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BMC Biology Sep 2015Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia...
BACKGROUND
Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date.
RESULTS
In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation.
CONCLUSIONS
Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.
Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Glucose; Glycerol; Methanol; Pichia
PubMed: 26400155
DOI: 10.1186/s12915-015-0186-5 -
BMC Biotechnology Jun 2021Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic...
BACKGROUND
Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others.
METHODS
A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K.
RESULTS
In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an E value of 41.40 KJ mol, a low variation of K and V, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K.
CONCLUSIONS
Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.
Topics: Enzyme Stability; Fungal Proteins; Kinetics; Laccase; Molecular Dynamics Simulation; Pichia; Recombinant Proteins
PubMed: 34088291
DOI: 10.1186/s12896-021-00698-3 -
Nanomedicine : Nanotechnology, Biology,... Apr 2017A genetically modified Pichia pastoris strain overexpressing a metal-resistant variant of cytochrome b5 reductase enzyme was developed for silver and selenium...
A genetically modified Pichia pastoris strain overexpressing a metal-resistant variant of cytochrome b5 reductase enzyme was developed for silver and selenium biosorption and for nanoparticle production. The maximum recombinant enzyme expression level was approximately 31 IU/ml in the intercellular fluid after 24 h of incubation, and the capacity of the recombinant biomass for the biosorption of silver and selenium in aqueous batch models were measured as 163.90 and 63.71 mg/g, respectively. The ions were reduced in the presence of enzyme, leading to the formation of stable 70-180 nm metal nanoparticles. Various instrumental analyses confirmed the well-dispersed and crystalline nature of the spherical nanometals. The purified silver and selenium nanoparticles exhibited at least 10-fold less cytotoxicity toward HDF, EPG85-257, and T47D cells than silver nitrate and selenium dioxide. These results revealed that the engineered Pichia strain is an eco-friendly, rapid, high-throughput, and versatile reduction system for nanometal production.
Topics: Biotransformation; Cytochrome-B(5) Reductase; Genetic Engineering; High-Throughput Screening Assays; Industrial Microbiology; Nanoparticles; Nanotechnology; Pichia; Selenium; Silver; Up-Regulation
PubMed: 27789260
DOI: 10.1016/j.nano.2016.10.009 -
Scientific Reports Feb 2023Following the rising concern on environmental issues caused by conventional fossil-based plastics and depleting crude oil resources, polyhydroxyalkanoates (PHAs) are of...
Following the rising concern on environmental issues caused by conventional fossil-based plastics and depleting crude oil resources, polyhydroxyalkanoates (PHAs) are of great interest by scientists and biodegradable polymer market due to their outstanding properties which include high biodegradability in various conditions and processing flexibility. Many polyhydroxyalkanoate-synthesizing microorganisms, including normal and halophilic bacteria, as well as algae, have been investigated for their performance in polyhydroxyalkanoate production. However, to the best of our knowledge, there is still limited studies on PHAs-producing marine yeast. In the present study, a halophilic yeast strain isolated from Spratly Island in Vietnam were investigated for its potential in polyhydroxyalkanoate biosynthesis by growing the yeast in Zobell marine agar medium (ZMA) containing Nile red dye. The strain was identified by 26S rDNA analysis as Pichia kudriavzevii TSLS24 and registered at Genbank database under code OL757724. The amount of polyhydroxyalkanoates synthesized was quantified by measuring the intracellular materials (predicted as poly(3-hydroxybutyrate) -PHB) by gravimetric method and subsequently confirmed by Fourier transform infrared (FTIR) spectroscopic and nuclear magnetic resonance (NMR) spectroscopic analyses. Under optimal growth conditions of 35 °C and pH 7 with supplementation of glucose and yeast extract at 20 and 10 gL, the isolated strain achieved poly(3-hydroxybutyrate) content and concentration of 43.4% and 1.8 gL after 7 days of cultivation. The poly(3-hydroxybutyrate) produced demonstrated excellent biodegradability with degradation rate of 28% after 28 days of incubation in sea water.
Topics: Polyhydroxyalkanoates; Pichia; Vietnam; Magnetic Resonance Spectroscopy
PubMed: 36823427
DOI: 10.1038/s41598-023-28220-z -
Biotechnology Journal Apr 2014Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different...
Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h(-1) . At very slow growth (μ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes.
Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Models, Biological; Pichia; Recombinant Proteins; Unfolded Protein Response
PubMed: 24323948
DOI: 10.1002/biot.201300334 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jul 2022Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue...
Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant GS115 strain capable of synthesizing chondroitin (Ch) from glycerol by introducing the Ch synthase coding genes , and UDP-glucose dehydrogenase coding gene into the . chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase (C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant . cells. Eventually, controllable synthesis of 0-40% CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.
Topics: Animals; Batch Cell Culture Techniques; Chondroitin Sulfates; Pichia; Polysaccharides; Recombinant Proteins; Saccharomycetales
PubMed: 35871627
DOI: 10.13345/j.cjb.220147 -
Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform.Biotechnology and Bioengineering Mar 2019Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free...
Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml . Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.
Topics: Biosensing Techniques; Cell-Free System; Genetic Engineering; Pichia; Protein Biosynthesis; Recombinant Proteins; Synthetic Biology
PubMed: 30552674
DOI: 10.1002/bit.26901 -
Journal of Applied Microbiology Apr 2013Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic...
AIMS
Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic acids, precursors of these phenolic compounds has been described in D. bruxellensis, no such pathway has been described in P. guilliermondii.
METHODS AND RESULTS
A molecular and physiological characterization of 14 D. bruxellensis and 15 P. guilliermondii phenol-producing strains was carried out. Both p-coumarate decarboxylase (CD) and vinyl reductase (VR) activities, responsible for the production of volatile phenols, were quantified and the production of 4-vinylphenol and 4-ethylphenol were measured. All D. bruxellensis and some P. guilliermondii strains showed the two enzymatic activities, whilst 11 of the 15 strains of this latter species showed only CD activity and did not produce 4-EP in the assay conditions. Furthermore, PCR products obtained with degenerated primers showed a low homology with the sequence of the gene for a phenyl acrylic acid decarboxylase activity described in Saccharomyces cerevisiae.
CONCLUSIONS
D. bruxellensis and P. guilliermondii may share a similar metabolic pathway for the degradation of cinnamic acids.
SIGNIFICANCE AND IMPACT OF THE STUDY
This is the first work that analyses the CD and VR activities in P. guilliermondii, and the results suggest that within this species, there are differences in the metabolization of cinnamic acids.
Topics: Carboxy-Lyases; Cinnamates; Dekkera; Food Contamination; Oxidoreductases; Phenols; Pichia; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique; Wine
PubMed: 23311591
DOI: 10.1111/jam.12134 -
BMC Infectious Diseases Jan 2018Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to...
BACKGROUND
Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and haematological malignancies.
METHODS
MALDI-TOF was performed to identify yeasts isolated from the oral cavity of 350 cancer patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3).
RESULTS
Among 162 yeasts and yeast-like fungi isolated from the oral cavity of cancer patients, Candida albicans was the most common species (50.6%), followed by Candida glabrata (24.7%), Pichia kudriavzevii (Candida krusei (9.9%)), Candida tropicalis (4.3%), Candida dubliniensis (3.7%), Kluyveromyces marxianus (Candida kefyr (3.7%)) and Candida parapsilosis (1%). In addition, uncommon yeast species i.e., Saprochaete capitata, Saccharomyces cerevisiae, Clavispora lusitaniae (C. lusitaniae) and Pichia kluyveri (C. eremophila) were recovered from oral lesions. Oral colonization by C. albicans, non-albicans Candida species and uncommon yeasts were as follow; 55%, 44% and 1%, whereas oral infection due to C. albicans was 33.3%, non-albicans Candida species 60.6%, and uncommon yeasts 6.1%. Poor oral hygiene and xerostomia were identified as independent risk factors associated with oral yeast colonization. The overall resistance to fluconazole was 11.7% (19/162). Low MIC values were observed for anidulafungin for all Candida and uncommon yeast species.
CONCLUSIONS
This current study provides insight into the prevalence and susceptibility profiles of Candida species, including emerging Candida species and uncommon yeasts, isolated from the oral cavity of Iranian cancer patients. The incidence of oral candidiasis was higher amongst patients with hematological malignancies. The majority of oral infections were caused by non-albicans Candida species which were often more resistant to anti-fungal agents. Our findings suggest that anidulafungin should be used as antifungal of choice for prophylaxis in clinically high-risk patients with documented oral colonization or infection.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antifungal Agents; Candida; Candidiasis, Oral; Female; Fluconazole; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Mouth; Neoplasms; Pichia; Prevalence; Risk Factors; Saccharomycetales; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xerostomia; Young Adult
PubMed: 29310582
DOI: 10.1186/s12879-017-2916-5 -
Nature Communications Aug 2023Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic...
Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L), yield (0.134 g g glucose) and productivity (0.511 g L h) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.
Topics: Saccharomyces cerevisiae; Pichia; Sorbic Acid
PubMed: 37652930
DOI: 10.1038/s41467-023-41064-5