Efficient virus detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis.

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization.

A plea for the integration of Green Toxicology in sustainable bioeconomy strategies - Biosurfactants and microgel-based pesticide release systems as examples.

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish, individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.

A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis.

Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3.

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

Perspectives for the application of Ustilaginaceae as biotech cell factories.

Basidiomycetes fungi of the family Ustilaginaceae are mainly known as plant pathogens causing smut disease on crops and grasses. However, they are also natural producers of value-added substances like glycolipids, organic acids, polyols, and harbor secretory enzymes with promising hydrolytic activities. These attributes recently evoked increasing interest in their biotechnological exploitation. The corn smut fungus Ustilago maydis is the best characterized member of the Ustilaginaceae. After decades of research in the fields of genetics and plant pathology, a broad method portfolio and detailed knowledge on its biology and biochemistry are available. As a consequence, U. maydis has developed into a versatile model organism not only for fundamental research but also for applied biotechnology. Novel genetic, synthetic biology, and process development approaches have been implemented to engineer yields and product specificity as well as for the expansion of the repertoire of produced substances. Furthermore, research on U. maydis also substantially promoted the interest in other members of the Ustilaginaceae, for which the available tools can be adapted. Here, we review the latest developments in applied research on Ustilaginaceae towards their establishment as future biotech cell factories.

A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis.

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified. Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial ß-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant. In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

A Straightforward Assay for Screening and Quantification of Biosurfactants in Microbial Culture Supernatants.

A large variety of microorganisms produces biosurfactants with the potential for a number of diverse industrial applications. To identify suitable wild-type or engineered production strains, efficient screening methods are needed, allowing for rapid and reliable quantification of biosurfactants in multiple cultures, preferably at high throughput. To this end, we have established a novel and sensitive assay for the quantification of biosurfactants based on the dye Victoria Pure Blue BO (VPBO). The assay allows the colorimetric assessment of biosurfactants directly in culture supernatants and does not require extraction or concentration procedures. Working ranges were determined for precise quantification of different rhamnolipid biosurfactants; titers in culture supernatants of recombinant Pseudomonas putida KT2440 calculated by this assay were confirmed to be the same ranges detected by independent high-performance liquid chromatography (HPLC)-charged aerosol detector (CAD) analyses. The assay was successfully applied for detection of chemically different anionic or non-ionic biosurfactants including mono- and di-rhamnolipids (glycolipids), mannosylerythritol lipids (MELs, glycolipids), 3-(3-hydroxyalkanoyloxy) alkanoic acids (fatty acid conjugates), serrawettin W1 (lipopeptide), and N-acyltyrosine (lipoamino acid). In summary, the VPBO assay offers a broad range of applications including the comparative evaluation of different cultivation conditions and high-throughput screening of biosurfactant-producing microbial strains.

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1.

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis, for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.

Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis.

Eukaryotic microorganisms use monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. During translation of these peptides the ribosome stalls, the peptide chain is released and the ribosome resumes translation. Thus, two independent polypeptide chains can be encoded from a single mRNA when a 2A peptide sequence is placed inbetween the two open reading frames. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

Transport and kinase activities of CbrA of Pseudomonas putida KT2440.

The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. CbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase CbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. The results demonstrate that CbrA functions as a L-histidine transporter and sensor kinase.

Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid.

Microbial valorization of plant biomass is a key target in bioeconomy. A promising candidate for consolidated bioprocessing is the dimorphic fungus Ustilago maydis. It harbors hydrolytic enzymes to degrade biomass components and naturally produces valuable secondary metabolites like itaconic acid, malic acid or glycolipids. However, hydrolytic enzymes are mainly expressed in the hyphal form. This type of morphology should be prevented in industrial fermentation processes. Genetic activation of these enzymes can enable growth on cognate substrates also in the yeast form. Here, strains were engineered for growth on polygalacturonic acid as major component of pectin. Besides activation of intrinsic enzymes, supplementation with heterologous genes for potent enzymes was tested. The presence of an unconventional secretion pathway allowed exploiting fungal and bacterial enzymes. Growth of the engineered strains was evaluated by a recently developed method for online determination of residual substrates based on the respiration activity. This enabled the quantification of the overall consumed substrate as a key asset for the assessment of the enzyme degradation potential even on polymeric substrates. Co-fermentation of endo- and exo-polygalacturonase overexpression strains resulted in efficient growth on polygalacturonic acid. In the future, the approach will be extended to establish efficient degradation and valorization of pectin.

A Potential Lock-Type Mechanism for Unconventional Secretion in Fungi.

Protein export in eukaryotes can either occur via the classical pathway traversing the endomembrane system or exploit alternative routes summarized as unconventional secretion. Besides multiple examples in higher eukaryotes, unconventional secretion has also been described for fungal proteins with diverse functions in important processes such as development or virulence. Accumulating molecular insights into the different export pathways suggest that unconventional secretion in fungal microorganisms does not follow a common scheme but has evolved multiple times independently. In this study, we review the most prominent examples with a focus on the chitinase Cts1 from the corn smut Ustilago maydis. Cts1 participates in cell separation during budding growth. Recent evidence indicates that the enzyme might be actively translocated into the fragmentation zone connecting dividing mother and daughter cells, where it supports cell division by the degradation of remnant chitin. Importantly, a functional fragmentation zone is prerequisite for Cts1 release. We summarize in detail what is currently known about this potential lock-type mechanism of Cts1 secretion and its connection to the complex regulation of fragmentation zone assembly and cell separation.

The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis.

Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear. Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.

Online evaluation of the metabolic activity of Ustilago maydis on (poly)galacturonic acid.

BACKGROUND: Pectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall. Thus, many different pectinases are required for an efficient microbial conversion of biomass waste streams with a high pectin content like citrus peel, apple pomace or sugar beet pulp. The screening and optimization of strains growing on pectic substrates requires both, quantification of the residual substrate and an accurate determination of the enzymatic activity. Galacturonic acid, the main sugar unit of pectin, is an uncommon substrate for microbial fermentations. Thus, growth and enzyme production of the applied strain has to be characterized in detail to understand the microbial system. An essential step to reach this goal is the development of online monitoring tools. RESULTS: In this study, a method for the online determination of residual substrate was developed for the growth of the plant pathogenic fungus Ustilago maydis on pectic substrates such as galacturonic acid. To this end, an U. maydis strain was used that expressed a heterologous exo-polygalacturonase for growth on polygalacturonic acid. The growth behavior on galacturonic acid was analyzed by online measurement of the respiration activity. A method for the online prediction of the residual galacturonic acid concentration during the cultivation, based on the overall oxygen consumption, was developed and verified by offline sampling. This sensitive method was extended towards polygalacturonic acid, which is challenging to quantify via offline measurements. Finally, the enzymatic activity in the culture supernatant was calculated and the enzyme stability during the course of the cultivation was confirmed. CONCLUSION: The introduced method can reliably predict the residual (poly)galacturonic acid concentration based on the overall oxygen consumption. Based on this method, the enzymatic activity of the culture broth of an U. maydis strain expressing a heterologous exo-polygalacturonase could be calculated. It was demonstrated that the method is especially advantageous for determination of low enzymatic activities. In future, it will be applied to U. maydis strains in which the number of produced hydrolytic enzymes is increased for more efficient degradation.

Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis.

The eukaryotic microorganism Ustilago maydis is currently being developed as an alternative protein expression platform. Protein fusion with an unconventionally secreted chitinase mediates export of heterologous proteins. The unique feature of this pathway is the circumvention of N-glycosylation. Different heterologous proteins could already be secreted via this novel mechanism in their active state. However, the system still suffers from low yields mainly attributed to the degradation of exported recombinant proteins by proteases. Here, we combined optimization steps on the level of cultivation conditions and strain engineering to further improve the system. Using the Respiration Activity Monitoring System we discovered that a pH drop during prolonged incubation results in loss of activity and degradation of the target protein. This problem can be reduced by buffering the cultivation medium. However, we still observed significant proteolysis even in buffered cultures. Hence, we revisited strain engineering to reduce the proteolytic activity. Secreted proteases were discovered using mass spectrometry. Then, genes for three identified proteases of a serine-carboxypeptidase family were deleted in an existing quintuple protease deletion mutant. This further diminished proteolytic activity and target protein degradation. The two approaches overall strongly improved the stability of heterologous proteins in this fungal system.

Dual function of a secreted fungalysin metalloprotease in Ustilago maydis.

Fungalysins from several phytopathogenic fungi have been shown to be involved in cleavage of plant chitinases. While fungal chitinases are responsible for cell wall remodeling during growth and morphogenesis, plant chitinases are important components of immunity. This study describes a dual function of the Ustilago maydis fungalysin UmFly1 in modulation of both plant and fungal chitinases. Genetic, biochemical and microscopic experiments were performed to elucidate the in vitro and in planta functions of U. maydis UmFly1. U. maydis ∆umfly1 mutants show significantly reduced virulence, which coincides with reduced cleavage of the maize chitinase ZmChiA within its chitin-binding domain. Moreover, deletion of umfly1 affected the cell separation of haploid U. maydis sporidia. This phenotype is associated with posttranslational activation of the endogenous chitinase UmCts1. Genetic complementation of the ∆umfly1 mutant with a homologous gene from closely related, but nonpathogenic, yeast fully rescued the cell separation defect in vitro, but it could not recover the ∆umfly1 defect in virulence and cleavage of the maize chitinase. We report on the dual function of the secreted fungalysin UmFly1. We hypothesize that co-evolution of U. maydis with its host plant extended the endogenous function of UmFly1 towards the modulation of plant chitinase activity to promote infection.

Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies.

Exploiting secretory pathways for production of heterologous proteins is highly advantageous with respect to efficient downstream processing. In eukaryotic systems the vast majority of heterologous proteins for biotechnological application is exported via the canonical endoplasmic reticulum-Golgi pathway. In the endomembrane system target proteins are often glycosylated and may thus be modified with foreign glycan patterns. This can be destructive for their activity or cause immune reactions against therapeutic proteins. Hence, using unconventional secretion for protein expression is an attractive alternative. In the fungal model Ustilago maydis, chitinase Cts1 is secreted via an unconventional pathway connected to cell separation which can be used to co-export heterologous proteins. Here, we apply this mechanism for the production of nanobodies. First, we achieved expression and unconventional secretion of a functional nanobody directed against green fluorescent protein (Gfp). Second, we found that Cts1 binds to chitin and that this feature can be applied to generate a Gfp-trap. Thus, we demonstrated the dual use of Cts1 serving both as export vehicle and as purification tag. Finally, we established and optimized the production of a nanobody against botulinum toxin A and hence describe the first pharmaceutically relevant target exported by Cts1-mediated unconventional secretion.

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology.

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on "Unconventional Protein and Membrane Traffic" (UPMT) during 4-7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.