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INTRODUCTION: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. METHODS: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. RESULTS: One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. DISCUSSION: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system.

Coronavirus disease 2019 (COVID-19) has caused the ongoing COVID-19 pandemic and there is a growing demand for safe and effective vaccines. The thermophilic Thermothelomyces heterothallica filamentous fungal host, C1-cell, can be utilized as an expression platform for the rapid production of large quantities of antigens for developing vaccines. The aim of this study was to evaluate the local tolerance and the systemic toxicity of a C1-cell expressed receptor-binding domain (C1-RBD) vaccine, following repeated weekly intramuscular injections (total of 4 administrations), in New Zealand White rabbits. The animals were sacrificed either 3 days or 3 weeks following the last dose. No signs of toxicity were observed, including no injection site reactions. ELISA studies revealed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G antibodies in the sera of C1-RBD-treated animals starting from day 13 post injection, that were further elevated. Histopathology evaluation and immunohistochemical staining revealed follicular hyperplasia, consisting of B-cell type, in the spleen and inguinal lymph nodes of the treated animals that were sustained throughout the recovery phase. No local or systemic toxicity was observed. In conclusion, the SARS-CoV-2 C1-RBD vaccine candidate demonstrated an excellent safety profile and a lasting immunogenic response against receptor-binding domain, thus supporting its further development for use in humans.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI–MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2–8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80–90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319–541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs. Graphical abstract: [Figure not available: see fulltext.]

Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

We investigated how a genetically engineered resilin fusion protein modifies cellulose surfaces. We characterized the pH-responsive behavior of a resilin-like polypeptide (RLP) having terminal cellulose binding modules (CBM) and showed its binding to cellulose nanofibrils (CNF). Characterization of the resilin fusion protein at different pHs revealed substantial conformational changes of the protein, which were observed as swelling and contraction of the protein layer bound to the nanocellulose surface. In addition, we showed that employment of the modified resilin in cellulose hydrogel and nanopaper increased their modulus of stiffness through a cross-linking effect.

Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.

Hypocrea jecorina is an industrially important filamentous fungus due to its effective production of hydrolytic enzymes. It has received increasing interest because of its ability to convert lignocellulosic biomass to monomeric sugars, which can be converted into biofuels or platform chemicals. Genetic engineering of strains is a highly important means of meeting the requirements of tailor-made applications. Therefore, we report the development of a transformation system that allows highly efficient gene targeting by using a tmus53 (human LIG4 homolog) deletion strain. Moreover, it permits the unlimited reuse of the same marker by employing a Cre/loxP-based excision system. Both marker insertion and marker excision can be positively selected for by combining resistance to hygromycin B and loss of sensitivity to fluoroacetamide. Finally, the marker pyr4 , also positively selectable for insertion and loss, can be used to remove the cre gene.

Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions.We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9. Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100. b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties.

Summary The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so-called secretion stress. A two-component regulatory system, named CssR-CssS was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha -amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface. This view is supported by the observation that transcription of the htrA gene encoding a predicted membrane-bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.

Aims: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. Methods and Results: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of α-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1·5-fold). Increasing the net negative charge of the cell wall because of lack of the d-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1·5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. Conclusions: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. Significance and Impact of the Study: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.

Background: The soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei. Results: We have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes. Conclusions: Transcriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

The present invention relates to a method for genetically modifying a filamentous fungus host for improved protein production. The method comprises that a filamentous fungus host is genetically modified to overexpress or to be deficient of specific genes. The invention relates also to the modified hosts. Furthermore, the invention relates to a method for improved production or for producing an improved composition of proteins, such as cellulases, hemicellulases, other proteins involved in the degradation of lignocellulosic material, or other proteins, in a filamentous fungus host.Patent family as of 23.9.2021DE602011050554 D1 20180830 DE201160050554T 20110530 DK2576792 T3 20181126 DK20110726858T 20110530 EP2576792 A2 20130410 EP20110726858 20110530 EP2576792 B1 20180801 EP20110726858 20110530 FI20105632 A0 20100604 FI20100005632 20100604 US2013078674 AA 20130328 US20110701894 20110530 US9512415 BB 20161206 US20110701894 20110530 WO11151512 A2 20111208 WO2011FI50492 20110530 WO11151512 A3 20120308 WO2011FI50492 20110530Link to current patent family on right

Background Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general. Results We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production. Conclusions aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

Summary The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ α-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in thed-alanylation of teichoic acids. The inactivation ofdlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA + strain. Furthermore, the dltmutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins.