Copper-inducible Promoter Research Articles

Gelatin expression from an engineered Saccharomyces cerevisiae CUP1 promoter in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper-inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S.cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose-dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper-containing fermentation basal salts media. Removal of a S.cerevisiae heat shock factor (Hsf1p) binding site reduced copper-dependent gelatin induction suggesting that a similar protein may regulate this promoter in P.pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

Isolation of two novel promoters from ramie (Boehmeria nivea L. Gaudich) and its functional characterization in Arabidopsis thaliana

The development of tissue-specific or inducible promoters is important for plant genetic engineering. In this study, we isolated two novel promoters (named BnGDSpro and BnSTMpro) from ramie and analyzed their functions in Arabidopsis. The results show that BnGDSpro and its 5′-truncated fragments drive GUS dominantly expressed in shoot meristem while BnSTMpro and its 5′-truncated fragments have apical meristem specificity in Arabidopsis seedlings and nodule specificity in the flowers and siliques. The − 1233 to − 914 region and − 425 to 0 region of BnSTMpro are responsible for apical root specificity and root-knot specificity, respectively. In particular, CuSO4 treatment reveals that both BnGDSpro and BnSTMpro are copper-inducible promoters. Our findings suggest that BnGDSpro and BnSTMpro and their truncated fragments have potential applications in plant genetic transformation.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72h) and temperature (28°C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus

BackgroundThe CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites.ResultsIn this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects.ConclusionsOur results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast

BackgroundAssembly of heterologous metabolic pathways is commonly required to generate microbial cell factories for industrial production of both commodity chemicals (including biofuels) and high-value chemicals. Promoter-mediated transcriptional regulation coordinates the expression of the individual components of these heterologous pathways. Expression patterns vary during culture as conditions change, and this can influence yeast physiology and productivity in both positive and negative ways. Well-characterized strategies are required for matching transcriptional regulation with desired output across changing culture conditions.ResultsHere, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production of trans-nerolidol, an acyclic sesquiterpene alcohol, in yeast. The choice of regulatory system significantly affected physiological features (growth and productivity) over batch cultivation. Use of constitutive promoters resulted in poor growth during the exponential phase. Delaying expression of the assembled metabolic modules using the copper-inducible CUP1 promoter resulted in a 1.6-fold increase in the exponential-phase growth rate and a twofold increase in productivity in the post-exponential phase. However, repeated use of the CUP1 promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineered GAL regulatory circuit and a set of four different GAL promoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L−1 using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through the GAL system; final nerolidol titres of 4–5.5 g L−1 were achieved, depending on the conditions.ConclusionDirect comparison of different transcriptional regulatory mechanisms clearly demonstrated that coupling the output strength to the fermentation stage is important to optimize the growth fitness and overall productivities of engineered cells in industrially relevant processes. Applying different well-characterized promoters with the same induction behaviour mitigates against the risks of homologous sequence-mediated genetic instability. Using these approaches, we significantly improved sesquiterpene production in yeast.

Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in Pichia pastoris

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (PPPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (PAOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of PPPLCC1 was compared with those of PAOX1 and PCUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for PPPLCC1 and PCUP1, respectively, after induction with 0.2 mM CuSO4 at OD600 = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For PAOX1 activity, yeast cells harboring PAOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, PPPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. PPPLCC1 is additionally superior to PAOX1 since it does not require laborious feeding with a carbon source.

Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography

Arginase (EC 3.5.3.1; l-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)6-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni–NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600μmolmin−1mg−1 protein.

Oral administration of myostatin-specific whole recombinant yeast Saccharomyces cerevisiae vaccine increases body weight and muscle composition in mice

Myostatin negatively regulates skeletal muscle growth. It was found that active immunization with myostatin-specific vaccine blocked myostatin function in vivo, which resulted in increase of body weight and muscle composition in mice. However, traditional vaccine and its administration method are expensive and laborious. In this study, we investigated the possibility of using heat-killed whole recombinant yeast Saccharomyces cerevisiae vaccine to modulate myostatin function in mice. The CDS of myostatin was obtained from a pig genome by PCR and subcloned into a yeast expression vector, which was driven by a copper-inducible promoter. Expression of recombinant myostatin was induced by CuSO(4) and confirmed by western blot. We vaccinated mice by oral feeding and subcutaneous injection as comparison. We found that oral feeding resulted in the similar effective immune response than injection, which was measured by the presence of myostatin-specific antibodies in mouse serum. Interestingly, animals vaccinated by both methods demonstrated enhanced growth performance compared to control. All animals were healthy looking throughout the course of experiment, suggesting that whole recombinant yeast vaccine is nontoxic and therefore safe to use. Given the simplicity of its nature, heat-killed myostatin-specific whole recombinant yeast vaccine holds a promise to treat human muscle-wasting diseases in the future.

ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy

The ubiquitin–proteasome pathway and autophagy are the two major mechanisms responsible for the clearance of cellular proteins. We have used the yeast Saccharomyces cerevisiae as a model system and the cystic fibrosis transmembrane conductance regulator (CFTR) as a model substrate to study the interactive function of these two pathways in the degradation of misfolded proteins. EGFP-tagged human CFTR was introduced into yeast and expressed under a copper-inducible promoter. The localization and degradation of EGFP-CFTR in live cells were monitored by time-lapse imaging following its de novo synthesis. EGFP-CFTR first appears within the perinuclear and sub-cortical ER and is mobile within the plane of the membrane as assessed by fluorescence recovery after photobleaching (FRAP). This pool of EGFP-CFTR is subsequently degraded through a proteasome-dependent pathway that is inhibited in the pre1-1 yeast strain defective in proteasomal degradation. Prolonged expression of EGFP-CFTR leads to the sequestration of EGFP-CFTR molecules into ER structures called ER-associated complexes (ERACs). The sequestration of EGFP-CFTR into ERACs appears to be driven by aggregation since EGFP-CFTR molecules present within ERACs are immobile as measured by FRAP. Individual ERACs are cleared from cells through the autophagic pathway that is blocked in the atg6Δ and atg1Δ yeast strains defective in autophagy. Our results suggest that the proteasomal and the autophagic pathways function together to clear misfolded proteins from the ER.

Vectors for lactobacilli and other Gram-positive bacteria based on the minimal replicon of pRV500 from Lactobacillus sakei

The low-copy-number plasmid pRV500, belonging to the pUCL287 group of θ-type plasmids, was previously isolated from Lactobacillus sakei and characterized. We show here that the replicon of this plasmid enables replication also in Enterococcus faecalis and Bacillus subtilis but not in Lactococcus lactis. A 1.25 kb region encompassing the iterons and the repA gene was sufficient for replication, copy-number control and relative stable maintenance in L. sakei. Functional implications of host or plasmid-borne factors in the maintenance of pUCL287-type plasmids are discussed. The minimal replicon from pRV500 was fused to pBluescript for constructing the shuttle E. coli/lactobacilli cloning vector pRV610. pRV610 enables the white/blue lacZ α-complementation in E. coli. The cassettes for selection (erythromycin resistance) and replication (iterons and repA gene) are each bordered by unique restriction sites for easy replacement if needed. Derivatives in which chloramphenicol or tetracycline resistance replaced erythromycin resistance were constructed. In order to allow inducible gene expression, a copper-inducible promoter was placed on the pRV613 derivative. Expression of the downstream reporter gene lacZ was shown to be induced by 30 μM CuSO 4.

A Bipartite Recombinant Yeast System for the Identification of Subtype-Selective Estrogen Receptor Ligands

Since the estrogen receptor alpha (ERalpha) and beta (ERbeta) are thought to mediate different biological effects, there is intense interest in designing or screening subtype-selective ER ligands. Here, we constructed a biosensor identified as bipartite recombinant yeast system (BRYS) to screen and evaluate subtype-selective ER ligands. Uniform design and immunoblotting was used to determine an optimal dose of copper which control the expression of ERs through a copper inducible metallothionine promoter (CUP1). Some chemicals including natural estrogen (17beta-estradiol), specific estrogen receptor agonist (PPT and DPN), and phytoestrogens (genistein) were tested to validate this system. There was obvious and anticipative discrimination between the agonistic activities when these chemicals were identified and characterized. Furthermore, antagonist (ICI 182,780), which could antagonize the transactivation of estrogen, and chromatin immunoprecipitation (ChIP) were used to confirm that the agonistic effects were mediated through ERs. Comparative studies showed that this system was reproducible and sensitive. 4.7 pM (1.3 ng/l) estrogen was the lowest concentration that could be detected to ERalpha and 0.12 nM (33.5 ng/l) for ERbeta. The results from our study showed that in vitro screening for subtype-selective ER ligands could be conducted in a simple yeast system that is rapid, sensitive, reliable, and quantitative.

Detection of Ubiquitylated Proteins in Yeast

INTRODUCTIONThis assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting.

Characterization of Copper-Inducible Promoters Regulated by CpxA/CpxR inEscherichia coli

The copper stimulon in Escherichia coli consists of four regulons, the CueR-, CusS/CusR-, CpxA/CpxR-, and YedV/YedW regulons. E. coli mutants defective in cpxRA showed higher sensitivity to copper than the wild type. A total of 15 promoters were found to be induced in E. coli culture upon exposure to copper in a CpxA/CpxR-dependent manner. After gel-shift and DNase I foot-printing analyses, a conserved tandem repeat of pentanucleotide sequence, GTAAA(N)(4-8)GTAAA, with a conserved A of 4-bp upstream of each pentamer, was identified to be the CpxR-binding site. The difference in the orientation and location of the CpxR box is discussed with respect to the regulation mechanism among CpxR-regulon genes.

Identification of a copper-inducible promoter for use in ectopic expression in the fungal pathogen Histoplasma capsulatum.

Despite the existence of a number of genetic tools to study the fungal pathogen Histoplasma capsulatum, strategies for conditional gene expression have not been developed. We used microarray analysis to identify genes that are transcriptionally induced or repressed by the addition of copper sulfate (CuSO(4)) to H. capsulatum yeast cultures. One of these genes, CRP1, encodes a putative copper efflux pump that is significantly induced in the presence of CuSO(4). The upstream regulatory region of CRP1 was sufficient to drive copper-regulated expression of two reporter genes, lacZ and the gene encoding green fluorescent protein. Microarray experiments were performed to determine a copper concentration that triggers accumulation of the CRP1 transcript without significant perturbation of global gene expression. These studies show that the CRP1 upstream regulatory region can be used for ectopic expression of heterologous genes in H. capsulatum. Furthermore, they demonstrate the strategic use of microarrays to identify conditional promoters that confer induction in the absence of large-scale shifts in gene expression.

Harnessing a tiny hairy beast

With the goal of using the ciliate Tetrahymena thermophila for biotechnology applications-as a heterologous protein expression system-researchers have identified a copper-inducible and repressible promoter.

Metallothionein Gene from Tetrahymena thermophila with a Copper-Inducible-Repressible Promoter

We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

Development of food-grade cloning and expression vectors for Lactococcus lactis

To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA.

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an 'all-or-none' behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3′-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.