Komagataella Pastoris Research Articles

The antidepressant effect of Komagataella pastoris KM 71 H in maternal separation mice model mediated by the microbiota-gut-brain axis

BackgroundThe intestinal microbiota plays a fundamental role in maintaining host health, especially during childhood, a critical period for its establishment. Early life stress can lead to shifts in gut microbiota composition, thus increasing the risk of major depressive disorder (MDD) in adulthood. The supplementation with probiotics restores intestinal permeability and the health of gut microbial communities, therefore being potential study targets for the treatment of MDD. In this sense, the yeast Komagataella pastoris was reported as a promising probiotic with antidepressant effect. MethodsHence, the present study aims to investigate this effect in mice submitted to maternal separation (MS) 3 h per day from PND2 to PND14. Adult mice and mothers were treated with K. pastoris KM71H (8 log UFC.g−1/per animal, i.g.) or PBS (500 µl, i.g.) for 14 days. After behavioral tests, the animals were euthanized, followed by hippocampi and intestines removal for biochemical analysis. ResultsOn behavioral tests, K. pastoris KM71H treatment reduced the immobility time in TST of adult mice and increased the grooming activity in splash test of adult mice and mothers induced by MS. The probiotic treatment restored plasma corticosterone levels and glucocorticoid receptor expression in hippocampi, alongside nitrate/nitrite levels and superoxide dismutase activity in intestine, in addition to reducing reactive species levels in both structures. Moreover, it also normalized the fecal pH and water content of feces. ConclusionThus, we conclude that K. pastoris KM71H is a promising therapeutic strategy for the treatment of MDD.

Optimizing Pichia pastoris protein secretion: Role of N-linked glycosylation on the α-mating factor secretion signal leader

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions.Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.

Komagataella pastoris KM71H Mitigates Depressive-Like Phenotype, Preserving Intestinal Barrier Integrity and Modulating the Gut Microbiota in Mice

The role of intestinal microbiota in the genesis of mental health has received considerable attention in recent years, given that probiotics are considered promising therapeutic agents against major depressive disorder. Komagataella pastoris KM71H is a yeast with probiotic properties and antidepressant-like effects in animal models of depression. Hence, we evaluated the antidepressant-like effects of K. pastoris KM71H in a model of antibiotic-induced intestinal dysbiosis in male Swiss mice. The mice received clindamycin (200 μg, intraperitoneal) and, after 24 h, were treated with K. pastoris KM71H at a dose of 8 log CFU/animal by intragastric administration (ig) or PBS (vehicle, ig) for 14 consecutive days. Afterward, the animals were subjected to behavioral tests and biochemical analyses. Our results showed that K. pastoris KM71H administration decreased the immobility time in the tail suspension test and increased grooming activity duration in the splash test in antibiotic-treated mice, thereby characterizing its antidepressant-like effect. We observed that these effects of K. pastoris KM71H were accompanied by the modulation of the intestinal microbiota, preservation of intestinal barrier integrity, and restoration of the mRNA levels of occludin, zonula occludens-1, zonula occludens-2, and toll-like receptor-4 in the small intestine, and interleukin-1β in the hippocampi of mice. Our findings provide solid evidence to support the development of K. pastoris KM71H as a new probiotic with antidepressant-like effects.

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration.

This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-β-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson’s disease. Currently, Parkinson’s disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography—IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and −80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

Heterologous Expression, Characterization, and Comparison of Laccases from the White Rot Causing Basidiomycete Cerrena Unicolor

Lignin is the most abundant renewable source of aromatics on earth, and conversion of it to chemicals and fuels is needed to build an economically viable renewable biofuels industry. Biological routes to converting lignin to fuels and chemicals involve depolymerizing lignin using lignin-degrading enzymes that catalyze the breaking of ether and carbon-carbon bonds in the phenolic and non-phenolic subunits of lignin. Laccases are a crucial class of lignin-degrading enzymes and are copper-containing enzymes capable of oxidizing electron-rich organic substrates such as lignin using molecular oxygen as an electron acceptor. The genome of <em>Cerrena unicolor</em> was recently added to the JGI MycoCosm database and has eight laccases. Two of these laccases, designated Lc1 and Lc2, predicted to have the highest likelihood for successful expression in soluble, active form were selected for characterization. Lc1 and Lc2, which share 65% sequence identity, were heterologously expressed in <em>Komagataella pastoris</em> (formerly <em>Pichia pastoris</em>), allowing characterization and comparison of their purified forms. Lc1 and Lc2 had half-lives of 16 min and 185 min at 60°C, respectively, and, based on molecular dynamics simulations, the longer half-life of Lc2 was due to an increased number and persistence of salt bridges compared to Lc1. Using model lignin-like dimers and a nanostructure-initiator mass spectrometry assay to quantify catalysis of specific bond-breaking events, both Lc1 and Lc2 had their highest activity at pH 3 and in combination with syringaldehyde as a mediator, with Lc1 having a higher catalytic efficiency of β-O-4' ether and C-C bond breaking. This comparative study demonstrates the diversity, including thermostability differences, of laccases from the same fungus, and improves our understanding of laccase catalyzed breaking of bonds commonly found in lignin, which will facilitate the developing this important class of enzymes for applications in the conversion of lignin to valuable bioproducts.

Spt5‐nucleic acid interactions are directly involved in promoter proximal pausing of RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We use Drosophila melanogasteras a model system to address this question. We studied DSIF‐nucleic acid contacts made by the Spt5 NGN and KOW1 domains, which form a part of the upstream DNA exit tunnel, and the Spt5 KOW4 domain, which, along with the KOW5 domain, forms a clamp around the nascent RNA. Biochemical and structural studies have implicated these domains in pausing. We hypothesize that electrostatic interactions between Spt5 and the nucleic acid scaffold contribute to pausing by inhibiting the translocation of Pol II. We disrupted these electrostatic interactions by reversing the charge of key basic residues in Spt5 and used an electrophoretic mobility shift assay to measure Pol II binding and NELF recruitment. We then tested the pausing activity of the DSIF mutants in Drosophilanuclear extract depleted of wild‐type DSIF. Reversing the charge of six basic residues in the KOW1 domain resulted in weaker binding to Pol II. Though this mutant was still able to recruit NELF, it showed greatly reduced pausing activity. Reversing the charge of four basic residues in the KOW4 domain only modestly reduced binding to Pol II, had no impact on NELF recruitment, and significantly impaired DSIF’s pausing function. Thus, nucleic acid contacts made by the KOW1 domain of Spt5 promote pausing by mediating Pol II‐DSIF interactions (and therefore NELF recruitment) while the KOW4 domain interactions with the nascent transcript contribute to pausing directly. We also reversed the charge of two basic residues in the NGN domain and found that these changes had no effect on DSIF binding and NELF recruitment but did result in a modest decrease in pausing activity. One of these basic residues is an arginine located in an alpha helix sequence that is conserved in eukaryotes with NELF and promoter proximal pausing but is absent in eukaryotes lacking NELF. To test whether this alpha helix is required for promoter proximal pausing, we replaced the Drosophila helix sequence with unstructured loop sequences from Komagataella pastoris, Saccharomyces cerevisiae, and Caenorhabditis elegans, none of which have NELF. These mutants were able to bind Pol II and recruit NELF but exhibited significantly reduced pausing activity, suggesting that a short alpha helix in the NGN domain is critical for stabilizing the promoter proximal pause. This is likely achieved by optimally positioning a conserved arginine to interact with the DNA scaffold.

Overcoming Obstacles in Protein Expression in the Yeast Pichia pastoris: Interviews of Leaders in the Pichia Field.

The yeast Pichia pastoris (also known as Komagataella pastoris) has been used for over 30 years to produce thousands of valuable, heterologous proteins, such as insulin to treat diabetes and antibodies to prevent migraine headaches. Despite its success, there are some common, stubborn problems encountered by research scientists when they try to use the yeast to produce their recombinant proteins. In order to provide those working in this field with strategies to overcome these common obstacles, nine experts in P. pastoris protein expression field were interviewed to create a written review and video (https://www.youtube.com/watch?v=Q1oD6k8CdG8). This review describes how each respected scientist addressed a specific challenge, such as identifying high expression strains, improving secretion efficiency and decreasing hyperglycosylation. Their perspective and practical advice can be a tool to help empower others to express challenging proteins in this popular recombinant host.

Komagataella pastoris KM71H modulates neuroimmune and oxidative stress parameters in animal models of depression: A proposal for a new probiotic with antidepressant-like effect

Many studies have suggested that imbalance of the gut microbial composition leads to an increase in pro-inflammatory cytokines and promotes oxidative stress, and this are directly associated with neuropsychiatric disorders, including major depressive disorder (MDD). Clinical data indicated that the probiotics have positive impacts on the central nervous system and thus may have a key role to treatment of MDD. This study examined the benefits of administration of Komagataella pastoris KM71H (8 log UFC·g–1/animal, intragastric route) in attenuating behavioral, neurochemical, and neuroendocrine changes in animal models of depressive-like behavior induced by repeated restraint stress and lipopolysaccharide (0.83 mg/kg). We demonstrated that pretreatment of mice with this yeast prevented depression-like behavior induced by stress and an inflammatory challenge in mice. We believe that this effect is due to modulation of the permeability of the blood-brain barrier, restoration in the mRNA levels of the Nuclear factor kappa B, Interleukin 1β, Interferon γ, and Indoleamine 2 3-dioxygenase, and prevention of oxidative stress in the prefrontal cortices, hippocampi, and intestine of mice and of the decrease the plasma corticosterone levels. Thus, we conclude that K. pastoris KM71H has properties for a new proposal of probiotic with antidepressant-like effect, arising as a promising therapeutic strategy for MDD.

Biovalorization of Lignocellulosic Materials for Xylitol Production by the Yeast Komagataella pastoris

The main goal of this study was to screen different lignocellulosic materials for their ability to support the cell growth of the yeast Komagataella pastoris and the production of xylitol. Several lignocellulosic materials, namely banana peels, brewer’s spent grains (BSGs), corncobs, grape pomace, grape stalks, and sawdust, were subjected to dilute acid hydrolysis to obtain sugar rich solutions that were tested as feedstocks for the cultivation of K. pastoris. Although the culture was able to grow in all the tested hydrolysates, a higher biomass concentration was obtained for banana peels (15.18 ± 0.33 g/L) and grape stalks (14.58 ± 0.19 g/L), while the highest xylitol production (1.51 ± 0.07 g/L) was reached for the BSG hydrolysate with a xylitol yield of 0.66 ± 0.39 g/g. Cell growth and xylitol production from BSG were improved by detoxifying the hydrolysate using activated charcoal, resulting in a fourfold increase of the biomass production, while xylitol production was improved to 3.97 ± 0.10 g/L. Moreover, concomitant with arabinose consumption, arabitol synthesis was noticed, reaching a maximum concentration of 0.82 ± 0.05 g/L with a yield on arabinose of 0.60 ± 0.11 g/g. These results demonstrate the feasibility of using lignocellulosic waste, especially BSG, as feedstock for the cultivation of K. pastoris and the coproduction of xylitol and arabitol. Additionally, it demonstrates the use of K. pastoris as a suitable microorganism to integrate a zero-waste biorefinery, transforming lignocellulosic waste into two high-value specialty chemicals with high market demand.

Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter.

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.

Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate

The present work shows the application of nickel- and magnesium-crosslinked gellan microspheres in ionic and affinity capture strategies to directly extract hSCOMT from the complex Komagataella pastoris lysate through a simple batch method. Both formulations present similar morphology, but nickel–crosslinked microspheres present higher crosslinker content and smaller diameters. Four different capture strategies were established, by manipulating the ionic strength, pH, temperature and competing agents' presence. The most promising results for hSCOMT capture and clarification were obtained employing an ionic strategy with nickel-crosslinked microspheres and an affinity strategy with magnesium-crosslinked microspheres at 4 °C. The bioactivity results (200%) and purification degree (70%) of hSCOMT captured by the ionic strategy were more satisfactory probably due to the soft ionic conditions used (100 mM NaCl). For the first time, the gellan polysaccharide versatility was demonstrated in the microsphere application for the direct capture of hSCOMT from a complex lysate, simplifying isolation biotechnological procedures.

Profiling of Fungal Diversity and Fermentative Yeasts in Traditional Chinese Xiaoqu.

To increase the safety and quality of baijiu and rice wine in China, controlling the use of traditional Xiaoqu by studying the beneficial yeasts present has recently been considered. The fungal diversity of six Chinese Xiaoqu including five traditional and one commercial samples was investigated to screen fermentative yeasts with low yields of higher alcohols. A high throughput sequencing approach detected fifteen fungal species with relative abundance higher than 1%, and displayed dissimilarities of fungal diversity among Xiaoqu samples. The 15 fungal species were composed of 11 filamentous fungi with Rhizopus arrhizus as the most common specie and four yeast species, containing Hyphopichia burtonii, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, and Saccharomycopsis malanga. Classic culture-dependent approaches, including 5.8S-ITS-RFLP analysis and D1/D2 sequencing of the 26S rRNA gene, identified nine yeast species in the five traditional Chinese Xiaoqu. In addition to the four yeast species also detected by high throughput sequencing approach, the other five yeast species isolated were Clavispora lusitaniae, Cryptococcus neoformans, Komagataella pastoris, Trichosporon asahii, and Wickerhamomyces anomalus. Further micro-fermentations of rice wine were performed using 19 single yeast isolates, and after the fermentation of rice wine, higher alcohols and ethanol were analyzed by gas chromatography. Two yeast strains, Saccharomyces cerevisiae FBKL2.8022 and Wickerhamomyces anomalus FBKL2.8023, were found to have low yields of higher alcohols and could produce 11.70%vol and 7.10%vol ethanol separately. This study for the first time, to the best of our knowledge, explored the fungal resources in traditional Xiaoqu from different regions of Guizhou, China. The screened S. cerevisiae and W. anomalus strains could be used to establish specific starters to promote the standardization of the production of baijiu and rice wine.

Update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA 12: suitability of taxonomic units notified to EFSA until March 2020.

The qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. It is based on an assessment of the taxonomic identity, the body of knowledge, safety concerns and antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are where possible to be confirmed at strain or product level, reflected by ‘qualifications’. No new information was found that would change the previously recommended QPS TUs of the 39 microorganisms notified to EFSA between October 2019 and March 2020, 33 were excluded, including five filamentous fungi, five Escherichia coli, two Enterococcus faecium, two Streptomyces spp. and 19 TUs already evaluated. Six TUs were evaluated. Akkermansia muciniphila was not recommended for QPS status due to safety concerns. Clostridium butyricum was not recommended because some strains contain pathogenicity factors. This TU was excluded for further QPS evaluation. Galdieria sulphuraria and Pseudomonas chlororaphis were also rejected due to a lack of body of knowledge. The QPS status of Corynebacterium ammoniagenes (with the qualification ‘for production purposes only’) and of Komagataella pastoris (with the qualification ‘for enzyme production’) was confirmed. In relation to the taxonomic revision of the Lactobacillus genus, previously designated Lactobacillus species will be reassigned to the new species and both the old and new names will be retained in the QPS list.

Chitin-glucan complex – Based biopolymeric structures using biocompatible ionic liquids

This work explores the novelty of dissolving chitin-glucan complex (CGC), from two fungal strains, Komagataella pastoris (CGCP) and Aspergillus niger (CGCKZ) (KiOnutrime-CG™), using biocompatible ionic liquids (ILs). Three cholinium-based ILs were tested, choline acetate, choline propionate and choline hexanoate. Although all tested ILs resulted in the dissolution of the co-polymer at a concentration of 5 % (w/w), distinct polymeric structures, films or gels, were obtained from CGCP and CGCKZ, respectively. CGCP films were dense, flexible and elastic, with high swelling capacity (> 200 %). The IL anion alkyl chain length influenced the polymeric structures’ properties, namely, the CGCP films elongation at break and swelling degree. CGCKZ resulted in weak gels. For both polymeric structures, exposure to the ILs under the dissolution conditions caused significant changes in the co-polymers’ chemical structure, namely, reduction of their glucan moiety and reduction of the degree of acetylation, thus yielding chitosan-glucan complexes (ChGC) enriched in glucosamine (53.4 ± 0.3–60.8 ± 0.3 %).

Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control

Rhipicephalus microplusis the most important ectoparasite of livestock in tropical and subtropical areas around the world. Research focused on developing an efficient vaccine for cattle tick control is a high priority. The aim of this study was to evaluate the rSBm7462® peptide (Bm86-B and T cell epitopes) regarding its properties of immunogenicity, protective effect in cattle and efficacy against R. microplus. This peptide was produced by a fermentative process in the yeast culture system of Komagataella (Pichia) pastoris strain Km 71. The vaccination assay was conducted in a tick-free area using non-splenectomised Holstein Friesian calves, separated into immunisation and control groups. These animals individually received the recombinant peptide and the inoculum without peptide using saponin as an adjuvant at three time points. The calves were challenged 21 days after the last immunisation using 4500 larvae per animal. An indirect ELISA was used to identify the IgG kinetics of serum samples from the calves studied. The qPCR was performed to determine the cytokine gene expression from the total RNA of the cultured PBMCs. Histomorphometry of the germinal centres (GCs) was performed measuring slides with haematoxylin-eosin staining of surgically removed lymph nodes from immunised calves. The antibody response showed a significant induction of high-affinity IgGs in calves immunised with the recombinant peptide in comparison to the controls. The kinetics of antibodies in immunised calves showed a significant increase during the experiment. This increase in high-affinity IgGs correlated with a gradual increase of the GC diameter following each peptide vaccination. Cytokine expression profiles demonstrating an adaptive immune response in calves immunised with rSBm7462® confirmed the T-dependent response. Vaccine efficacy was calculated at 72.4 % following the analysis and fecundity of collected adult female ticks, compared between control and vaccinated groups. These findings demonstrate that this new recombinant peptide is an option for control of R. microplus infestations.

Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation.

Invertases catalyze the hydrolysis of sucrose into fructose and glucose and can be employed as an alternative in producing high fructose syrup. In this study, we reported the heterologous expression of an invertase gene (GspInv) of Gongronella sp. w5 in Komagataella pastoris. GspInv activity reached 147.6 ± 0.4 U/mL after 5 days of methanol induction. GspInv is invertase with a high specific activity of 2,776.1 ± 124.2 U/mg toward sucrose. GspInv showed high tolerance to sucrose (IC50 = 1.2 M), glucose (IC50 > 2 M), fructose (IC50 = 1.5 M), and a variety of metal ions that make it an ideal candidate for high fructose syrup production. A carbohydrate-binding module was sequence-optimized and fused to the N-terminus of GspInv. The fusion protein had the highest immobilization efficiency at room temperature within 1 h adsorption, with 1 g of cellulose absorption up to 8,000 U protein. The cellulose-immobilized fusion protein retained the unique properties of GspInv. When applied in high fructose syrup preparation by using 1 M sucrose as the substrate, the sucrose conversion efficiency of the fused protein remained at approximately 95% after 50 h of continuous hydrolysis on a packed bed reactor. The fused protein can also hydrolyze completely the sucrose in sugarcane molasses. Our results suggest that GspInv is an unusual invertase and a promising candidate for high fructose syrup preparation.

Low Temperature Dissolution of Yeast Chitin-Glucan Complex and Characterization of the Regenerated Polymer.

Chitin-glucan complex (CGC) is a copolymer composed of chitin and glucan moieties extracted from the cell-walls of several yeasts and fungi. Despite its proven valuable properties, that include antibacterial, antioxidant and anticancer activity, the utilization of CGC in many applications is hindered by its insolubility in water and most solvents. In this study, NaOH/urea solvent systems were used for the first time for solubilization of CGC extracted from the yeast Komagataella pastoris. Different NaOH/urea ratios (6:8, 8:4 and 11:4 (w/w), respectively) were used to obtain aqueous solutions using a freeze/thaw procedure. There was an overall solubilization of 63–68%, with the highest solubilization rate obtained for the highest tested urea concentration (8 wt%). The regenerated polymer, obtained by dialysis of the alkali solutions followed by lyophilization, formed porous macrostructures characterized by a chemical composition similar to that of the starting co-polymer, although the acetylation degree decreased from 61.3% to 33.9–50.6%, indicating that chitin was converted into chitosan, yielding chitosan-glucan complex (ChGC). Consistent with this, there was a reduction of the crystallinity index and thermal degradation temperature. Given these results, this study reports a simple and green procedure to solubilize CGC and obtain aqueous ChGC solutions that can be processed as novel biomaterials.