Nepeta cataria L. (catnip) can serve as a chassis for the engineering of secondary metabolic pathways.

OBJECTIVE: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences. RESULTS: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 µmol g-1 s-1, N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 µmol g-1 s-1 and 18 ± 4 µmol g-1 s-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides. CONCLUSION: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.

Transcription factors enhancing synthesis of recombinant proteins and resistance to stress in Yarrowia lipolytica.

Resistance to environmental stress and synthesis of recombinant proteins (r-Prots) are both complex, strongly interconnected biological traits relying on orchestrated contribution of multiple genes. This, in turn, makes their engineering a challenging task. One of the possible strategies is to modify the operation of transcription factors (TFs) associated with these complex traits. The aim of this study was to examine the potential implications of selected five TFs (HSF1-YALI0E13948g, GZF1-YALI0D20482g, CRF1-YALI0B08206g, SKN7-YALI0D14520g, and YAP-like-YALI0D07744g) in stress resistance and/or r-Prot synthesis in Yarrowia lipolytica. The selected TFs were over-expressed or deleted (OE/KO) in a host strain synthesizing a reporter r-Prot. The strains were subjected to phenotype screening under different environmental conditions (pH, oxygen availability, temperature, and osmolality), and the obtained data processing was assisted by mathematical modeling. The results demonstrated that growth and the r-Prot yields under specific conditions can be significantly increased or decreased due to the TFs' engineering. Environmental factors "awakening" individual TFs were indicated, and their contribution was mathematically described. For example, OE of Yap-like TF was proven to alleviate growth retardation under high pH, while Gzf1 and Hsf1 were shown to serve as universal enhancers of r-Prot production in Y. lipolytica. On the other hand, KO of SKN7 and HSF1 disabled growth under hyperosmotic stress. This research demonstrates the usefulness of the TFs engineering approach in the manipulation of complex traits and evidences newly identified functions of the studied TFs. KEY POINTS: • Function and implication in complex traits of 5 TFs in Y. lipolytica were studied. • Gzf1 and Hsf1 are the universal r-Prots synthesis enhancers in Y. lipolytica. • Yap-like TF's activity is pH-dependent; Skn7 and Hsf1 act in osmostress response.

Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii.

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.

How can we develop an effective subunit vaccine to achieve successful malaria eradication?

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.

Biotechnological applications of Bacillus licheniformis.

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.

Gene regulation by a glycine riboswitch singlet uses a finely tuned energetic landscape for helical switching.

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.

Riboswitches: choosing the best platform.

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.

Recent advances and future trends of riboswitches: attractive regulatory tools.

Bacterial genomes contain a huge amount of different genes. These genes are spatiotemporally expressed to accomplish some required functions within the organism. Inside the cell, any step of gene expression may be modulated at four possible places such as transcription initiation, translation regulation, mRNA stability and protein stability. To achieve this, there is a necessity of strong regulators either natural or synthetic which can fine-tune gene expression regarding the required function. In recent years, riboswitches as metabolite responsive control elements residing in the untranslated regions of certain messenger RNAs, have been known to control gene expression at transcription or translation level. Importantly, these control elements do not prescribe the involvement of protein factors for metabolite binding. However, they own their particular properties to sense intramolecular metabolites (ligands). Herein, we highlighted current important bacterial riboswitches, their applications to support genetic control, ligand-binding domain mechanisms and current progress in synthetic riboswitches.

Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn2+-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA7942) and its cognate SmtB7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA6803) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster ß-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. IMPORTANCE: This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA7942 operator/promoter and smtB7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B.

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.

Microalgae as Solar-Powered Protein Factories.

Microalgae have an enormous ecological relevance as they contribute significantly to global carbon fixation. But also for biotechnology microalgae became increasingly interesting during the last decades as many algae provide valuable natural products. Especially the high lipid content of some species currently attracts much attention in the biodiesel industry. A further application that emerged some years ago is the use of microalgae as expression platform for recombinant proteins. Several projects on the production of therapeutics, vaccines and feed supplements demonstrated the great potential of using microalgae as novel low-cost expression platform. This review provides an overview on the prospects and advantages of microalgal protein expression systems and gives an outlook on potential future applications.

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

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.

Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery.

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.

An Adenoviral Vector as a Versatile Tool for Delivery and Expression of miRNAs.

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.

Immunological implications of diverse production approaches for Chikungunya virus-like particle vaccines.

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.

A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy.

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.

DIVERSIFY: A Fungal Multispecies Gene Expression Platform.

Recent sequencing of numerous fungal species revealed large repertoires of putative biotechnologically relevant genes and secondary metabolite gene clusters. However, often the commercial potential of these species is impeded by difficulties to predict host physiological and metabolic compatibility with a given product, and lack of adequate genetic tools. Consequently, most heterologous production is performed in standard hosts where genetic tools and experience are in place. However, these species may not be suitable for all products. To increase chances of successful heterologous production, we have created a flexible platform, DIVERSIFY, for multispecies heterologous gene expression. This reduces the workload to construction of a single gene expression cassette, used to transform all DIVERSIFY strains in order to identify the optimal cell factory host. As proof of principle of the DIVERSIFY concept, we present the first version of our platform, DIVERSIFY 1.0, which we have successfully used for the production of three proteins and a metabolite in four different Aspergilli species, and for the identification of the best producer for each of the products. Moreover, we show that DIVERSIFY 1.0 is compatible with marker-free gene targeting induced by the CRISPR nucleases Cas9 and MAD7.

Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.

Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins.

The production of peptides as active pharmaceutical ingredients (APIs) by recombinant technologies is of emerging interest. A reliable production platform, however, is still missing due the inherent characteristics of peptides such as proteolytic sensitivity, aggregation and cytotoxicity. We have developed a new technology named Numaswitch solving present limitations. Numaswitch was successfully employed for the production of diverse peptides and small proteins varying in length, physicochemical and functional characteristics, including Teriparatide, Linaclotide, human ß-amyloid and Serum amyloid A3. Additionally, the potential of Numaswitch for a cost-efficient commercial production is demonstrated yielding > 2 g Teriparatide per liter fermentation broth in a quality meeting API standard.