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There is a growing market for craft beverages with unique flavors. This study aimed to obtain a palate-pleasing mead derived from Pichia kudriavzevii 4A as a monoculture. Different culture media were evaluated to compare the fermentation kinetics and final products. The crucial factors in the medium were ~200 mg L-1 of yeast assimilable nitrogen and a pH of 3.5-5.0. A panel of judges favored the mead derived from Pichia kudriavzevii 4A (fermented in a medium with honey initially at 23 °Bx) over a commercial sample produced from Saccharomyces cerevisiae, considering its appearance, fruity and floral flavors (provided by esters, aldehydes, and higher alcohols), and balance between sweetness (given by the 82.91 g L-1 of residual sugars) and alcohol. The present mead had an 8.57% v/v ethanol concentration, was elaborated in 28 days, and reached a maximum biomass growth (2.40 g L-1) on the same fermentation day (6) that the minimum level of pH was reached. The biomass growth yield peaked at 24 and 48 h (~0.049 g g-1), while the ethanol yield peaked at 24 h (1.525 ± 0.332 g g-1), in both cases declining thereafter. The Gompertz model adequately describes the kinetics of sugar consumption and the generation of yeast biomass and ethanol. Pathogenic microorganisms, methanol, lead, and arsenic were absent in the mead. Thus, Pichia kudriavzevii 4A produced a safe and quality mead with probable consumer acceptance.
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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.
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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.
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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
Palm kernel cake (PKC), a byproduct of palm oil extraction, serves an important role in Ecuador's animal feed industry. The emergence of yellow-orange fungal growth in PKC on some cattle farms in Ecuador sparked concerns within the cattle industry regarding a potential mycotoxin-producing fungus on this substrate. Due to the limited availability of analytical chemistry techniques in Ecuador for mycotoxin detection, we chose to isolate and identify the fungus to determine its association with mycotoxin-producing genera. Through molecular identification via ITS region sequencing, we identified the yellow-orange fungus as the yeast Candida ethanolica. Furthermore, we isolated two other fungi-the yeast Pichia kudriavzevii, and the fungus Geotrichum candidum. Molecular identification confirmed that all three species are not classified as mycotoxin-producing fungi but in contrast, the literature indicates that all three have demonstrated antifungal activity against Aspergillus and Penicillium species, genera associated with mycotoxin production. This suggests their potential use in biocontrol to counter the colonization of harmful fungi. We discuss preventive measures against the fungal invasion of PKC and emphasize the importance of promptly identifying fungi on this substrate. Rapid recognition of mycotoxin-producing and pathogenic genera holds the promise of mitigating cattle intoxication and the dissemination of mycotoxins throughout the food chain.
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Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50 °C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50 °C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50 °C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.
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Pichia , Madeira , Xilanos , Madeira/microbiologia , Xilanos/metabolismo , Candida tropicalis/genética , Candida tropicalis/metabolismo , Peptonas/metabolismo , Fermentação , Leveduras , Carbono/metabolismo , Nitrogênio/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismoRESUMO
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.
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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.
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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.
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Glicerol , Saccharomycetales , Glicerol/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Regiões Promotoras Genéticas , Carbono/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
There is an expanding market for beer of different flavors. This study aimed to prepare a craft Belgian-style pale ale with a non-Saccharomyces yeast. Pichia kudriavzevii 4A was used as a sole starter culture, and malted barley as the only substrate. The ingredients and brewing process were carefully monitored to ensure the quality and innocuousness of the beverage. During fermentation, the yeast consumed 89.7% of total sugars and produced 13.8% v/v of ethanol. The product was fermented and then aged for 8 days, adjusted to 5% v/v alcohol, and analyzed. There were no traces of mycotoxins, lead, arsenic, methanol, or microbiological contamination that would compromise consumer health. According to the physicochemical analysis, the final ethanol concentration (5.2% v/v) and other characteristics complied with national and international guidelines. The ethyl acetate and isoamyl alcohol present are known to confer sweet and fruity flavors. The sensory test defined the beverage as refreshing and as having an apple and pear flavor, a banana aroma, and a good level of bitterness. The judges preferred it over a commercial reference sample of Belgian-style pale ale made from S. cerevisiae. Hence, P. kudriavzevii 4A has the potential for use in the beer industry.
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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.