High-level Expression Of Heterologous Genes Research Articles

A Universal Gene Expression System for Novel Yeast Species.

The current progress in sequencing of genomes and characterization of new species provides an increasing list of yeasts that show interesting physiological properties; however, the lack of expression tools for these new hosts is prohibiting their broader use in research or industry. Recently, we developed a universal expression system (SES) functional in broad spectrum of fungal species, which represent a solution for feasible gene expression control and genetic manipulation in these novel hosts. Here, we describe three example approaches for DNA transformation and high-level heterologous gene expression, using the SES system, in three yeast species, where minimal knowledge or prior experience in genetic modifications is available.

Identification of Loci Enabling Stable and High-Level Heterologous Gene Expression.

Efficient and reliable genome engineering technologies have yet to be developed for diatoms. The delivery of DNA in diatoms results in the random integration of multiple copies, quite often leading to heterogeneous gene activity, as well as host instability. Transgenic diatoms are generally selected on the basis of transgene expression or high enzyme activity, without consideration of the copy number or the integration locus. Here, we propose an integrated pipeline for the diatom, Phaeodactylum tricornutum, that accurately quantifies transgene activity using a β-glucuronidase assay and the number of transgene copies integrated into the genome through Droplet Digital PCR (ddPCR). An exhaustive and systematic analysis performed on 93 strains indicated that 42% of them exhibited high β-glucuronidase activity. Though most were attributed to high transgene copy numbers, we succeeded in isolating single-copy clones, as well as sequencing the integration loci. In addition to demonstrating the impact of the genomic integration site on gene activity, this study identifies integration sites for stable transgene expression in Phaeodactylum tricornutum.

Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 7002 mirrors the protein overproduction phenotype from E. coli BL21(DE3)

With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. KEY POINTS: • Promoter driving T7-RNA polymerase was optimized. • Up to 60-fold dynamic range in expression, depending on CO2 conditions. • Two-thirds of total protein is T7-RNA polymerase dependent.

PS1454 CAG PROMOTER DRIVEN C‐MYC OVEREXPRESSION CAUSES RAPIDLY LETHAL CARDIOMYOPATHY

Background:We have previously reported that mice expressing elevated levels of NFE2 display a myeloproliferative phenotype which spontaneously transforms to acute leukemia. Upon leukemic transformation, a high percentage of mice acquire duplications or amplification of the murine c‐myc locus. The CMV early enhancer/chicken‐beta‐actin (CAG) promoter has been reported to direct high level heterologous gene expression specifically to hematopoietic stem cells and myeloid progenitors.Aims:We hypothesized that elevated expression of both NFE2 and c‐myc synergize to induce leukemic transformation. Therefore, we compared the hematological phenotype of mice carrying either augmented NFE2 or c‐myc levels alone to mice overexpressing both genes.Methods:We crossed mice expressing a CAG‐driven tetracycline activator (CAG‐rtTA3) with the tet‐O‐myc mouse strain, in order to achieve high levels of doxycycline‐inducible c‐myc expression in the hematopoietic compartment. Double transgenic CAG‐myc mice were subsequently bred to heterozygous knock‐in mice expressing the NFE2‐D297–300 mutation of the endogenous promoter, generating both CAG‐myc‐NFE2‐wt and CAG‐myc‐NFE2mut mice. C‐myc expression was induced by doxycycline treatment administered through various routes, including drinking water, oral gavage and food pellets.Results:Unexpectedly both CAG‐myc and CAG‐myc‐NFE2 mice died rapidly, between 6 and 7 days following c‐myc induction, independent of the route of doxycycline administration. Autopsy and histological analysis revealed marked cardiomyocyte hypertrophy, with heart volume more than double the wt control (Fig. 1). In addition to hematopoietic stem and progenitor cells, the CAG promoter has been reported to direct expression to cardiomyocytes. C‐myc overexpression in adult cardiomyocytes by tet‐mediated c‐myc induction has previously been shown to cause cell cycle re‐entry that leads to cardiomyopathy and hypertrophy. However, it was not expected that the CAG promoter, seen as a “highly effective tool for probing hematopoietic development and disease”, would have a similarly cardiotoxic effect.Fig. 1: Pathology and histology of hearts following doxycycline induction of CAG‐myc expression. (A) representative autopsy finding from a wt control mouse (left) and a CAG‐myc‐NFE2‐wt mouse. Results are representative of n = 16 CAG‐myc mice. (B) Hematoxylin Eosin (H&E) staining of histological sections from CAG‐myc‐NFE2 wt and CAG‐myc‐NFE2mut mice before and after doxycycline induction. 200x magnification, size bars represent 50 mm.Summary/Conclusion:While our murine model did not allow us to investigate our hypothesized synergistic effect between c‐myc and NFE2 in leukemic transformation, the CAG‐myc mouse strain generated constitutes an excellent model for studying rapidly lethal cardiomyopathy. Moreover, our data serve to alert researchers in the hematopoietic field to possible side effects of using the CAG promoter to drive gene expression in hematopoietic stem and progenitor cells.image

Targeted and Repetitive Chromosomal Integration Enables High-Level Heterologous Gene Expression in Lactobacillus casei.

Lactobacillus casei is a potential cell factory for the production of enzymes and bioactive molecules using episomal plasmids, which suffer from genetic instability. While chromosomal integration strategies can provide genetic stability of recombinant proteins, low expression yields limit their application. To address this problem, we developed a two-step integration strategy in Lb. casei by combination of the LCABL_13040-50-60 recombineering system (comprised of LCABL_1340, LCABL_13050, and LCABL_13060) with the Cre/loxP site-specific recombination system, with an efficiency of ∼3.7 × 103 CFU/µg DNA. A gfp gene was successfully integrated into six selected chromosomal sites, and the relative fluorescence intensities (RFUs) of the resulting integrants varied up to ∼3.7-fold depending on the integrated site, among which the LCABL_07270 site gfp integration showed the highest RFU. However, integrants with gfp gene(s) integrated into the LCABL_07270 site showed various RFUs, ranging from 993 ± 89 to 7,289 ± 564 and corresponding to 1 to 13.68 ± 1.08 copies of gfp gene integration. Moreover, the integrant with 13.68 ± 1.08 copies of the gfp gene had a more stable RFU after 63 generations compared to that of a plasmid-engineered strain. To investigate the feasibility of this system for bioactive molecules with high expression levels, the fimbrial adhesin gene, faeG, from Escherichia coli was tested and successfully integrated into the LCABL_07270 site with 5.51 ± 0.25 copies, and the integrated faeG achieved stable expression. All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location in Lb. caseiIMPORTANCE Lactic acid bacteria (LAB), including Lactobacillus casei, have the potential for overexpression of heterologous proteins, such as bioactive molecules and enzymes. However, traditional genetic tools for expression of these proteins show genetic instability or low yields of the desired product. In this study, we provide a procedure for repetitive integration of genes at various chromosomal locations, achieving high-level and stable expression of proteins in Lb. casei without selective pressure. The protocol developed in this study provides an essential reference for chromosomal overexpression of proteins or bioactive molecules in LAB.

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum.

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.

Design and Optimization of Short DNA Sequences That Can Be Used as 5′ Fusion Partners for High-Level Expression of Heterologous Genes in Escherichia coli

The 5' terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. The ifn-α2bS gene is poorly expressed in Escherichia coli unless a translocation signal sequence (pelB) is fused to the 5' end of the gene. A combined in silico and in vivo analysis reported here further indicates that the ifn-α2bS 5' coding sequence is suboptimal for efficient gene expression. ifn-α2bS therefore presents a suitable model gene for describing properties of 5' fusions promoting expression. We show that short DNA sequences corresponding to the 5' end of the highly expressed celB gene, whose protein product is cytosolic, can functionally replace pelB as a 5' fusion partner for efficient ifn-α2bS expression. celB fusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of a celB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5' fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of the celB fusion, and one of these (celBD11) could be used to additionally increase ifn-α2bS expression more than 4-fold at the protein level. Interestingly, celBD11 also stimulated greater protein production of three other medically important human genes than the wild-type celB fragment.

Molecular characterization of Candida boidinii MIG1 and its role in the regulation of methanol-inducible gene expression

Methanol-inducible gene promoters in methanol-utilizing yeasts are used in high-level heterologous gene expression systems. Generally, expression of methanol-inducible genes is completely repressed by the presence of glucose. In this study we identified the MIG1 gene in Candida boidinii, which encodes a homologue of the glucose repressor Mig1p of Saccharomyces cerevisiae. Disruption of the CbMIG1 gene had no growth effect on various carbon sources. Activation of the methanol-inducible AOD1 gene, which encodes alcohol oxidase, was increased in the early stage of methanol induction when cells of the CbMIG1-disrupted strain were transferred from glucose medium to methanol medium. Furthermore, CbMig1p tagged with yellow fluorescent protein was primarily localized in the nucleus of glucose-grown cells, but was diffuse in the cytosol of methanol-grown cells. This cytosolic diffusion in methanol-grown cells occurred in a CbMsn5p-dependent manner. These results suggest that CbMig1p is involved in negative regulation of methanol-inducible gene expression in C. boidinii.

Alphavirus Vectors in Vaccine Development

The high-level heterologous gene expression provided by alphavirus vectors has accelerated their applications in vaccine development. The versatility of alphavirus vectors has allowed their use in the form of recombinant viral particles, naked RNA and layered DNA molecules for immunization. The most commonly used alphaviruses have been Semliki Forest virus, Sindbis virus and Venezuelan Equine Encephalitis virus. Numerous viral structural proteins have been used as antigens to generate neutralizing antibodies in immunized animals. Vaccination has demonstrated protection against challenges with lethal doses of viruses. Moreover, vaccination with tumor antigens has demonstrated prophylactic protection against cancer. Novel approaches include the application of RNA interference and microRNA. The other side of the coin is the development of vaccines against alphaviruses themselves, and typically the Chikungunya virus.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants

The functional and biological significance of translation initiation context sequence in determining high-level expression of modified synthetic human alpha(1)-proteinase inhibitor (alpha(1)-PI) gene was documented in stable transgenic tomato plants. Context sequence of initiator ATG codon derived from statistical analysis of databases was identified as taaA(A/C)aATGGCt in highly expressed dicot plant genes. Removal of initiator ATG context sequence reduced the expression of recombinant alpha(1)-PI protein to fourfolds. The mutation of consensus base at +4 position to a pyrimidine either alone or with substitution at -3 position eliminated most of the alpha(1)-PI expression, while mutation at -3 alone resulted in about sevenfold reduction. The presence of steady-state levels of alpha(1)-PI transcript in transgenic plants indicated that the variation in expression is entirely due to the point mutations incorporated in translation initiation context. These results indicated the significance of conserved nucleotide sequence around initiator ATG codon in augmenting post-transcriptional events and high-level expression of heterologous genes in transgenic plants.

Methanol‐inducible gene expression and heterologous protein production in the methylotrophic yeast Candida boidinii

Methylotrophic yeasts can obtain all of their required carbon and energy from methanol, a promising feedstock for biotechnological and chemical processes and a potential replacement for coal and petroleum. When these yeast cells are cultured in the presence of methanol, the enzymes involved in methanol metabolism are massively produced, indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using these promoters, high-level heterologous gene expression systems have been developed in several methylotrophic yeast strains, including Pichia pastoris, Hansenula polymorpha, Pichia methanolica and Candida boidinii. In the present minireview we describe recent insights into the molecular basis of methanol-inducible gene expression in C. boidinii. In addition, the utility of C. boidinii gene expression systems for the production of various enzymes is also reviewed.

Molecular Basis of Methanol-Inducible Gene Expression and Its Application in the Methylotrophic YeastCandida boidinii

Methanol is a promising feedstock for biotechnological and chemical processes as well as a primary source of energy to replace coal and petroleum. Methylotrophic yeasts, that can utilize methanol as the sole source of carbon and energy, have been studied intensively in terms of both physiological activities and potential applications. During growth on methanol, the enzymes involved in methanol metabolism are massively produced in these yeasts, indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using these promoters, high-level heterologous gene expression systems have been developed in several methylotrophic yeast strains, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii. To achieve efficient industrial use of methanol and efficient protein production by methylotrophic yeasts, it is important to elucidate the molecular basis of methanol-inducible gene expression in these yeasts. This review describes recent advances in understanding of the regulation of methanol-inducible gene expression and the molecular mechanism of transcriptional activation in the methylotrophic yeast C. boidinii. Application of this gained knowledge led to successful production of useful enzymes in this yeast, which is also reviewed.

Two-Cistronic Expression Plasmids for High-Level Gene Expression in Escherichia coli Preventing Translational Initiation Inhibition Caused by the Intramolecular Local Secondary Structure of mRNA

Two-cistronic expression plasmids for the wild-type solubilized domain of porcine NADPH-cytochrome P450 reductase (PsCPR) gene in Escherichia coli were systematically constructed using a solubilized domain of porcine cytochrome b5 gene (Psb5 gene) or a derivative of it as the first cistron to examine their utility for second gene expression preventing the translational inhibition caused by the intramolecular local secondary structure of mRNA at the ribosome-binding site (RBS). The mRNAs from the plasmids lacking an RBS for the second cistron (SD2) accumulated very low levels of PsCPR, while those from the plasmids having an SD2 accumulated higher levels of PsCPR. The level of accumulation of PsCPR by the mRNA from plasmid pCbSD-T-CPR-3, which has an SD2 upstream of the termination codon of the first cistron, was higher than for those with an SD2 in the intercistronic region. The predicted intramolecular local secondary structures at the SD2 of mRNAs from these plasmids were stable enough to cause translational initiation inhibition. These results indicate that the use of a two-cistronic expression plasmid is an effective way to overcome translational initiation inhibition. Improved plasmids, pCP1 and pCP2P, were constructed from pCbSD-T-CPR-3. Using these plasmids, the solubilized donain of porcine NADH-cytochrome b5 reductase was also highly accumulated on prevention of the translational initiation inhibition. These plasmids are expected to be useful tools for the comprehensive high-level expression of heterologous genes in E. coli cells.

Alphavirus vectors for cancer gene therapy (Review)

Alphaviruses have several characteristics that make them attractive as gene therapy vectors such as transient and high-level expression of a heterologous gene. Alphavirus vectors, Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEE) have been developed as gene expression vectors. Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in mammalian cells. The alphavirus RNA replication machinery has been engineered for high level heterologous gene expression. Since an RNA virus vector cannot integrate into chromosomal DNA, concerns about cell transformation are reduced. Alphavirus vectors demonstrate promise for the safe tumor-killing and tumor-specific immune responses. Recombinant alphavirus RNA replicons may facilitate gene therapy of cancer.

The effect of ethanol and acetate on protein expression in Pichia pastoris

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut +, Mut s, or Mut −). We have investigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut +) and MC100-3 (Mut −), expressing an AOX1-lacZ fusion. The addition of 10 mg l −1 ethanol at the start of methanol induction delayed β-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.

The Effect of Ethanol and Acetate on Protein Expression in Pichia pastoris.

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut+, Mut(s), or Mut-). We have nvestigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut+) and MC100-3 (Mut-), expressing an AOX1-lacZ fusion. The addition of 10 mg l(-1) ethanol at the start of methanol induction delayed beta-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.

Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance.

To construct a vector for high-level expression of heterologous genes in Lentinus edodes, the L. edodes GPD promoter, which is expressed constitutively and strongly, was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selective marker. Using the resulting pLG-hph construct, L. edodes was efficiently transformed to hygromycin resistance by restriction enzyme-mediated integration (REMI). The restriction enzyme concentrations yielding the maximal numbers of transformants by the REMI method were 10 U per transformation in the case of BglII and 25-50 U in the case of HindIII. Southern analysis of the transformants indicated that some 50% of plasmid integrations were REMI-mediated events. These results indicate that pLG is a useful vector for transformation of L. edodes. Deletion analysis of the GPD promoter region suggested that the segment between positions -442 bp and -270 bp relative to the transcription start point may be essential for function.

Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors.

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high ( approximately 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-beta-Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.

A Regulatory Element of the Human Keratin 18 Gene with AP-1-dependent Promoter Activity

The human keratin 18 (K18) gene is expressed in a restricted but diverse subset of differentiated epithelial tissues and carcinomas. The 10-kilobase pair K18 gene contains all of the genetic information necessary for tissue-specific, copy number-dependent and integration site-independent expression in transgenic mice. We identified a 100-base pair regulatory element that activates the K18 proximal promoter in the presence of the previously identified first intron enhancer. Deletion of the element greatly diminished K18 expression. This regulatory element also has cryptic, AP-1-dependent promoter activity in the absence of the normal promoter, which results in 10-40-fold higher levels of K18 RNA expression in transgenic mice. The high activity of this cryptic promoter is dependent upon the first intron enhancer. These experiments define interactive regulatory regions of the K18 gene that modulate expression in diverse epithelial cell types and identify an unusual regulatory element with promoter activity that may be useful for high level heterologous gene expression in transgenic animals.