 2001/ Accepted in revised form 29 March 2002     

Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli

Aim: To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P_BAD promoter in Escherichia coli. Method and Results: The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose P_BAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring P_BAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions: The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study: We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes.     

An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of TnΩP_BAD to place the highly regulable, arabinose inducible P_BAD promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P_BAD promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.     

Unbalanced phospholipid composition of <Emphasis Type="Italic">Escherichia coli</Emphasis> membranes affects P<Subscript>PHO</Subscript> promoter activity

The activity of phosphatase P_PHO or arabinose P_BAD promoters of Escherichia coli has been studied as dependent on the content of zwitterionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or upon a significant decrease in the content of anionic phospholipids, the activity of the P_PHO promoter (but not that of the P_BAD promoter) was inhibited. Since the P_PHO promoter belongs to the Pho regulon, a member of the family of two-component regulatory systems of signal transduction that have membrane sensors, the regulation of gene expression by phospholipids is presumed to be accomplished through a membrane sensor.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 294–302.Original Russian Text Copyright © 2005 by Golovastov, Krasovskaya, Anissimova, Shchukin, Badyakina, Nesmeyanova.Deceased.     

Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems

During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial systems remain most attractive due to low cost, high productivity, and rapid use. However, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. This review gives an overview of the most commonly used systems: As hosts, Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Caulobacter crescentus, other strains, and, most importantly, Escherichia coli BL21 and E. coli K12 and their derivatives are presented. On the promoter side, the main features of the l-arabinose inducible araBAD promoter (P_BAD), the lac promoter, the l-rhamnose inducible rhaP _BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter p _L , and the anhydrotetracycline-inducible tetA promoter/operator are summarized.     

Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria

We describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the P_BAD promoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, using Pseudomonas aeruginosa as the model host.     

Targeted deletion of the ara operon of Salmonella typhimurium enhances L-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents

Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the P_BAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the P_BAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (～30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives P_BAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.     

Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

Cytokinin regulates differentially expression of PAHK-GUS constructs in transgenic Arabidopsis thaliana plants

The expression regulation by cytokinin of genetic constructs P _AHK2 -GUS, P _AHK3 -GUS, and P _AHK4 -GUS in transgenic Arabidopsis thaliana (L.) Heynh plants bearing the gene encoding β-glucuronidase (GUS) under the control of the promoter of one of three genes encoding histidine protein kinases, which are membrane receptors of cytokinin was studied. In 4–5-day-old etiolated A. thaliana seedlings, treatment with cytokinin resulted in the strongest expression activation of the constructs P _AHK2 -GUS and P _AHK3 -GUS. The same constructs were activated by cytokinin also at the seedling transit from scoto- to photomorphogenesis. Long-term seedling growing in darkness on medium containing cytokinin resulted in the substantial promoter activation of the gene encoding the histidine kinase AHK2. In the leaves of three-week-old plants with actively functioning chloroplasts, treatment with cytokinin mainly stimulated expression of the construct P _AHK3 -GUS. In detached senescing leaves, treatment with cytokinin retarded the loss of chlorophyll but did not affect significantly GUS activity under both light and darkness conditions in either of tested lines containing GUS gene under the control of promoters of histidine kinase genes. At the same time, cytokinin activated the promoter of the gene of primary response to cytokinin in the construct P _ARR5 -GUS. Thus, in the studied test-system, treatment with cytokinin of A. thaliana plant grown in darkness or in the light affected differently the expression of histidine kinase genes in dependence of plant age, conditions of plant cultivation, and plant physiological state.     

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors

Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.

Significance and Impact of the Study

A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.     

Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P_BAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P_BAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.     

Two tandem promoters to increase gene expression in Lactococcus lactis

Two plasmids, pPAH and pAH, containing a staphylokinase variant gene (sakXH) under the control of two tandem promoters (P₃₂-P_lacA) or promoter P_lacA alone were constructed and introduced into Lactococcus lactis MG5267. The expression of sakXH in the strain MG5267(pPAH) was approximately twice as high as that in the strain MG5267(pAH), according to the formation of fibrinolytic halos on fibrinolytic plates detected at the same conditions, indicating that the two tandem promoters were stronger than one alone. Difference between the expressions of sakXH under the inducible and non-inducible conditions suggested that P_lacA retained its feature as an inducible promoter when fused to promoter P₃₂.     

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P _lac , P _tac , or P _BAD derived from Escherichia coli, or promoter regions of phaC1 (P _phaC ) or phaP1 (P _phaP ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P _lac , P _tac , P _phaC , and P _phaP mediated constitutive gene expression, among which P _tac was the strongest promoter. lacI-P _tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P _BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P _phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P _phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.