RESUMEN

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.

Asunto(s)

Proteína C-Reactiva , Proteínas Recombinantes , Animales , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Ratas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Expresión Génica , Saccharomycetales/genética , Saccharomycetales/metabolismo , Pichia/genética , Pichia/metabolismo

RESUMEN

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.

Asunto(s)

Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Animales , Ratones , COVID-19/prevención & control , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Glicosilación , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Humanos , Anticuerpos Neutralizantes/inmunología , Ratones Endogámicos BALB C , Anticuerpos Antivirales/inmunología , Saccharomycetales/genética , Saccharomycetales/inmunología , Saccharomycetales/metabolismo , Femenino , Pichia/genética , Pichia/metabolismo

RESUMEN

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Asunto(s)

Colágeno Tipo II , Humanos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ratones , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Fermentación , Pichia/genética , Pichia/metabolismo , Movimiento Celular/genética , Fibroblastos/metabolismo , Diferenciación Celular , Reactores Biológicos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Nanofibras/química

RESUMEN

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-ß-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-ß-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-ß-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.

Asunto(s)

Endo-1,4-beta Xilanasas , Estabilidad de Enzimas , Xilanos , Xilanos/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/química , Hidrólisis , Actinobacteria/enzimología , Actinobacteria/genética , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Clonación Molecular/métodos , Especificidad por Sustrato , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Pichia/genética , Pichia/metabolismo , Actinomycetales/enzimología , Actinomycetales/genética , Secuencia de Aminoácidos , Saccharomycetales

RESUMEN

The Pichia kluyveri, a proliferation commonly found in Sichuan pickles (SCPs), can accelerate the growth and reproduction of spoilage bacteria, causing off-odor development and decay. Although D-limonene, a common natural preservative, effectively restricts P. kluyveri, its inhibitory mechanism remains unclear. This study aimed to elucidate this molecular mechanism by investigating the impact on basic P. kluyveri metabolism. The findings revealed that D-limonene inhibited P. kluyveri growth and disrupted the transcription of the genes responsible for encoding the enzymes involved in cell wall and membrane synthesis, oxidative phosphorylation, glycolysis, and the tricarboxylic acid (TCA) cycle pathway. The results indicated that these events disrupted crucial metabolism such as cell wall and membrane integrity, adenosine triphosphate (ATP) synthesis, and reactive oxygen species (ROS) balance. These insights provided a comprehensive understanding of the inhibitory effect of D-limonene on the growth and reproduction of P. kluyveri while highlighting its potential application in the SCP industry.

Asunto(s)

Limoneno , Pichia , Limoneno/farmacología , Pichia/metabolismo , Pichia/genética , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

BACKGROUND: Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. RESULTS: To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. CONCLUSION: These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pichia/genética , Pichia/metabolismo

RESUMEN

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Asunto(s)

Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Recombinantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Ingeniería Genética/métodos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Pichia/genética , Pichia/metabolismo

RESUMEN

As a highly toxic mycotoxin, ochratoxin A (OTA) is widely contaminating agricultural products and has various toxicological effects. Bioenzymes for OTA degradation have shown promising potential for detoxification. Other than the efficient amidohydrolase ADH3 previously, two novel amidohydrolases ADH1 and AMD3 were obtained in this study. During Escherichia coli expression, the expressed protein solubility was very low and will limit future industrial application. Here, high copy number integrations were screened, and the amidohydrolases were efficiently secretory expressed by Pichia pastoris GS115. The protein yields from 1.0 L of fermentation supernatant were 53.5 mg for ADH1, 89.15 mg for ADH3, and 79.5 mg for AMD3. The catalytic efficiency (Kcat/Km) of secretory proteins was 124.95 s-1 mM-1 for ADH3, 123.21 s-1 mM-1 for ADH1, and 371.99 s-1 mM-1 for AMD3. In comparison to E. coli expression, the active protein yields substantially increased 15.78-51.53 times. Meanwhile, two novel amidohydrolases (ADH1 and AMD3) showed much higher activity than ADH3 that produced by secretory expression.

Asunto(s)

Amidohidrolasas , Expresión Génica , Ocratoxinas , Ocratoxinas/metabolismo , Ocratoxinas/química , Hidrólisis , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Amidohidrolasas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomycetales/genética , Saccharomycetales/enzimología , Saccharomycetales/metabolismo , Cinética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Fermentación , Pichia/genética , Pichia/metabolismo

RESUMEN

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

Asunto(s)

Anticuerpos de Cadena Única , Animales , Ratones , Conejos , Secuencia de Aminoácidos , Animales Ponzoñosos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/farmacología , Expresión Génica , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/química , Neurotoxinas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Venenos de Escorpión/antagonistas & inhibidores , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/farmacología

RESUMEN

Komagataella phaffii (K. phaffii) (Pichia pastoris), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered significant interest as a platform for the production of recombinant proteins. Numerous benefits include effective secretory expression that facilitates the easy purification of heterologous proteins, high cell density with rapid growth, post-translational changes, and stable gene expression with integration into the genome. In the last thirty years, K. phaffii has also been refined as an adaptable cell factory that can produce hundreds of biomolecules in a laboratory setting and on an industrial scale. Indeed, over 5000 recombinant proteins have been generated so far using the K. phaffii expression method, which makes up 30% of the total cell protein or 80% of the total released protein. K. phaffii has been used to manufacture more than 70 commercial products in addition to over 300 industrial processes that have been granted licenses. Among these are useful enzymes for industrial biotechnology, including xylanase, mannanase, lipase, and phytase. The others are biopharmaceuticals, which include human serum albumin, insulin, hepatitis B surface antigen, and epidermal growth factor. Compared to other expression systems, this yeast is also considered a special host for synthesizing subunit vaccines, which have recently been supplanted by alternative vaccination types, such as inactivated/killed and live attenuated vaccines. Moreover, efficient production of recombinant proteins is achieved through multi-level optimization methods, such as codon bias, gene dosage, promoters, signal peptides, and environmental factors. Therefore, although K. phaffii expression systems are efficient and simple with clearly established process procedures, it is still necessary to determine the ideal conditions since these vary depending on the target protein to ensure the highest recombinant protein generation. This review addresses the K. phaffii expression system, its importance in industrial and biopharmaceutical protein production, and some bioprocessing and genetic modification strategies for efficient protein production. K. phaffii will eventually continue contributing as a potent expression system in research areas and industrial applications.

Asunto(s)

Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Productos Biológicos/metabolismo , Biotecnología/métodos , Pichia/genética , Pichia/metabolismo

RESUMEN

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

Asunto(s)

Aminas Biogénicas , Fermentación , Glicina , Glicina/metabolismo , Aminas Biogénicas/metabolismo , Sales (Química) , Putrescina/metabolismo , Tiramina/metabolismo , Microbiología de Alimentos , Lactobacillus/metabolismo , Lactobacillus/genética , Alimentos Fermentados/microbiología , Pichia/metabolismo , Pichia/genética

RESUMEN

PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Asunto(s)

6-Fitasa , Reactores Biológicos , Fermentación , Glucosa , Metanol , Proteínas Recombinantes , 6-Fitasa/genética , 6-Fitasa/metabolismo , Glucosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Metanol/metabolismo , Reactores Biológicos/microbiología , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/crecimiento & desarrollo , Pichia/genética , Pichia/metabolismo , Pichia/crecimiento & desarrollo , Expresión Génica

RESUMEN

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40 ℃ and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (Ki) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226riceXIP@HZ1-E136AnXylA10@OE2 and K226riceXIP@HZ3-E242AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was -59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

Asunto(s)

Antioxidantes , Aspergillus niger , Endo-1,4-beta Xilanasas , Proteínas Fúngicas , Glucuronatos , Oligosacáridos , Proteínas Recombinantes , Xilanos , Glucuronatos/metabolismo , Glucuronatos/química , Xilanos/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/química , Aspergillus niger/enzimología , Aspergillus niger/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Oligosacáridos/metabolismo , Antioxidantes/metabolismo , Antioxidantes/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Simulación de Dinámica Molecular , Oryza , Fagus , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Madera , Pichia/genética , Pichia/metabolismo , Hidrólisis , Dominio Catalítico

RESUMEN

Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified 60 yeast strains from the natural fermentation broth of Sidamo coffee beans to screen for indigenous beneficial yeasts. Among them, 25 strains were obtained through morphological characterization on nutritional agar medium from Wallerstein Laboratory (WL), with molecular biology identifying Saccharomyces cerevisiae strain YBB-47 and the remaining 24 yeast strains identified as Pichia kudriavzevii. We investigated the fermentation performance, alcohol tolerance, SO2 tolerance, pH tolerance, sugar tolerance, temperature tolerance, ester production capacity, ethanol production capacity, H2S production capacity, and other brewing characteristics of YBB-33 and YBB-47. The results demonstrated that both strains could tolerate up to 3% alcohol by volume at a high sucrose mass concentration (400 g/L) under elevated temperature conditions (40 ℃), while also exhibiting a remarkable ability to withstand an SO2 mass concentration of 300 g/L at pH 3.2. Moreover, S. cerevisiae YBB-47 displayed a rapid gas production rate and strong ethanol productivity. whereas P. kudriavzevii YBB-33 exhibited excellent alcohol tolerance. Furthermore, this systematic classification and characterization of coffee bean yeast strains from the Sidamo region can potentially uncover additional yeasts that offer high-quality resources for industrial-scale coffee bean production.

Asunto(s)

Etanol , Fermentación , Pichia , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/aislamiento & purificación , Pichia/metabolismo , Pichia/aislamiento & purificación , Pichia/genética , Pichia/clasificación , Etanol/metabolismo , Concentración de Iones de Hidrógeno , Café/microbiología , Coffea/microbiología , Temperatura , Semillas/microbiología , Sulfuro de Hidrógeno/metabolismo

RESUMEN

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Asunto(s)

Técnicas de Cultivo Celular por Lotes , Biomasa , Reactores Biológicos , Glicerol , beta-Fructofuranosidasa , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo , Reactores Biológicos/microbiología , Glicerol/metabolismo , Fermentación , Aspergillus niger/genética , Aspergillus niger/enzimología , Saccharomycetales/genética , Saccharomycetales/enzimología , Oxígeno/metabolismo , Regiones Promotoras Genéticas , Medios de Cultivo/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Oligosacáridos

RESUMEN

To reduce food spoilage and deterioration caused by microbial contamination, antimicrobial peptides (AMPs) have gradually gained attention as a biological preservative. Odorranain-C1 is an α-helical cationic antimicrobial peptide extracted from the skin of frogs with broad-spectrum antimicrobial activity. In this study, we achieved the expression of Odorranain-C1 in Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) by employing DNA recombination technology. The recombinant Odorranain-C1 showed broad-spectrum antibacterial activity and displayed a minimum inhibitory concentration within the range of 8-12 µg.mL-1. Meanwhile, Odorranain-C1 exhibited superior stability and lower hemolytic activity. Mechanistically, Odorranain-C1 disrupted the bacterial membrane's integrity, ultimately causing membrane rupture and subsequent cell death. In tilapia fillets preservation, Odorranain-C1 inhibited the total colony growth and pH variations, while also reducing the production of total volatile basic nitrogen (TVB-N) and thiobarbituric acid (TBA). In conclusion, these studies demonstrated the efficient recombinant expression of Odorranain-C1 in P. pastoris, highlighting its promising utilization in food preservation.

Asunto(s)

Conservación de Alimentos , Saccharomycetales , Animales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Conservación de Alimentos/métodos , Pruebas de Sensibilidad Microbiana , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/metabolismo , Antibacterianos/farmacología , Hemólisis/efectos de los fármacos , Pichia/genética , Pichia/metabolismo , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Proteínas Anfibias/metabolismo , Anuros/metabolismo

RESUMEN

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.

Asunto(s)

Proteínas Fúngicas , Mitocondrias , Peroxisomas , Pichia , Peroxisomas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Pichia/metabolismo , Pichia/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Peroxinas/metabolismo , Peroxinas/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Transporte de Proteínas

RESUMEN

Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L-1 with a specific activity of 96 ± 0.32 U mg-1, resulting in a total enzyme activity of 19137 ± 1131 U L-1, demonstrating significant potential for industrial applications.

Asunto(s)

Carboxipeptidasa B , Membrana Celular , Aparato de Golgi , Ingeniería de Proteínas , Proteínas Recombinantes , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ingeniería de Proteínas/métodos , Carboxipeptidasa B/genética , Carboxipeptidasa B/metabolismo , Membrana Celular/metabolismo , Membrana Celular/genética , Aparato de Golgi/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/enzimología , Saccharomycetales/genética , Saccharomycetales/enzimología , Mutación , Pichia/genética , Pichia/metabolismo , Señales de Clasificación de Proteína/genética , Transporte de Proteínas

RESUMEN

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Asunto(s)

Ingeniería Metabólica , Peroxisomas , Peroxisomas/metabolismo , Peroxisomas/genética , Ingeniería Metabólica/métodos , Señales de Direccionamiento al Peroxisoma/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo

RESUMEN

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.

Asunto(s)

6-Fitasa , Estabilidad de Enzimas , Calor , 6-Fitasa/genética , 6-Fitasa/metabolismo , 6-Fitasa/química , Concentración de Iones de Hidrógeno , Mutación , Pichia/genética , Pichia/metabolismo , Temperatura , Alimentación Animal/análisis , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química