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The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression. • We report the first use of dMAD7 in K. phaffii to regulate the expression of enhanced green fluorescent protein (eGFP). • A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. • Whole genome sequencing and RNA-sequencing were used to assess the integration events and to characterize the strains. • Our findings conclusively demonstrate the utility of the dMAD7 system in K. phaffii. [ABSTRACT FROM AUTHOR]

Glycogen, a branched polysaccharide organized into glycogen granules (GGs), is delivered from the cytoplasm to the lysosomes of hepatocytes by STBD1-driven selective autophagy (glycophagy). Recently, we developed Komagataella phaffii yeast as a simple model of GG autophagy and found that it proceeds non-selectively under nitrogen starvation conditions. However, another group, using Saccharomyces cerevisiae as a model, found that glycogen is a non-preferred cargo of nitrogen starvation-induced bulk autophagy. To clarify cargo characteristics of K. phaffii GGs, we used the same glycogen synthase-based reporter (Gsy1-GFP) of GG autophagy in K. phaffii as was used in S. cerevisiae. The K. phaffii Gsy1-GFP marked the GGs and reported on their autophagic degradation during nitrogen starvation, as expected. However, unlike in S. cerevisiae, glycogen synthase-marked GGs were delivered to the vacuole and degraded there with the same efficiency as a cytosolic glycogen synthase in glycogen-deficient cells, suggesting that glycogen is a neutral cargo of bulk autophagy in K. phaffii. We verified our findings with a new set of reporters based on the glycogen-binding CBM20 domain of human STBD1. The GFP-CBM20 and mCherry-CBM20 fusion proteins tagged GGs, reported about the autophagy of GGs, and confirmed that GGs in K. phaffii are neither preferred nor non-preferred substrates of bulk autophagy. They are its neutral substrates. [ABSTRACT FROM AUTHOR]

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application. [ABSTRACT FROM AUTHOR]

Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties. Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter. Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca2+, Mg2+, SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n-hexane and n-heptane. Enzymatic analysis revealed a Km of 2.89 ± 0.09 μM and a Vmax of 14.20 ± 0.12 nM•min−1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP. Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research. [ABSTRACT FROM AUTHOR]

Objective: Der f 2, a major allergen derived from Dermatophagoides farinae, is a leading cause of allergic asthma. IL-6 and GM–CSF play essential roles in the exacerbation of asthma. However, the mechanical act by which Der f 2 mediates the expression of IL-6, IL-8, and GM–CSF in airway epithelial cells remains incompletely elucidated. Herein, we aimed to explore the effect of Der f 2 on IL-6 and GM–CSF expression in the human airway epithelial cell BEAS-2B and A549. Methods: Recombinant Der f 2 (rDf2) was acquired using Pichia pastoris. BEAS-2B and A549 cells were used as cell model. The expression of genes and proteins and the involvement of the signaling cascade were assessed using RT–PCR, quantitative real-time PCR (qPCR), Western blotting, and ELISA, respectively. Results: Our findings showed that rDf2 significantly induced mRNA expression and protein production of IL-6 and GM-CSF in BEAS-2B and A549 cells. In contrast, rDf2 did not influence IL-8 expression or production in both cells. Mechanistic studies revealed that rDf2 triggered activation of the p38 MAPK and JNK. Inhibition of p38, but not JNK, significantly attenuated rDf2-induced IL-6 and GM–CSF expression and production. Conclusion: This study demonstrates that Der f 2 promotes the expression and production of the pro-inflammatory cytokines IL-6 and GM–CSF in airway epithelial cells via activation of the p38 signaling pathway. These findings provide insights into the molecular mechanisms that Der f 2 may exacerbate airway inflammation. [ABSTRACT FROM AUTHOR]

The statin is the primary cholesterol-lowering drug. Monacolin J (MJ) is a key intermediate in the biosynthetic pathway of statin. It was obtained in industry by the alkaline hydrolysis of lovastatin. The hydrolysis process resulted in multiple by-products and expensive cost of wastewater treatment. In this work, we used Pichia pastoris as the host to produce the MJ. The biosynthesis pathway of MJ was built in P. pastoris. The stable recombinant strain MJ2 was obtained by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome-editing tool, and produced the MJ titer of 153.6 ± 2.4 mg/L. The metabolic engineering was utilized to enhance the production of MJ, and the fermentation condition was optimized. The MJ titer of 357.5 ± 5.0 mg/L was obtained from the recombinant strain MJ5-AZ with ATP-dependent citrate lyase (ACL), glucose-6-phosphate dehydrogenase (ZWF1) and four lovB genes, 132.7% higher than that from the original strain MJ2. The recombinant strain MJ5-AZ was cultured in a 7-L fermenter, and the MJ titer of 1493.0 ± 9.2 mg/L was achieved. The results suggested that increasing the gene dosage of rate-limiting step in the biosynthesis pathway of chemicals could improve the titer of production. It might be applicable to the production optimization of other polyketide metabolites. [ABSTRACT FROM AUTHOR]

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1–97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H6-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H6-trC1INH(MGS)-MSA), and H6-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H6-trC1INH(MGS)-MSA was significantly increased versus that of H6-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H6-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H6-trC1INH(MGS)-MSA by 33% relative to that of H6-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H6-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE. [ABSTRACT FROM AUTHOR]

The demand for food-grade β-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for β-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of β-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (w/v) methanol, 0.56% (w/v) sorbitol, and 0.48% (w/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications. [ABSTRACT FROM AUTHOR]

Background: Pichia pastoris-secreted delta sleep inducing peptide and crossing the blood-brain barrier peptides (DSIP-CBBBP) fusion peptides holds significant promise for its potential sleep-enhancing and neurotransmitter balancing effects. This study investigates these properties using a p-chlorophenylalanine (PCPA) -induced insomnia model in mice, an approach akin to traditional methods evaluating sleep-promoting activities in fusion peptides. Aim of the study: The research aims to elucidate the sleep-promoting mechanism of DSIP-CBBBP, exploring its impact on neurotransmitter levels and sleep regulation, and to analyze its composition and structure. Materials and methods: Using a PCPA-induced insomnia mouse model, the study evaluates the sleep-promoting effects of DSIP-CBBBP. The peptide’s influence on neurotransmitters such as 5-HT, glutamate, dopamine, and melatonin is assessed. The functions of DSIP-CBBBP are characterized using biochemical and animal insomnia-induced behavior tests and compared without CBBBP. Results: DSIP-CBBBP demonstrates a capacity to modulate neurotransmitter levels, indicated by changes in 5-HT, glutamate, DA, and melatonin. DSIP-CBBBP shows a better restorative effect than DSIP on neurotransmitter imbalance and the potential to enhance sleep. Conclusion: The study underscores DSIP-CBBBP potential in correcting neurotransmitter dysregulation and promoting sleep, hinting at its utility in sleep-related therapies. [ABSTRACT FROM AUTHOR]

The control of fungal and bacterial plant diseases mainly relies on the application of mineral and chemical fungicides/bactericides, which are considered to be environmental pollutants and toxic to humans. Moreover, the prolonged use of bactericides and antibiotics has led to the development of resistance among pathogens, as well as an increase in environmental and health threats. Therefore, the development of non-toxic and non-polluting treatments to control plant diseases has been the focus of extensive research in agriculture. Synthetic antimicrobial peptides have recently received extensive attention as the potential alternatives to other conventional methods in terms of their strong broad-spectrum antimicrobial activity. In vitro assays using various chemically synthesized peptides showed that the broad-spectrum peptide thanatin derived from the spined soldier bug (Podisus maculiventris (Say)) had the greatest potential for eliminating aflatoxigenic fungi. However, the antibacterial effect of thanatin against bacterial plant pathogens was less studied so far. The thanatin encoding sequence was codon optimized for expression in Pichia pastoris. This coding sequence cloned into P. pastoris expression vector pPICZαA and used for synthetizing the recombinant thanatin. Then, antibacterial activities of the constructed peptide were studied under in vitro and in vivo condition. The result showed that, all construction, cloning and expression processes were successfully performed in yeast. The results of the MIC and MBC tests showed that the growth rate of the majority of bacterial plant pathogens including gram-negative and gram-positive bacteria was inhibited by recombinant thanatin under in vitro conditions. MIC values for different bacterial isolates ranged from 0.016 to 2.048, while the MBC values for the same bacterial isolates were determined to be between 0.064 and 4.096 μg/mL. These amounts were significantly lower than the MIC and MBC values obtained by applying copper oxychloride. The application of 4.09 μg/mL of recombinant thanatin against rice leaf blight splendidly controlled this bacterial disease under greenhouse conditions. However, this promising antimicrobial peptide requires further investigation for the development of novel molecules for the control of plant pathogens. [ABSTRACT FROM AUTHOR]

In Komagataella phaffii (Pichia pastoris), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self‐induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF‐C1 metabolism, and it cannot be converted into carbon dioxide in a FdhKO strain. Under non‐repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF‐C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for a FdhKO strain on sorbitol and a non‐disrupted strain on sorbitol‐methanol. Considering a K. Phaffii FdhKO strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol‐free processes in K. phaffii. [ABSTRACT FROM AUTHOR]

Laccase is a copper‐containing polyphenol oxidase that can oxidize phenolic and non‐phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L−1protein. The highest production level of the recombinant laccase was 2760 U L−1. The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'‐azino‐bis‐(3‐ethylbenzothiazoline)−6‐sulphonate (ABTS) as the substrate, the Km and Vmax values were 0.136 mM and 9778 μM min−1 mg−1, respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco‐crystal violet (LCV) and leuco‐malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution. Summary: This study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea using synthetic biology methods, which could be successfully expressed in Pichia pastoris.The recombinant laccase possessed better substrate affinity, reaction rate and capable of efficient degradation of triphenylmethane dyes.In the current context of increasingly severe organic pollution, the degradation model of dyes constructed in this study can be widely applied to environmental remediation dealing with various pollutantsCompared with traditional methods, the advantages including higher efficiency, lower cost, easier operation, and without secondary pollution, providing a new solution for environmental pollution. [ABSTRACT FROM AUTHOR]

Background: Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. Results: High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. Conclusions: These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii. [ABSTRACT FROM AUTHOR]

Fusing insect derived neurotoxic peptides with Galanthus nivalis agglutinin (GNA) has been shown to enhance the insecticidal activity of the neuropeptides, especially when administered orally. This study produced a recombinant scorpion insect specific neurotoxin BjαIT, GNA, and a fusion protein BjαIT/GNA using Pichia pastoris as an expression host. Recombinant rBjαIT/GNA was found to be easily degraded during expression in yeast which and produced a main protein product with a molecular weight of approximately 14 kDa. Cytotoxicity results showed that rBjαIT, rGNA, and rBjαIT/GNA had no toxicity to mammalian NIH/3T3 cells. Adding rBjαIT or rBjαIT/GNA at a concentration as low as 1 ng/mL to insect cell culture medium inhibited the proliferation of insect Sf9 cells, with rBjαIT exhibiting stronger cytotoxicity, while 20 ng/mL rGNA did not inhibit the proliferation of Sf9 cells. Silkworm larval injection results showed that rBjαIT/GNA was the most toxic of the three proteins, followed by rBjαIT, and rGNA. When rBjαIT/GNA was injected at a concentration of 0.129 nmol/g body weight 46.7% of silkworm died within 48 h. Feeding newly hatched silkworms with rBjαIT/GNA at a leaf surface concentration of 40 µg/cm2 resulted in 76.7% mortality within 24 h. However, rBjαIT/GNA showed almost no oral insecticidal activity against second instar silkworms. The results indicated that rBjαIT/GNA has stronger injection insecticidal activity and feeding insecticidal activity than rBjαIT and rGNA individually, making it more suitable for biological control. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process. [ABSTRACT FROM AUTHOR]

Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1 g/L, and its kcat was 3237.6 s−1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200 g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application. [ABSTRACT FROM AUTHOR]

Pichia pastoris is a methylotrophic yeast with remarkable characteristics such as lacking endotoxin, producing high amounts of recombinant protein, performing post-translational modifications, and so on. Influenza A virus, a member of the Orthomyxoviridae family, is the cause of avian influenza. Three avian influenza virus subtypes, H5, H7 and H9, are commercially and physiologically significant in the poultry industry. Some researchers considered influenza to be the next pandemic disease. Nowadays, researchers have paid attention to producing novel and effective recombinant vaccines, especially in the poultry industry. Due to the advantages of P. pastoris yeast, it can be used as an ideal expression system for producing subunit vaccines. Although several studies have been conducted in this field, there is no comprehensive review of using P. pastoris to produce recombinant influenza vaccines. This review explains the different strains, phenotypes, and advantages of this yeast and then the production of recombinant influenza vaccines using this expression system is discussed in detail. [ABSTRACT FROM AUTHOR]

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest—more than two-fold higher compared with K—implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs. [ABSTRACT FROM AUTHOR]

(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0–8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) plays a crucial role in the diagnosis and monitoring of the non-specific acute phase response in humans. In contrast, rat CRP (rCRP) is an atypical acute-phase protein that possesses unique features, such as a possible incapacity to trigger the complement system and markedly elevated baseline plasma concentrations. To facilitate in vitro studies on these unique characteristics, obtaining high-quality pure rCRP is essential. Here we explored various strategies for rCRP purification, including direct isolation from rat plasma and recombinant expression in both prokaryotic and eukaryotic systems. Our study optimized the recombinant expression system to enhance the secretion and purification efficiency of rCRP. Compared to traditional purification methods, we present a streamlined and effective approach for the expression and purification of rCRP in the Pichia pastoris system. This refined methodology offers significant improvements in the efficiency and effectiveness of rCRP purification, thereby facilitating further structural and functional studies on rCRP. [ABSTRACT FROM AUTHOR]

The experiment aims to develop xylanase XynA and provide a new resource for feeding xylanase. The XynA of Geobacillus stearothermophilus was heterologous expressed in Pichia pastoris, the optimal fermentation conditions, enzymatic properties and antioxidant properties of the hydrolysis product Xylo-oligosaccharide (XOS) were studied. The results showed that the crude XynA with enzyme activity of about 33.7 U/mL could be produced after 96 h induced by 1.0% methanol concentration. The optimum pH value of XynA is 9.0, the optimum temperature is 65 °C, moreover, XynA remains above 80% of the activity at pH value ranging from 5.0 to 10.5 and temperatures between 45 and 75 °C. Thin-layer chromatography (TLC) analysis revealed that the main products of the hydrolysis of bagasse xylan by XynA were xylotriose and xylohexaose. The scavenging rate of DPPH free radical reached 72.04% and the scavenging rate of hydroxylradical reached 99.16% with 1 000 mg/L XOS. The study indicates that XynA exhibits robust activity and stability, efficiently hydrolyzing xylan to produce XOS, thereby facilitating its application in the feed industry. [ABSTRACT FROM AUTHOR]

Human complement factor H (CFH) plays a central role in regulating activated C3b to protect host cells. CFH contain 20 short complement regulator (SCR) domains and eight N-glycosylation sites. The N-terminal SCR domains mediate C3b degradation while the C-terminal CFH domains bind to host cell surfaces to protect these. Our earlier study of Pichia-generated CFH fragments indicated a self-association site at SCR-17/18 that comprises a dimerization site for human factor H. Two N-linked glycans are located on SCR-17 and SCR-18. Here, when we expressed SCR-17/18 without glycans in an Escherichia coli system, analytical ultracentrifugation showed that no dimers were now formed. To investigate this novel finding, full-length CFH and its C-terminal fragments were purified from human plasma and Pichia pastoris respectively, and their glycans were enzymatically removed using PNGase F. Using size-exclusion chromatography, mass spectrometry, and analytical ultracentrifugation, SCR-17/18 from Pichia showed notably less dimer formation without its glycans, confirming that the glycans are necessary for the formation of SCR-17/18 dimers. By surface plasmon resonance, affinity analyses interaction showed decreased binding of deglycosylated full-length CFH to immobilized C3b, showing that CFH glycosylation enhances the key CFH regulation of C3b. We conclude that our study revealed a significant new aspect of CFH regulation based on its glycosylation and its resulting dimerization. [ABSTRACT FROM AUTHOR]

Xylanase, an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat, has been found to improve the organizational structure of dough and thus increase its volume. In our past work, one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P. pastoris. It has shown significant potential in improving the quality of whole wheat bread, making it become a candidate for development as a new flour improver. After optimization of expression elements and gene dose, the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P. pastoris. In addition, 12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in threecopies strain, and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL. Nevertheless, combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone. To further evaluate the industrial potential, the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter. These engineering strategies improved the expression of xylanase FXYL by more than 104-fold, providing valuable insights for the costeffective industrial application of FXYL in the baking field. [ABSTRACT FROM AUTHOR]

Accurate and reliable estimation of specific growth rate (μ) in real-time is pivotal for reliable process monitoring of a bioprocess and subsequent implementation of advanced control strategies. Gibbs free energy dissipation is imminent for any biological system, and the metabolic heat flow measurements (calorimetry) formed the basis for estimating μ. However, the rationale behind selecting a suitable μ estimator model based on calorimetric perspective remains unexplored. The present investigation addresses the notion behind the selection of an appropriate estimator for μ and the assessment of the estimator models was illustrated using different types of energy metabolism, namely, high exothermic and low exothermic processes. The results indicated that the μ values from the instantaneous heat flow estimator significantly deviated (10-fold higher) from the offline values for highly exothermic process. Notably, the cumulative heat-based estimator accurately estimated μ values on both types of energy metabolism with performance metrics <0.005 h−1. [Display omitted] [ABSTRACT FROM AUTHOR]

Methanol, being electron rich and derivable from methane or CO 2 , is a very promising feedstock for biomanufacturing. Nevertheless, challenges still remain in methanol metabolism for value-added chemicals overproduction. Here, we performed gene mining and revealed the endogenous homoserine cycle for methanol conversion in Pichia pastoris for the first time. In addition, by manipulation of the concentration of methanol, adding the amino acids, and overexpressing the pathway genes, we identified the key substrates and rate-limiting enzymes (SDA and TS) affecting the homoserine cycle. Hence, the metabolic fluxes were rewired toward homoserine cycle and the methanol conversion was stepwise promoted through the introduction of cytoplasmic methanol oxidation pathway, overexpression of the key genes in the glycine and serine synthesis routes, and multicopy integration of the SDA and TS expression cassettes. Eventually, in combination with pathway revelation and metabolic engineering, the rebuilding methylotroph with strengthened homoserine cycle was able to produce 33.9 mg/L/OD β-carotene with a titer of 173.9 mg/L from methanol. The unit cell titer of β-carotene was 145.7 % higher than that of the initial strain (Kp16). This study will lay a good foundation for optimizing methylotrophic platform to promote industrial utilization. [Display omitted] • Revelation of the endogenous homoserine cycle in P. pastoris. • Identifying and manipulating the key substrates and enzymes. • The methanol conversion was greatly improved. [ABSTRACT FROM AUTHOR]

The constitutive and strong GAP promoter (PGAP) from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene has emerged as a suitable option for protein production in methanol-free Komagataella phaffii (syn. Pichia pastoris) expression systems. Nevertheless, the effect of the transcriptional terminator from the alcohol oxidase 1 gene (TAOX1) or GAPDH gene (TGAP) within the heterologous gene structure on the transcriptional activity in a PGAP-based strain and the impact on the extracellular product/biomass yield (Yp/x) has not yet been fully characterized. In this study, we engineered two K. phaffii strains, each harboring a single copy of a different combination of regulatory DNA elements (i.e., PGAP-TAOX1 or PGAP-TGAP pairs) within the heterologous gene structure. Moreover, we assessed the impact of the regulatory element combinations, along with the carbon source (glucose or glycerol) and the stage of cell growth, on the transcript levels of the reporter gene and the endogenous GAPDH gene in the yeast cells, as well as the extracellular Yp/x values. The results indicate that the regulation of transcription for both heterologous and endogenous GAPDH genes, the extracellular Yp/x values, and translation and/or heterologous protein secretion were influenced by the PGAP-transcriptional terminator combination, with the carbon source and the stage of cell growth acting as modulatory factors. The highest transcript levels for the heterologous and endogenous GAPDH genes were observed in glucose cultures at a high specific growth rate (0.253 h−1). Extracellular Yp/x values showed an increasing trend as the culture progressed, with the highest values observed in glucose cultures, and in the PGAP-TAOX1-based strain. The presence of TAOX1 or TGAP within the heterologous gene structure activated distinct gene regulatory elements in each strain, leading to differential modulation of gene regulation for the heterologous and the GAPDH genes, even though both genes were under the control of the same promoter (PGAP). TAOX1 induced competitive regulation of transcriptional activity between the two genes, resulting in enhanced transcriptional activity of the GAPDH gene. Moreover, TAOX1 led to increased mRNA stability and triggered distinct metabolic downregulation mechanisms due to carbon source depletion compared to TGAP. TAOX1 enhanced translation and/or heterologous protein secretion activity at a high specific growth rate (0.253 h−1), while TGAP was more effective in enhancing post-transcriptional activity at a low specific growth rate (0.030 h−1), regardless of the carbon source. The highest extracellular Yp/x was obtained with the PGAP-TAOX1-based strain when the culture was carried out at a low specific growth rate (0.030 h−1) using glucose as the carbon source. The optimization of regulatory elements and growth conditions presents opportunities for enhancing the production of biomolecules of interest. [ABSTRACT FROM AUTHOR]

Poliovirus (PV) is on the brink of eradication due to global vaccination programs utilizing live-attenuated oral and inactivated polio vaccines. Recombinant PV virus-like particles (VLPs) are emerging as a safe next-generation vaccine candidate for the impending polio-free era. In this study, we investigate the production, antigenicity, thermostability, immunogenicity, and structures of VLPs derived from PV serotype 2 (PV2) wildtype strain and thermally stabilized mutant (wtVLP and sVLP, respectively). Both PV2 wtVLP and sVLP are efficiently produced in Pichia pastoris yeast. The PV2 sVLP displays higher levels of D-antigen and significantly enhanced thermostability than the wtVLP. Unlike the wtVLP, the sVLP elicits neutralizing antibodies in mice at levels comparable to those induced by inactivated polio vaccine. The addition of an aluminum hydroxide adjuvant to sVLP results in faster induction and a higher magnitude of neutralizing antibodies. Furthermore, our cryo-EM structural study of both sVLP and wtVLP reveals a native conformation for the sVLP and a non-native expanded conformation for the wtVLP. Our work not only validates the yeast-produced PV2 sVLP as a promising vaccine candidate with high production potential but also sheds light on the structural mechanisms that underpin the assembly and immunogenicity of the PV2 sVLP. These findings may expedite the development of sVLP-based PV vaccines. [ABSTRACT FROM AUTHOR]

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large. [ABSTRACT FROM AUTHOR]

Abstract Background Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii. Results High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time. Conclusions These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.

Methanol is a promising substrate for sustainable biomanufacturing, and Pichia pastoris has become a commonly used yeast for methanol utilization due to its powerful methanol metabolic pathways and methanol inducible promoter. Previous reconstruction of gene circuits highly improved transcriptional activity, but excessive expression of chimeric transactivator damaged cell growth on methanol. Here we employed transcriptome analysis to investigate the effects of chimeric transactivator overexpression on cellular metabolism and regulatory networks. The results showed that strong expression of chimeric transactivator unexpectedly downregulated methanol metabolism, especially the alcohol oxidase 1 (AOX1), but without remarkable changes in expression of transcriptional factors. Meanwhile, the synthesis of peroxisomes also varied with chimeric transactivator expression. In addition, the enrichment analysis of differentially expressed genes revealed their impact on cellular metabolism. The gene expression patterns caused by different expression levels of chimeric transactivators have also been clarified. This work provides useful information to understand the transcriptional regulation of the AOX1 promoter and methanol signaling. It revealed the importance of balancing transcription factor expression for the host improvement.

In order to solve the problems of low efficiency and cumbersome process of Pichia pastoris genetic manipulation, secretory genetic manipulation tools were constructed and optimized to improve their industrial production and application value. Firstly, the α-amylase was employed as the reporter protein, and the non-integrated expression plasmid was constructed by fusing the self-replicating sequence. Secondly, endogenous signal peptides that best match the target protein were quickly screened and evaluated using universal primers and POE-PCR. Finally, a nutrition-dependent screening marker was constructed with phosphite as the sole phosphorus source. The results indicate that non-integrated plasmids express α-amylase with 2-fold and 1.6-fold higher enzyme activity compared to integrated plasmids, respectively; The use of FLO10, EXG1, and MSB2 signal peptides results in 2.5-fold, 1.94-fold, and 1.75-fold higher α-amylase activity than the conventional signal peptide α-MF, respectively; Arecombinant strain for heterologous expression of α-amylase is successfully screened using the phosphite dehydrogenase gene (ptxD). The process of gene operation is simplified, the genetic operation tools are improved, and green, safe, and low-cost nutrition-dependent screening markers are provided, which lays a foundation for the application and promotion of the Pichia pastoris secretory expression system.

Abstract Background Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. Results At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. Conclusions We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Abstract Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.

Spent fermentation media from bioprocessing represent a significant waste stream, and interest in recycling them as part of the developing circular bioeconomy is growing. The potential to reuse yeast spent culture media (YSM) to feed secondary bacterial fermentations producing recombinant protein was investigated in this study. Elemental and amino acid compositional analysis using inductively coupled plasma mass spectrometry (ICP-MS) and LC-MS/MS identified significant differences in the concentrations of 6 elements and 18/20 amino acids in YSM compared with rich microbiological media (LB). Restoration of levels of magnesium and sodium through addition of their salts and amino acids from tryptone supplementation led to the expression of equivalent titres of recombinant proteins by E. coli (0.275 g/L), compared to that in LB media (0.296 g/L) and BMMY media (0.294 g/L) in shake flask culture. When this supplementation strategy was employed in a bioreactor system, we observed a significant increase in recombinant protein titre using the supplemented YSM (2.29 (±0.02) g/L) over that produced using LB media (1.29 (±0.09) g/L). This study demonstrates through highly sensitive compositional analysis and identification of supplementation strategies the potential to valorise spent media from yeast fermentations that underpin industrial processes of significant scale, creating a circular approach to waste stream management.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines. Author summary: SARS-CoV-2 continuously circulates, resulting in infections and re-infections globally. COVID-19 vaccines can provide protection against disease and death caused by SARS-CoV-2. However, the immune responses induced by vaccination will wane gradually. In addition, new SARS-CoV-2 sub-variants emerge and show immune evasion. Therefore, inoculating with the updated COVID-19 vaccines is recommended for enhancing protection against the circulating SARS-CoV-2 sub-variants. Pichia pastoris is an eukaryotic system that enables fast and low-cost producing proteins with easy scale-up. In this study, we developed a strategy for producing SARS-CoV-2 RBD-dimer immunogens in glycoengineered Pichia pastoris with homogeneous glycosylation and high stability. The RBD antigen can be updated with the sequence of circulating sub-variants. In addition, mouse experiments demonstrated that the yeast-produced RBD-dimer vaccines induced robust and protective immune responses. This study provides a simple and promising approach to produce yeast-derived SARS-CoV-2 RBD-dimers as a boosting vaccine and guides the basis for the production of the other vaccines in Pichia pastoris. [ABSTRACT FROM AUTHOR]

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria. [ABSTRACT FROM AUTHOR]

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production. [ABSTRACT FROM AUTHOR]

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. • α-MF secretion signal leaders from K. pastoris and K. phaffii exhibit lower secretion efficiency compared to that from S. cerevisiae in the P. pastoris system. • N-linked glycosylation on the α-MF secretion signal leader enhances protein secretion. • N-linked glycosylation on the α-MF secretion signal leader reduces ER stress and enhanced protein folding mediated by calnexin. [ABSTRACT FROM AUTHOR]

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (K i) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE. • Ecallantide is a small protein engineered to inhibit plasma kallikrein. • Overactive kallikrein production leads to Hereditary Angioedema (HAE). • Ecallantide does not stay in circulation long enough for prophylactic use. • Ecallantide-albumin fusion proteins circulate longer than ecallantide. • Ecallantide-albumin fusion protein clearance involves a scavenger receptor. [ABSTRACT FROM AUTHOR]

Background: Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. Results: At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. Conclusions: We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future. [ABSTRACT FROM AUTHOR]

Komagataella phaffii (previously described as Pichia pastoris) is a yeast that produces high-level heterologous proteins with a wide range of applications in medicine and industry. The methanol-induced alcohol oxidase I promoter (PAOX1) is frequently used for protein expression in this yeast. However, limitations on the use of methanol have been observed in large-scale production, including its flammability, toxicity, and need for special handling. Here, we propose to develop a system using recombinant cells constitutively expressing pectinmethyl esterase for expression of two reporter proteins, GFP and azurin, under the control of PAOX1 using pectin in production medium. So, this system is coherent with yeast culture medium containing pectin and heterologous gene inserted downstream of PAOX1 can be successfully expressed without the addition of methanol. Therefore, this novel Self-inducibLe heterologous protein EXpression (SILEX) system, which does not require the addition of methanol, can be used for the production of any protein. It can also be adapted for large-scale production. [ABSTRACT FROM AUTHOR]

Introduction: Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. Methods: The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera™), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. Results: The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Conclusion: Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply. [ABSTRACT FROM AUTHOR]

Previous data established 4′‐deoxyflavone glycosides (4′‐DFGs) as important pharmaceutical components in the roots of rare medical plants like Scutellaria baicalensis Georgi. Extracting these compounds from plants involves land occupation and is environmentally unfriendly. Therefore, a modular ("plug‐and‐play") yeast‐consortium platform is developed to synthesize diverse 4′‐DFGs de novo. By codon‐optimizing glycosyltransferase genes from different organisms for Pichia pastoris, six site‐specific glycosylation chassis are generated to be capable of biosynthesizing 18 different 4′‐DFGs. Cellular factories showed increased 4′‐DFG production (up to 18.6‐fold) due to strengthened synthesis of UDP‐sugar precursors and blocked hydrolysis of endogenous glycosides. Co‐culturing upstream flavone‐synthesis‐module cells with downstream glycoside‐transformation‐module cells alleviated the toxicity of 4′‐deoxyflavones and enabled high‐level de novo synthesis of 4′‐DFGs. Baicalin is produced at the highest level (1290.0 mg L−1) in a bioreactor by controlling the consortium through carbon‐source shifting. These results provide a valuable reference for biosynthesizing plant‐derived 4′‐DFGs and other glycosides with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Serine protease is an extracellular protease secreted by biocontrol fungi that can effectively control nematode diseases by degrading nematode eggshells and enhancing plant resistance. Trichoderma longibrachiatum T6, an important biocontrol fungus, has been demonstrated to effectively parasitize and degrade Heterodera avenae cysts, eggs, and second-stage juveniles (J2s). However, the genes that encoding serine protease and their functions in T. longibrachiatum T6 have not been thoroughly investigated. In this study, we successfully cloned and sequenced the serine protease gene TlSP1 in T. longibrachiatum T6. Our results revealed that the expression level of the TlSP1 gene was induced and significantly increased in T. longibrachiatum T6 after inoculation with H. avenae cysts. The full-length sequence of the coding region (CDS) of TlSP1 gene was 1230 bp and encoded a protein consisting of 409 amino acids. Upon the transformation of the TlSP1 gene into Pichia pastoris X33, the purified recombinant TlSP1 protein exhibited optimal activity at a temperature of 50 °C and pH 8.0. Following 4–10-day of treatment with the purified recombinant TlSP1 protein, the eggshells and content were dissolved and exuded. The number of nematodes invading wheat roots was reduced by 38.43% in the group treated with both TlSP1 and eggs on one side (P1+N) compared to the control group, while the number of nematodes invading wheat roots was reduced by 30.4% in the TlSP1 and eggs two-sided treatment group (P1/N). Furthermore, both the P1+N and P1/N treatments significantly upregulated genes associated with defense enzymes (TaPAL, TaCAT, TaSOD, and TaPOD), genes involved in the lignin synthesis pathway (TaC4H, Ta4CL2, TaCAD1, and TaCAD12), and salicylic acid (SA)-responsive genes (TaNPR1, TaPR1, and TaPR2) and led to the high expression of jasmonic acid (JA)-responsive genes (TaPR4, TaOPR3, and TaAOS2). This study has highlighted the significant role of the TlSP1 gene in facilitating H. avenae eggshells' dissolution, preventing nematode invasion in the host plant, and boosting plant resistance in wheat. [ABSTRACT FROM AUTHOR]

Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two‐stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q‐membrane filter and we demonstrate sufficient removal of key supernatant impurities including host‐cell proteins (HCPs) and DNA with yields of 1–2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS‐CoV‐2 subunit protein antigen and compared the purified protein to a conventional two‐step chromatographic process. We then applied this method to compare several SARS‐CoV‐2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single‐domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early‐stage protein therapeutics and vaccine candidates produced in K. phaffii. [ABSTRACT FROM AUTHOR]

Dust mite allergen is one of the most common allergens that cause allergies in humans. Der p 1 protein, the first allergen of house dust mite (Dermatophagoides pteronyssinus), is the most important allergen. IgE antibody specifically binding to Der p 1 can be detected in serum of over 80% allergic organism. The allergy detection kit based on Der p 1 protein can be used for allergy specific diagnosis. At present, the allergy detection kit in China is mainly depends on imports, so it is urgent to establish an efficient production system of recombinant dust mite allergen to replace imported raw materials. In this study, recombinant expression of Der p 1 was performed based on Pichia pastoris expression system. First, after codon optimization, the full-length coding gene of PreProDer p 1 was expressed in Pichia pastoris GS115, and the yield was up to 100 mg/L. Further co-expression of molecular chaperones increased the yield to 140 mg/L. And the fermentation yield of 3 L reactor was increased to 1 g/L, which was the reported highest level. Afterwards, the purification process of the recombinant protein by cation exchange chromatography and affinity chromatography was optimized, and realized the purpose protein yield reached 60.7%. Finally, the bioactivity analysis showed that the purified protein had lower antigenic activity than the commercial product. The molecular weight analysis of the protein suggested that the cleavage of PreProDer p 1 might be uneven during the secretion, and the cleavage conditions of the leader peptide requires subsequent optimization. [ABSTRACT FROM AUTHOR]

Toxoplasmosis represents a significant public health and veterinary concern due to its widespread distribution, zoonotic transmission, and potential for severe health impacts in susceptible individuals and animal populations. The ability to design and produce recombinant proteins with precise antigenic properties is fundamental, as they serve as tools for accurate disease detection and effective immunization strategies, contributing to improved healthcare outcomes and disease control. Most commonly, a prokaryotic expression system is employed for the production of both single antigens and multi-epitope chimeric proteins; however, the cloning strategies, bacterial strain, vector, and expression conditions vary. Moreover, literature reports show the use of alternative microbial systems such as yeast or Leishmania tarentolae. This review provides an overview of the methods and strategies employed for the production of recombinant Toxoplasma gondii antigenic proteins for the serological detection of T. gondii infection and vaccine development. [ABSTRACT FROM AUTHOR]

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The goal of the current work was to optimize the growth parameters needed to manufacture agarase enzyme from a non-marine PI strain of Bacillus subtilis on an agar-based medium. Using Plackett–Burman design (PBD), nine process parameters were evaluated, and agar, peptone, and yeast-extract were identified as the most significant independent factors influencing agarase production with confidence levels more than 90%. To evaluate the optimal concentrations of the indicated process parameters on agarase production, the Box–Behnken design (BBD) was applied. After optimization, B. subtilis strain PI produced 119.8 U/ml of agarase, representing a 1.36-fold increase. In addition the agar hydrolysate fermented products contain the liberated oligosaccharide acts as strong antioxidant which has 62.4% scavenging activity. Also, the agarase yields increased (1141.12, 1350.253, 1684.854 and 1921.863 U/ml) after substitution the agar with algal biomass of Carolina officinalis at different concentrations (2, 5, 10 and 15%), respectively. After completing the saccharification process, the resulted hydrolysate was used to produce ethanol through fermentation with Pichia pastoris yeast strain as an economical method giving yields (6.68317, 7.09748, 7.75648 and 8.22332 mg/ml), that are higher than using yeast extract peptone dextrose (YPD) medium (4.461 mg/ml). [ABSTRACT FROM AUTHOR]

As an essential component of the fungal cell wall, β-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel β-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50℃, and showed great selectivity for continuous β-1,6-glycosidic bonding polysaccharides. Based on this, β-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 105/ml conidia with 0.03 μg/μl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure. [ABSTRACT FROM AUTHOR]