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Current Opinion in Biotechnology Dec 2013Biopharmaceuticals are an integral part of modern medicine and pharmacy. Both, the development and the biotechnological production of biopharmaceuticals are highly... (Review)
Review
Biopharmaceuticals are an integral part of modern medicine and pharmacy. Both, the development and the biotechnological production of biopharmaceuticals are highly cost-intensive and require suitable expression systems. In this review we discuss established and emerging tools for reengineering the methylotrophic yeast Pichia pastoris for biopharmaceutical production. Recent advancements of this industrial expression system through synthetic biology include synthetic promoters to avoid methanol induction and to fine-tune protein production. New platform strains and molecular cloning tools as well as in vivo glycoengineering to produce humanized glycoforms have made P. pastoris an important host for biopharmaceutical production.
Topics: Animals; Biological Products; Biotechnology; Glycoproteins; Glycosylation; Humans; Methanol; Pichia; Promoter Regions, Genetic; Synthetic Biology
PubMed: 23522654
DOI: 10.1016/j.copbio.2013.02.024 -
PloS One 2022Xylanase is one of industrial enzymes with diverse applications including the paper-bleaching industry and feed additives. Here, a strain having xylanolytic activity and...
Xylanase is one of industrial enzymes with diverse applications including the paper-bleaching industry and feed additives. Here, a strain having xylanolytic activity and identified as Bacillus sonorensis T6 was isolated from soil. A secretory enzyme was identified by mass-spectrometry as a xylanase of glycosyl hydrolase family 11, with a molecular weight of 23.3 kDa. The xylanase gene of Bacillus sonorensis T6 was cloned and expressed in Escherichia coli (yielding an enzyme designated as rXynT6-E) and in Pichia pastoris (yielding rXynT6-P). The recombinant xylanases were found to have optimal activity at 47-55°C and pH 6.0-7.0. The recombinant xylanase expressed in P. pastoris has 40% higher thermal stability than that expressed in E. coli. The recombinant xylanases retained 100% of activity after 10 h incubation in the pH range 3-11 and 68% of activity after 1 h at pH 2.0. The xylanase activities of rXynT6-E and rXynT6-P under optimal conditions were 1030.2 and 873.8 U/mg, respectively. The good stability in a wide range of pH and moderate temperatures may make the xylanase from Bacillus sonorensis T6 useful for various biotechnological applications, e.g., as an enzyme additive in the feed industry.
Topics: Bacillus; Cloning, Molecular; Endo-1,4-beta Xylanases; Enzyme Stability; Escherichia coli; Hydrogen-Ion Concentration; Pichia; Recombinant Proteins; Temperature
PubMed: 35298551
DOI: 10.1371/journal.pone.0265647 -
BMC Systems Biology Apr 2012Pichia stipitis and Pichia pastoris have long been investigated due to their native abilities to metabolize every sugar from lignocellulose and to modulate methanol...
BACKGROUND
Pichia stipitis and Pichia pastoris have long been investigated due to their native abilities to metabolize every sugar from lignocellulose and to modulate methanol consumption, respectively. The latter has been driving the production of several recombinant proteins. As a result, significant advances in their biochemical knowledge, as well as in genetic engineering and fermentation methods have been generated. The release of their genome sequences has allowed systems level research.
RESULTS
In this work, genome-scale metabolic models (GEMs) of P. stipitis (iSS884) and P. pastoris (iLC915) were reconstructed. iSS884 includes 1332 reactions, 922 metabolites, and 4 compartments. iLC915 contains 1423 reactions, 899 metabolites, and 7 compartments. Compared with the previous GEMs of P. pastoris, PpaMBEL1254 and iPP668, iLC915 contains more genes and metabolic functions, as well as improved predictive capabilities. Simulations of physiological responses for the growth of both yeasts on selected carbon sources using iSS884 and iLC915 closely reproduced the experimental data. Additionally, the iSS884 model was used to predict ethanol production from xylose at different oxygen uptake rates. Simulations with iLC915 closely reproduced the effect of oxygen uptake rate on physiological states of P. pastoris expressing a recombinant protein. The potential of P. stipitis for the conversion of xylose and glucose into ethanol using reactors in series, and of P. pastoris to produce recombinant proteins using mixtures of methanol and glycerol or sorbitol are also discussed.
CONCLUSIONS
In conclusion the first GEM of P. stipitis (iSS884) was reconstructed and validated. The expanded version of the P. pastoris GEM, iLC915, is more complete and has improved capabilities over the existing models. Both GEMs are useful frameworks to explore the versatility of these yeasts and to capitalize on their biotechnological potentials.
Topics: Biomass; Carbon; Fermentation; Genome, Fungal; Genomics; Models, Biological; Oxygen; Phenotype; Pichia; Recombinant Proteins; Xylose
PubMed: 22472172
DOI: 10.1186/1752-0509-6-24 -
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 -
Brazilian Journal of Microbiology :... 2018The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an...
The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14°C+1%M provided the most stable viability. By contrast, the temperature of 18°C+1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.
Topics: Culture Media; Fatty Acids, Nonesterified; Fermentation; Glycerol; Methanol; Pichia; Recombinant Proteins; Temperature
PubMed: 29705163
DOI: 10.1016/j.bjm.2018.03.008 -
Biotechnology and Bioengineering Jan 2022Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or...
Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor-intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.
Topics: Automation, Laboratory; Biomass; Bioreactors; Culture Media; Fermentation; Pichia
PubMed: 34596238
DOI: 10.1002/bit.27947 -
BMC Research Notes Sep 2019Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited,...
OBJECTIVE
Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost.
RESULTS
The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L) that could allow their direct application.
Topics: Batch Cell Culture Techniques; Esterases; Extracellular Space; Fermentation; Glucuronic Acid; Methanol; Pichia; Recombinant Proteins
PubMed: 31533815
DOI: 10.1186/s13104-019-4638-9 -
Biochemistry Aug 2016RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions,...
RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.
Topics: Pichia; RNA, Fungal; beta-Galactosidase
PubMed: 27456029
DOI: 10.1021/acs.biochem.6b00330 -
Molecules (Basel, Switzerland) Jul 2023Antibiotic resistance to pathogenic bacteria is becoming an increasing public health threat, and identifying alternatives to antibiotics would be an effective solution...
Antibiotic resistance to pathogenic bacteria is becoming an increasing public health threat, and identifying alternatives to antibiotics would be an effective solution to the problem of drug resistance. Antimicrobial peptides are small peptides produced by various organisms; they are considered to be adequate antibiotic substitutes because they have intense, broad-spectrum antibacterial activity and stability, are widely available, and target strains do not quickly develop resistance. Recent research on antimicrobial peptides has shown that they have broad potential for applications in medicine, agriculture, food, and animal feed. Turgencin A is a potent antimicrobial peptide isolated from the Arctic sea squirt. We established a His-tagged expression system for and developed a rTurgencin A using the recombinant expression in with nickel column purification. This antimicrobial peptide showed intense antimicrobial activity against Gram-positive and Gram-negative bacteria and a good stability at most temperatures and pHs, as well as in various protease and salt ion concentrations, but underwent a significant decrease in stability in high-temperature and low-pH environments. Turgencin A induced bacterial membrane rupture, resulting in content leakage and subsequent cell death. It was also shown to have low hemolytic activity. This study provides primary data for the industrial production and application of the antimicrobial peptide Turgencin A.
Topics: Animals; Anti-Bacterial Agents; Gram-Negative Bacteria; Pichia; Gram-Positive Bacteria; Antimicrobial Cationic Peptides; Bacteria; Microbial Sensitivity Tests
PubMed: 37513276
DOI: 10.3390/molecules28145405 -
Journal of Industrial Microbiology &... Feb 2023The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing...
UNLABELLED
The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products.
IMPORTANCE
The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms.
ONE-SENTENCE SUMMARY
Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.
Topics: Saccharomyces cerevisiae; Pichia; Biological Products; Dodecanol; Recombinant Proteins
PubMed: 37942557
DOI: 10.1093/jimb/kuad035