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Toxins Nov 2022Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial...
Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene encoding carboxylesterase (CarE) into pPICZαA and transformed it into GS115. The target protein was successfully obtained, and CarE could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) / ratios of 560.15, 600.25, and 713.46. The molecular formula was CHO, CHNO, and CHNO. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with / of 488.26 and molecular formula CHO. These properties render the CarE a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.
Topics: Animals; Carboxylesterase; Gossypol; Moths; Pichia; Biotransformation; Recombinant Proteins
PubMed: 36548713
DOI: 10.3390/toxins14120816 -
BMC Genomics Aug 2016Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein... (Comparative Study)
Comparative Study
BACKGROUND
Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species.
RESULTS
Here, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts.
CONCLUSIONS
Our studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.
Topics: Alternative Splicing; DNA, Fungal; Gene Expression Profiling; Genomics; Molecular Sequence Annotation; Mutation; Pichia; Species Specificity
PubMed: 27495311
DOI: 10.1186/s12864-016-2876-y -
Marine Drugs Jun 2020Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides...
Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in . The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2-4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.
Topics: Alginates; Animals; Cloning, Molecular; Pichia; Polysaccharide-Lyases; Temperature
PubMed: 32545157
DOI: 10.3390/md18060305 -
Microbial Cell Factories Dec 2013The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity.... (Review)
Review
The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potential success of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increase recombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the first reported use of multi-copy clones and it is still an active area of research to find the fastest and most efficient method for generating these strains. It has also become apparent that there is not always a linear correlation between copy number and protein titer, leading to in-depth investigations into how to minimize the negative impact of secretory stress and achieve clonal stability.
Topics: Clone Cells; Gene Dosage; Gene Expression Regulation, Fungal; Pichia; Unfolded Protein Response
PubMed: 24354594
DOI: 10.1186/1475-2859-12-128 -
Journal of Microbiology and... May 2013During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA... (Review)
Review
During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.
Topics: Antibodies; Humans; Industrial Microbiology; Membrane Proteins; Pichia; Recombinant Proteins
PubMed: 23648847
DOI: 10.4014/jmb.1210.10063 -
FEMS Microbiology Reviews Aug 2002Nitrate assimilation has received much attention in filamentous fungi and plants but not so much in yeasts. Recently the availability of classical genetic and molecular... (Review)
Review
Nitrate assimilation has received much attention in filamentous fungi and plants but not so much in yeasts. Recently the availability of classical genetic and molecular biology tools for the yeast Hansenula polymorpha has allowed the advance of the study of this metabolic pathway in yeasts. The genes YNT1, YNR1 and YNI1, encoding respectively nitrate transport, nitrate reductase and nitrite reductase, have been cloned, as well as two other genes encoding transcriptional regulatory factors. All these genes lie closely together in a cluster. Transcriptional regulation is the main regulatory mechanism that controls the levels of the enzymes involved in nitrate metabolism although other mechanisms may also be operative. The process involved in the sensing and signalling of the presence of nitrate in the medium is not well understood. In this article the current state of the studies of nitrate assimilation in yeasts as well as possible venues for future research are reviewed.
Topics: Anion Transport Proteins; Gene Expression Regulation, Fungal; Nitrate Reductases; Nitrate Transporters; Nitrates; Nitrite Reductases; Nitrites; Pichia; Transcription Factors
PubMed: 12165428
DOI: 10.1111/j.1574-6976.2002.tb00615.x -
Brazilian Journal of Microbiology :... Nov 2018Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are... (Review)
Review
Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.
Topics: Carbon; Industrial Microbiology; Pichia; Promoter Regions, Genetic; Recombinant Proteins; Temperature
PubMed: 29858140
DOI: 10.1016/j.bjm.2018.03.010 -
BMC Genomics May 2022Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole...
BACKGROUND
Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).
RESULTS
The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.
CONCLUSIONS
This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA-protein crosslinking.
Topics: Formaldehyde; Glutathione; Metabolome; Methanol; Pichia; Saccharomycetales; Transcriptome
PubMed: 35549850
DOI: 10.1186/s12864-022-08592-8 -
PloS One 2023Oxalate oxidase is an enzyme that degrades oxalate and is used in commercial urinary assays to measure oxalate levels. The objective of this study was to establish an...
Oxalate oxidase is an enzyme that degrades oxalate and is used in commercial urinary assays to measure oxalate levels. The objective of this study was to establish an enhanced expression system for secretion and purification of oxalate oxidase using Pichia pastoris. A codon optimized synthetic oxalate oxidase gene derived from Hordeum vulgare (barley) was generated and cloned into the pPICZα expression vector downstream of the N-terminal alpha factor secretion signal peptide sequence and used for expression in P. pastoris X-33 strain. A novel chimeric signal peptide consisting of the pre-OST1 sequence fused to pro-αpp8 containing several amino acid substitutions was also generated to enhance secretion. Active enzyme was purified to greater than 90% purity using Q-Sepharose anion exchange chromatography. The purified oxalate oxidase enzyme had an estimated Km value of 256μM, and activity was determined to be 10U/mg. We have developed an enhanced oxalate oxidase expression system and method for purification.
Topics: Hordeum; Pichia; Protein Sorting Signals; Oxalates; Recombinant Proteins
PubMed: 37167324
DOI: 10.1371/journal.pone.0285556 -
Brazilian Journal of Microbiology :... Dec 2013Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins.... (Review)
Review
Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.
Topics: Biotechnology; Health Care Sector; Humans; Industrial Microbiology; Organisms, Genetically Modified; Pichia; Technology, Pharmaceutical
PubMed: 24688491
DOI: 10.1590/s1517-83822013000400004