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This article discusses the use of seed plastids as a novel platform for expressing recombinant proteins. The authors compare different expression platforms, such as protein bodies in seeds and chloroplasts in leaves, and highlight the advantages of expressing proteins in seed plastids, including high-level protein accumulation and long-term stability. The authors describe a two-component system using an engineered PPR10 RNA-binding protein for seed-specific expression in plastids. They successfully expressed GFP, mScarlet, and GUS proteins in transplastomic tobacco plants, with GFP remaining stable for at least 1 year. The authors suggest that seed-based bioreactors could enable long-term storage of stable proteins at ambient temperature and discuss potential applications for seed plastids in expressing bacterial vaccine antigens and new metabolic pathways. [Extracted from the article]

Riboswitches are highly conserved RNA elements that located in the 5′-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications. [ABSTRACT FROM AUTHOR]

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously. [ABSTRACT FROM AUTHOR]

• Chikungunya virus (CHIKV) is a mosquito-borne pathogen capable of causing explosive, disruptive epidemics. • CHIKV infection causes acute febrile illness commonly associated with incapacitating polyarthalgia and myalgia and debilitating long term symptoms in many patients. • CHIKV virus-like particles (VLPs) are a promising vaccine modality. • Various genetic derivations and production systems have been used to generate CHIKV VLPs. • Nucleotide codon optimization improves production yields. • A CHIKV VLP of a single genotype is likely to generate protection against all other CHIKV genotypes. Chikungunya virus (CHIKV), an arbovirus from the Alphavirus genus, causes sporadic outbreaks and epidemics and can cause acute febrile illness accompanied by severe long-term arthralgias. Over 20 CHIKV vaccine candidates have been developed over the last two decades, utilizing a wide range of vaccine platforms, including virus-like particles (VLP). A CHIKV VLP vaccine candidate is among three candidates in late-stage clinical testing and has potentially promising data in nonclinical and clinical studies exploring safety and vaccine immunogenicity. Despite the consistency of the CHIKV VLP structure, vaccine candidates vary significantly in protein sequence identity, structural protein expression cassettes and their mode of production. Here, we explore the impact of CHIKV VLP coding sequence variation and the chosen expression platform, which affect VLP expression yields, antigenicity and overall vaccine immunogenicity. Additionally, we explore the potential of the CHIKV VLP platform to be modified to elicit protection against other pathogens. [ABSTRACT FROM AUTHOR]

The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis is a Gram positive spore-forming bacterial species of high biotechnological interest with numerous present and potential uses, including the production of bioactive compounds that are applied in a wide range of fields, such as aquaculture, agriculture, food, biomedicine, and pharmaceutical industries. Its use as an expression vector for the production of enzymes and other bioproducts is also gaining interest due to the availability of novel genetic manipulation tools. Furthermore, besides its widespread use as a probiotic, other biotechnological applications of B. licheniformis strains include: bioflocculation, biomineralization, biofuel production, bioremediation, and anti-biofilm activity. Although authorities have approved the use of B. licheniformis as a feed additive worldwide due to the absence of toxigenic potential, some probiotics containing this bacterium are considered unsafe due to the possible transference of antibiotic resistance genes. The wide variability in biological activities and genetic characteristics of this species makes it necessary to establish an exact protocol for describing the novel strains, in order to evaluate its biotechnological potential. [ABSTRACT FROM AUTHOR]

The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta , thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases. [ABSTRACT FROM AUTHOR]

The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana . This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform. [ABSTRACT FROM AUTHOR]

Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community. [ABSTRACT FROM AUTHOR]

pergilli have a long history in biotechnology as expression platforms for the production of food ingredients, pharmaceuticals and enzymes. The achievements made during the last years, however, have the potential to revolutionize Aspergillus biotechnology and to assure Aspergillus a dominant place among microbial cell factories. This mini-review will highlight most recent breakthroughs in fundamental and applied Aspergillus research with a focus on new molecular tools, techniques and products. New trends and concepts related to Aspergillus genomics and systems biology will be discussed as well as the challenges that have to be met to integrate omics data with metabolic engineering attempts. [ABSTRACT FROM AUTHOR]

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco ( Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7 lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform. [ABSTRACT FROM AUTHOR]

The cellular concentrations of certain metabolites are assiduously monitored to achieve appropriate levels of gene expression. Although proteins have long been known to act as sensors in this capacity, metabolite-binding RNAs, or riboswitches, also play an important role. More than 20 distinct classes of riboswitches have been identified to date, and insights to the molecular recognition strategies of a significant subset of these have been provided by detailed structural studies. This diverse set of metabolite-sensing RNAs is found to exploit a variety of distinct mechanisms to regulate genes that are fundamental to metabolism. [ABSTRACT FROM AUTHOR]

A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a ‘universal’ fungal vector appears to be feasible. [Copyright &y& Elsevier]

Malaria, a mosquito-borne infection, is the most widespread parasitic disease. Despite numerous efforts to eradicate malaria, this disease is still a health concern worldwide. Owing to insecticide-resistant vectors and drug-resistant parasites, available controlling measures are insufficient to achieve a malaria-free world. Thus, there is an urgent need for new intervention tools such as efficient malaria vaccines. Subunit vaccines are the most promising malaria vaccines under development. However, one of the major drawbacks of subunit vaccines is the lack of efficient and durable immune responses including antigen-specific antibody, CD4+, and CD8+ T-cell responses, long-lived plasma cells, memory cells, and functional antibodies for parasite neutralization or inhibition of parasite invasion. These types of responses could be induced by whole organism vaccines, but eliciting these responses with subunit vaccines has been proven to be more challenging. Consequently, subunit vaccines require several policies to overcome these challenges. In this review, we address common approaches that can improve the efficacy of subunit vaccines against malaria. [Display omitted] • Applying appropriate adjuvants can enhance the immunogenicity of subunit vaccines against malaria. • A proper expression platform needs to be selected based on the target antigen to produce the proper conformation of protein. • Antigenic regions are required to introduce to the immune system in a proper manner to induce the potent immune responses. • The application of a suitable immunization strategy could enhance the desired immune responses to malaria subunit vaccines. • The identification of new vaccine candidates by reverse vaccinology may facilitate the design of new subunit vaccines. [ABSTRACT FROM AUTHOR]

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required. [ABSTRACT FROM AUTHOR]

Riboswitch discovery and characterisation have come a long way since the term was first coined almost two decades ago. Riboswitches themselves are likely derived from ancient ligand-binding transcripts, which have evolved into sophisticated genetic control elements that are widespread in prokaryotes. Riboswitches are associated with a multitude of cellular processes including biosynthetic pathways, transport mechanisms and stress responses leading to an ever-increasing appreciation for an in-depth understanding of their triggers and functions in order to address physiological and regulatory questions. The majority of riboswitches exert their control via transcriptional or translational expression platforms depending on their genetic context. It remains, however, to be determined precisely why one platform is favoured over another. Is this a question of the layout of the gene expression machinery, ligand availability, the degree of control required, serendipity or various combinations of these? With this review, rather than providing answers, I am hoping to plant a seed for further scientific discussions about this puzzle., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Riboswitches are cis-acting, non-coding RNA elements found in the 5'UTR of bacterial mRNA and 3' UTR of eukaryotic mRNA, that fold in a complex manner to act as receptors for specific metabolites hence altering their conformation in response to the change in concentrations of a ligand or metabolite. Riboswitches function as gene regulators in numerous bacteria, archaea, fungi, algae and plants., Aim and Objective: This study identifies different classes of riboswitches in the Archaeal domain of life. Previous studies have suggested that riboswitches carry a conserved aptameric domain in different domains of life. Since Archaea are considered to be the most idiosyncratic organisms it was interesting to look for the conservation pattern of riboswitches in these obviously strange microorganisms., Materials and Methods: Completely sequenced Archaeal Genomes present in the NCBI repository were used for studying riboswitches and other ncRNAs. The sequence files in FASTA format were downloaded from NCBI Genome database and information related to these genomes was retrieved from GenBank. Three bioinformatics approaches were used namely, ab initio, consensus structure prediction and statistical model-based prediction for identifying riboswitches., Results: Archaeal genomes have a sporadic distribution of putative riboswitches like the TPP, FMN, Guanidine, Lysine and c-di-AMP riboswitches, which are known to occur in bacteria. Also, a class of riboswitch sensing c-di-GMP, a second messenger, has been identified in a few Archaeal organisms., Conclusion: This study clearly reveals that bioinformatics methods are likely to play a major role in identifying conserved riboswitches and in establishing how widespread these classes are in all domains of life, even though the final confirmation may come from wet lab methods., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2-3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events., (© 2018 Torgerson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Riboswitches are RNA elements that control the expression of genes through a variety of mechanisms in response to the specific binding of small-molecule ligands. Since their discovery, riboswitches have shown promise for the artificial control of transcription or translation of target genes, be it for industrial biotechnology, protein expression, metabolic engineering, antimicrobial target validation, or gene function discovery. However, natural riboswitches are often unsuitable for these purposes due to their regulation by small molecules which are already present within the cell. For this reason, research has focused on creating riboswitches that respond to alternative biologically inert ligands or to molecules which are of interest for biosensing. Here we present methods for the development of artificial riboswitches in Gram-negative and Gram-positive bacteria. These methods are based on reengineering natural aptamers to change their ligand specificity toward molecules which do not bind the original aptamer (ie, that are orthogonal to the original). The first approach involves targeted mutagenesis of native riboswitches to change their specificity toward rationally designed synthetic ligand analogs. The second approach involves the fusion of previously validated orthogonal aptamers with native expression platforms to create novel chimeric riboswitches for the microbial target. We establish the applicability of these methods both for the control of exogenous genes as well as for the control of native genes., (© 2016 Elsevier Inc. All rights reserved.)