Search Results (84)

Soybean molasses, a by-product of alcohol-based soy protein concentrate production, is rich in stachyose and other functional oligosaccharides, but its high sucrose content and other fermentable non-target sugars hinder the efficient purification of stachyose. In this study, the sugar-utilization patterns of four commonly used microbial chassis or production strains, Escherichia coli W, E. coli BL21, Saccharomyces pastorianus Weihenstephan 34/70, and Komagataella phaffii (formerly Pichia pastoris) GS115, were systematically compared to identify a suitable host for selective stachyose enrichment. Among them, E. coli W showed the best performance in rapidly consuming non-target sugars while retaining stachyose. Based on this strain, a CRISPR–Cas9 engineering strategy was applied by deleting the endogenous α-galactosidase gene melA and overexpressing the sucrose permease gene cscB. The resulting strain selectively and nearly completely removed sucrose and other non-target sugars from soybean molasses, increasing the proportion of stachyose from <30% to >90% of total soluble solids. Further optimization of nitrogen source level, inoculum size, and initial °Brix improved fermentation performance. These results demonstrate an effective biological pre-purification strategy for selective stachyose enrichment from soybean molasses. Full article

Komagataella phaffii (formerly Pichia pastoris) is a prominent platform for recombinant protein production, yet secretion efficiency often remains a critical bottleneck. In this study, we validated three candidate genes—WHI3, CLPP, and PMP20—previously identified through genome-wide CRISPR activation screening, for their potential to enhance heterologous protein secretion. Overexpression of these factors under the control of the methanol-inducible AOX1 promoter increased the secretion of human serum albumin (HSA), with WHI3 and CLPP yielding improvements of 18.3% and 17.9%, respectively. Furthermore, applying this strategy to human lactoferrin (hLF) revealed that WHI3 overexpression robustly enhanced hLF secretion by approximately 70%. Comparative analysis of different promoters (AOX1, GAP, and CAT) indicated that the AOX1 promoter remains the most effective driver for these enhancers, suggesting a threshold-dependent regulatory mechanism. These results demonstrate the protein-dependent nature of secretion optimization and identify WHI3, CLPP, and PMP20 as novel, effective co-expression factors for improving recombinant protein yields in K. phaffii. Full article

Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of Komagataella phaffii through multi-factorial optimization. Initially, the native bovine prochymosin gene (pcw) was codon-optimized (pcm14) and cloned, along with an mCherry-tag construct (clpcm14), into inducible vector pPIC9 for expression in Komagataella phaffii GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU/(mL·h), twice that of inducible strain clp2-91 (53.59 SU/(mL·h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0–6.0). This study demonstrates an efficient strategy for chymosin expression in K. phaffii, offering insights that may support the future development and optimization of heterologous protein production in this yeast. Full article

The increasing demand for recombinant proteins has driven innovation in bioprocessing strategies using Komagataella phaffii as a host organism. Conventional fed-batch cultivation with pure methanol induction remains widely used but presents challenges including high methanol consumption, extended downtime, and elevated operational costs. This study evaluates alternative strategies combining mixed induction (methanol/sorbitol) with continuous cultivation to enhance productivity, sustainability, and improved economic outcome. Using KEX2 protease as a model industrial recombinant protein, we compared four cultivation modes: fed-batch with methanol (benchmark), fed-batch with mixed induction, continuous with methanol, and continuous with mixed induction. Cell growth, volumetric yield, and specific productivity were evaluated at 5L scale and then modelled to simulate industrial scales (40 L and 400 L). Results demonstrate that continuous cultivation with mixed induction significantly improves yield up to 9-fold compared to conventional fed-batch and reduces methanol usage and oxygen demand. Techno-economic simulations reveal that a 40 L continuous process can match or exceed the output of two 400 L fed-batch runs, while lowering capital and operating costs and minimizing environmental footprint. This integrated strategy offers a scalable, low-cost, and safer method for recombinant protein production, supporting compact and sustainable manufacturing solutions. Full article

The COVID-19 pandemic accelerated vaccine innovation but also exposed weaknesses in global access and manufacturing. Yeast-based platforms, particularly Saccharomyces cerevisiae and Pichia pastoris, also known as Komagataella phaffii, offer a practical complement to vector systems. These eukaryotic microorganisms combine safety, scalability, and cost-effectiveness with the ability to express complex antigens and assemble virus-like particles. Building on the success of the recombinant hepatitis B vaccine, recent advances in glycoengineering, CRISPR-based host optimization, and surface display technologies have expanded the utility of yeast-based platforms for the rapid development of vaccines. Yeast-derived SARS-CoV-2 receptor-binding domain (RBD) subunit vaccines, such as Corbevax and Abdala (CIGB-66), demonstrate that affordable, immunogenic, and thermostable products are feasible at scale. Emerging innovations in glycan humanization, thermostable formulations, and oral or mucosal delivery highlight the potential of yeast-based vaccines for decentralized manufacturing and equitable pandemic preparedness. This review summarizes recent technical and clinical progress in yeast-based vaccine research, positioning these platforms as accessible and adaptable tools for future outbreak responses and global immunization strategies. Full article

Xylanases (EC 3.2.1.8) are value-added enzymes essential for biomass deconstruction and are widely used in the pulp and paper, food, feed, and biofuel sectors. This review provides a comprehensive analysis of the current state and future prospects of xylanase research and application. It begins by examining the structural diversity of xylan substrates and the corresponding classification of xylanase enzymes, their catalytic mechanisms, and methods for their functional study, such as inhibitor analysis. The discussion then covers the challenges and methods involved in the purification of xylanases from complex biological mixtures. While natural microbial sources (fungi and bacteria) remain important, the limitations of wild-type (WT) strains for industrial production are highlighted. The review assesses the most common recombinant production systems, including Escherichia coli, Bacillus subtilis, and Komagataella phaffii, comparing their advantages for high-yield enzyme production. Finally, the paper focuses on protein engineering strategies as powerful tools for enhancing key enzyme properties (thermostability, specific activity, and pH tolerance). By integrating fundamental knowledge with applied technological approaches, this review underscores the critical role of xylanases in industrial biotechnology and identifies future research directions for their optimization. Full article

This research characterized a novel Komagataella phaffii strain generated through intraspecific crossing between a wild isolate and a laboratory strain. This segregant, called S467, expressed 2.2-fold more secreted recombinant β-glucosidase than its parental strains in microtiter scale, which suggested that S467 could be an attractive host for bioprocess optimization. S467 was grown alongside the laboratory strain CBS7435 expressing β-glucosidase (CBS_BGL9), as a control, in a 1.5 L bioreactor to determine kinetics parameters, and similar cell growth rate (0.12 h⁻¹) but higher recombinant protein activity, measured as enzymatic activity, was observed in S467. The effect of specific cell growth rate was studied using continuous cultures (chemostat) at different dilution rates, identifying conditions that provided up to a twofold increase in enzymatic activity in S467. RT-qPCR was conducted on key genes associated with the genetic background of S467, in order to clarify differences at the transcriptomic level that render S467 as a potential superior host for recombinant protein production. Overall, this study provides quantitative evidence of the positive effect of the natural isolate IRA1 allele for the generation of recombinant β-glucosidase and highlights the usability of natural genetic diversity in K. phaffii. Full article

Fungal laccases oxidize a wide range of substrates with a diverse spectrum of subsequent non-specific free radical reactions, leading to the production of unwanted byproducts. This work describes a unique recombinant alkaliphilic laccase from Paramyrothecium roridum VKM F-3565 capable of performing specific oligomerization of phenylpropanoids (precursors of natural lignin and lignans) in a neutral environment, thus preventing the reverse reaction of depolymerization which occurs in an acidic environment. The recombinant alkaliphilic laccase from P. roridum VKM F-3565 with a specific enzyme activity of about 154.0 U/mg (in the reaction with 1 mM ABTS) was obtained using a Komagataella phaffii transformant with a yield of 20 ± 1.5 mg/L. The recombinant laccase had an increased degree of N-glycosylation (MW = 97 kDa), higher pH optimum in reaction with phenylpropanoids and a decreased temperature optimum, compared to the wild-type laccase. The enzyme exhibited great resistance to surfactants and the EDTA in the neutral conditions rather than the acidic ones, whereas its tolerance to mono- and divalent-metal ions was high at acidic conditions. This work demonstrates the important role of N-glycosylation of the alkaliphilic laccase of P. roridum VKM F-3565 in its functional activity. The presence of pH-dependent reactions makes the studied laccase attractive for the phenylpropanoid oligomerization with the production of novel oligomeric phenylpropanoid derivatives for industrial and pharmacological purposes. Full article

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important therapeutic cytokine. Methylotrophic yeasts such as Komagataella phaffii and Ogataea parapolymorpha are attractive hosts for recombinant protein production. In this study, these yeasts were engineered to produce GM-CSF in both secreted and cell-surface-anchored forms. Secreted GM-CSF accumulated to tens of milligrams per liter in culture supernatants following induction. Fluorescent antibody labeling confirmed that yeast strains expressing surface-displayed GM-CSF exhibited markedly increased fluorescence compared to parental strains. The highest signal was observed in K. phaffii and further validated by immunofluorescence microscopy. Functional assays demonstrated that K. phaffii cells displaying GM-CSF stimulated TF-1 cell proliferation 1.41-fold compared to control yeast lacking GM-CSF. These results confirm both the successful surface display and preserved biological activity of the cytokine. This work demonstrates the feasibility of engineering K. phaffii to present active human GM-CSF on the cell surface. Surface display was efficient and yielded biologically functional cytokine, as shown by fluorescence-based quantification and TF-1 proliferation assays. The study highlights yeast surface display as a promising platform for delivering therapeutic cytokines without requiring purification steps. Full article

Dengue is a rapidly spreading mosquito-borne viral infection, with increasing reports of outbreaks globally. According to the World Health Organization (WHO), by 30 April 2024, over 7.6 million dengue cases were reported, including 3.4 million confirmed cases, more than 16,000 severe cases, and over 3000 deaths. As dengue remains endemic in many regions, there is a critical need for the development of new vaccines and manufacturing processes that are efficient, cost-effective, and capable of meeting growing demand. In this study, we explore an alternative process development pathway for the future manufacturing of a dengue vaccine, utilizing Komagataella phaffii (Pichia pastoris) as the host organism, one of the most promising candidates for the expression of heterologous proteins in vaccine development. It combines the speed and ease of highly efficient prokaryotic platforms with some key capabilities of mammalian systems, making it ideal for scalable and cost-effective production. The key outcomes of our research include (i) demonstrating the versatility of the Komagataella phaffii platform in the production of dengue viral-like particles (VLPs); (ii) optimizing the culture process using Design of Experiments (DoE) approaches in small-scale bioreactors; (iii) developing a novel purification platform for enveloped VLPs (eVLPs), and (iv) establishing alternative biophysical characterization methods for the dengue vaccine prototype. These findings provide a promising foundation for efficient and scalable production of dengue vaccines, addressing both technical and operational challenges in vaccine manufacturing. Full article

Valorisation of spent yeast biomass post-fermentation requires energy-intensive autolysis or enzymatic hydrolysis that reduces the net benefit. Here, we present a simple and reproducible method for generating functional yeast extract recycled from Komagataella phaffii biomass without a requirement of a pre-treatment process. Spent yeast pellets from fermentations were freeze-dried to produce a fine powder that can be used directly at low concentrations, 0.0015% (w/v), together with 2% peptone (w/v), to formulate complete media ready for secondary fermentations. This media formulation supported growth rates of yeast culture that were statistically indistinguishable (p-value > 0.05) from cultures grown in standard YPD media containing commercial yeast extract, and these cultures produced equivalent titres of recombinant β-glucosidase (0.998 Abs_405nm commercial extract vs. 0.899 Abs_405nm recycled extract). Additionally, nutrient analyses highlight equivalent levels of sugars (~23 g/L), total proteins, and cell yield per carbon source (~2.17 g) with this recycled yeast extract media formulation when compared to commercial media. This method reduces process complexity and cost and enables the circular reuse of yeast biomass. The protocol is technically straightforward to implement, using freeze drying that is commonly available in research laboratories, representing a broadly applicable and sustainable alternative to conventional media supplementation that achieves a circular approach within the same fermentation system. Full article

A novel diamine oxidase (DAO) was discovered in the bacterium Glutamicibacter halophytocola (DAO-GH). The gene of DAO-GH was integrated into the genome of the yeast Komagataella phaffii and recombinantly produced under control of the methanol-inducible AOX1 promoter in a bioreactor cultivation. A high DAO activity of 70.2 ± 5.2 µkat/L_culture (5.25 ± 0.22 µkat/g_protein) was yielded after 90 h of cultivation. The DAO-GH was partially purified by the polyethyleneimine precipitation of nucleic acids, fractionated ammonium sulfate precipitation and hydrophobic interaction chromatography, resulting in a specific DAO activity of 19.7 µkat/g_Protein. The DAO-GH was then biochemically investigated regarding its potential for histamine and tyramine degradation in fermented foods and the human small intestine. Interestingly, the DAO-GH showed activity even at a low pH of 5 and low temperature of 6 °C. Both histamine and tyramine were effectively degraded and DAO-GH showed especially very high affinity towards tyramine (K_m of 0.009 mM). The DAO-GH was shown to be capable of degrading around 20% of the initially applied histamine in tuna paste (pH 5.6) at 5 °C within 24 h and completely degraded the histamine in a simulated intestinal fluid within 1.5 h in bioconversion experiments. The DAO-GH was spray-dried for the production of a storable enzyme preparation. Only around 17% of activity were lost in this process and the DAO-GH remained stable at room temperature for at least 3 months. The discovery of this DAO with its very advantageous biochemical properties allows the preparation of histamine-reduced or -free fermented foods by a simple enzymatic treatment or the treatment of histamine intolerance symptoms as a dietary supplement or medicine. Full article

The Pichia system has been exploited for decades as a host for recombinant protein production, but there is still an information gap regarding problems that may arise with its use. The application of strains based on the methanol-induced alcohol oxidase 1 (AOX1) promoter may represent a safety issue, and its performance varies among strains. In this study, the ability of a Komagataella phaffii Mut^S KM71H strain to produce recombinant cutinases was evaluated and compared to that of the more widely used Mut⁺ X-33 strain. The effects of the nature of the cutinase (ANCUT1 and ANCUT3, from Aspergillus nidulans), methanol level, and inoculum concentrations were evaluated in shake flasks containing a complex medium. Higher activities and volumetric cutinase productivity were observed at lower induction cell densities (0.5%) for the Mut^S KM71H aox1::pPICZα-A-ANCUT1 strain, while a higher one (2%) yielded better results in KM71H aox1::pPICZα-A-ANCUT3. The best inoculum and inducer conditions for both strains yielded similar results. The behavior of the different cutinases in the Mut^S or Mut⁺ genetic background was opposed: strain KM71H aox1::pPICZα-A-ANCUT3 produced 19% more activity than strain X-33 aox1::pPICZα-A-ANCUT3, while the ANCUT1 containing strain produced significantly higher activity in the X-33 Mut⁺ strain. These results indicate that Mut^S strains are viable host options without the complications of rapidly growing methanol strains. The effect of the gene structure being expressed is a phenomenon that needs further exploration. Full article

Industrial applications of xylanases in high-temperature settings are limited by enzyme instability. This study evaluated glycerol and phenolic compounds as modulators of the catalytic and structural properties of a recombinant Myceliophthora heterothallica endoxylanase (rMhXyn) expressed in Komagataella phaffii. Glycerol (20% v/v) significantly improved thermostability (5-fold increase in half-life at 55 °C), decreased the activation energy for catalysis, and enhanced structural rigidity as evidenced by molecular dynamics simulations (reduced RMSD and Rg). In contrast, phenolic acids provided only short-term stabilization at moderate temperatures and did not confer structural benefits. Enzyme kinetics revealed that glycerol enhanced catalytic turnover (↑V_max), while phenolic compounds modified both K′ and cooperativity (Hill coefficient). Thermodynamic analysis supported glycerol’s stabilizing effect, with increased ∆H_(D) and a positive shift in ∆S_(D). These results suggest glycerol as a superior stabilizer for rMhXyn in high-temperature bioprocesses such as lignocellulosic biomass hydrolysis. These findings highlight the potential of targeted additives to improve enzyme performance for biotechnological applications. Full article

Bilirubin is a key component in the preparation of two traditional Chinese medicines: Calculus bovis sativus and Calculus bovis artifactus. Currently, industrial-scale production of bilirubin is limited to extraction from pig bile in a very low yield and its market price is very high, so it is important to develop an alternative method for producing bilirubin. This study developed a potential process for bilirubin production through biotransformation of biliverdin. The codon-optimized gene for biliverdin reductase (BVR) from Synechocystis PCC6803 was recombinantly expressed in Komagataella phaffii GS115, resulting in the genetically modified strain GS115-bvdR, which successfully expressed BVR with intracellular activity. Whole cells of GS115-bvdR were capable of transforming biliverdin to bilirubin in vitro. The overexpression conditions were optimized to enhance BVR production by GS115-bvdR, and the optimal conditions for the biotransformation of biliverdin into bilirubin using resting GS115-bvdR cells were established (pH 5.0 buffer, at 30 °C for 24 h, with 200 mg/L biliverdin). Under these conditions, a bilirubin concentration of 153 mg/L was achieved, with a conversion of 76.2% from biliverdin. These findings provide valuable insights for future studies on the biosynthesis of bilirubin through metabolic engineering. Full article