RESUMO
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.
Assuntos
6-Fitase , Reatores Biológicos , Fermentação , Glucose , Metanol , Proteínas Recombinantes , 6-Fitase/genética , 6-Fitase/metabolismo , Glucose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Metanol/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Pichia/genética , Pichia/metabolismo , Pichia/crescimento & desenvolvimento , Expressão GênicaRESUMO
OBJECTIVE: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics. METHODS: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay. RESULTS: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100ËC and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4ËC. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells. CONCLUSION: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.
Assuntos
Conservação de Alimentos , Listeria monocytogenes , Pediocinas , Pediocinas/farmacologia , Pediocinas/genética , Animais , Conservação de Alimentos/métodos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Humanos , Antibacterianos/farmacologia , Clonagem Molecular , Suínos , Testes de Sensibilidade Microbiana , Bacteriocinas/farmacologia , Bacteriocinas/genética , Bacteriocinas/metabolismo , Pediococcus acidilactici/genética , Pediococcus acidilactici/metabolismo , Expressão Gênica , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/efeitos dos fármacosRESUMO
During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.
Assuntos
COVID-19 , Saccharomycetales , Vacinas , Humanos , COVID-19/diagnóstico , COVID-19/prevenção & controle , Teste para COVID-19 , Pichia/genética , Pichia/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Recombinantes/química , Vacinas/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos AntiviraisRESUMO
Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.
RESUMO
The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.
Assuntos
Glicerol , Saccharomycetales , Glicerol/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Regiões Promotoras Genéticas , Carbono/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: ⢠Transient hypoxia and high methanol trigger high level of recombinant protein synthesis ⢠In Pichia pastoris, pAOX1 induction is higher in flocculent cells ⢠Medium heterogeneity leads to morphological diversification.
Assuntos
Metanol , Pichia , Metanol/metabolismo , Pichia/genética , Pichia/metabolismo , Reatores Biológicos , Proteínas Recombinantes/metabolismo , HipóxiaRESUMO
BACKGROUND: Although Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products. RESULTS: The expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h. At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level. CONCLUSION: We demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.
Assuntos
Oligossacarídeos , Açúcares , Oligossacarídeos/metabolismo , Glucose , Monossacarídeos , Sacarose/metabolismo , Frutose/metabolismo , Frutanos/metabolismoRESUMO
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
Assuntos
Escorpiões , Peçonhas , Animais , Humanos , Escorpiões/química , Peçonhas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismoRESUMO
ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.
Assuntos
Isópteros , beta-Glucosidase , Animais , beta-Glucosidase/química , Celobiose/metabolismo , Isópteros/metabolismo , Pichia/metabolismo , Especificidade por Substrato , Cinética , Glucose/metabolismoRESUMO
Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbß3, responsible for the platelet aggregation and αvß3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 µM ADP, 10 nM thrombin, and 1 µg/mL collagen (IC50 of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 µM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC50 1.77 µM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.