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Methods in Molecular Biology (Clifton,... 2022During the past three decades, the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii) has gained widespread acceptance as a system of...
During the past three decades, the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii) has gained widespread acceptance as a system of choice for heterologous protein expression. One of the reasons that this yeast is used so frequently is the simplicity of techniques required for its molecular genetic manipulation. There are several different protocols available for introducing DNA into P. pastoris using electroporation or heat shock. We describe here a shortened protocol for cell preparation and transformation that works reliably with either prototrophic markers or antibiotic selection in this host. This procedure utilizes the most efficient portions of the electroporation and heat-shock transformation protocols to yield a method that is both time-saving and effective.
Topics: Electroporation; Heat-Shock Response; Pichia; Protein Processing, Post-Translational; Saccharomycetales
PubMed: 35781202
DOI: 10.1007/978-1-0716-2399-2_7 -
Biomolecules Feb 2023Since the mid-1960s, methylotrophic yeast (previously described as ) has received increasing scientific attention. The interest for the industrial production of... (Review)
Review
Since the mid-1960s, methylotrophic yeast (previously described as ) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with . at industrial levels by a variety of companies-describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLE™ FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)-is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported.
Topics: Humans; Pichia; Saccharomycetales; Yeasts; Recombinant Proteins
PubMed: 36979376
DOI: 10.3390/biom13030441 -
Methods in Enzymology 2021The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial...
The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial enzymes. The popularity of P. pastoris stems from its ability to grow to high cell densities, producing high titers of secreted heterologous protein with very low amounts of endogenous proteins. Its ability to express correctly folded proteins with post-translational modifications makes it an excellent candidate for the production of biopharmaceuticals. In addition, production in P. pastoris typically uses the strong, methanol-inducible and tightly regulated promoter (P), which can result in heterologous protein that constitutes up to 30% of total cell protein upon growth in methanol. In this chapter, we present methodology for the production of secreted recombinant proteins in P. pastoris, and we discuss alternatives to enhance protein production with the desired yield and quality.
Topics: Methanol; Pichia; Recombinant Proteins; Saccharomycetales
PubMed: 34742398
DOI: 10.1016/bs.mie.2021.07.004 -
International Journal of Molecular... Jan 2021The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non- yeasts such as ,... (Review)
Review
The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non- yeasts such as , , , and have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non- species. stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as is indispensable to achieve a proper fermented alcohol. The impact of is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no species other than is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future.
Topics: Bioengineering; Ecology; Fermentation; Food Quality; Industrial Microbiology; Pichia; Vitis; Wine
PubMed: 33530422
DOI: 10.3390/ijms22031196 -
FEMS Yeast Research Dec 2021The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii... (Review)
Review
The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the 'conventional' yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.
Topics: Biotechnology; Methanol; Pichia; Saccharomyces cerevisiae; Saccharomycetales
PubMed: 34849756
DOI: 10.1093/femsyr/foab059 -
Biotechnology Advances 2023Recombinant proteins (RP) are widely used as biopharmaceuticals, industrial enzymes, or sustainable food source. Yeasts, with their ability to produce complex proteins... (Review)
Review
Recombinant proteins (RP) are widely used as biopharmaceuticals, industrial enzymes, or sustainable food source. Yeasts, with their ability to produce complex proteins through a broad variety of cheap carbon sources, have emerged as promising eukaryotic production hosts. As such, the prevalence of yeasts as favourable production organisms in commercial RP production is expected to increase. Yet, with the selection of a robust production host on the one hand, successful scale-up is dependent on a thorough understanding of the challenging environment and limitations of large-scale bioreactors on the other hand. In the present work, several prominent yeast species, including Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis and Kluyveromyces marxianus are reviewed for their current state and performance in commercial RP production. Thereafter, the impact of principal process control parameters, including dissolved oxygen, pH, substrate concentration, and temperature, on large-scale RP production are discussed. Finally, technical challenges of process scale-up are identified. To that end, process intensification strategies to enhance industrial feasibility are summarized, specifically highlighting fermentation strategies to ensure sufficient cooling capacity, overcome oxygen limitation, and increase protein quality and productivity. As such, this review aims to contribute to the pursuit of sustainable yeast-based RP production.
Topics: Yeasts; Recombinant Proteins; Saccharomyces cerevisiae; Bioreactors; Yarrowia; Fermentation; Pichia
PubMed: 36775001
DOI: 10.1016/j.biotechadv.2023.108121 -
Letters in Applied Microbiology Sep 2023Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming...
Film-forming yeasts are potential sources of defects in alcoholic beverages. The aim of this study is to assess the growth capacity of Pichia and Candida film-forming yeasts in cider and wine and the effects on their chemical composition. Cider, partially and fully fermented wine were inoculated with strains of C. californica, P. fermentans, P. kluyveri, P. kudriavzevii, P. manshurica, and P. membranifaciens to simulate a post-fermentative contamination. The former three species grew only in cider. Pichia manshurica and P. kudriavzevii displayed high viability in wine up to 13.18% (v v-1) ethanol. Significant changes in odour-active molecules from different chemical groups were observed in cider and wine in the inoculated samples, compared to the non-inoculated ones. Cider is more susceptible to contamination by all of the species tested, due to its low alcohol content, while P. membranifaciens, P. manshurica, and P. kudriavzevii are additionally potential spoilage agents of wine. This study highlights the risk of cider and wine contamination by film-forming yeasts. Their impact on aroma profiles depends on their ability to grow and their metabolism. This study contributes to an understanding of the possible physiological and metabolic mechanisms responsible for film formation and chemical changes in alcoholic beverages.
Topics: Wine; Fermentation; Pichia; Alcoholic Beverages; Candida
PubMed: 37656878
DOI: 10.1093/lambio/ovad099 -
Biotechnology Advances 2019Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins,... (Review)
Review
Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.
Topics: Industrial Microbiology; Pichia; Protein Engineering; Recombinant Proteins; Silk
PubMed: 30902728
DOI: 10.1016/j.biotechadv.2019.03.012 -
Methods in Molecular Biology (Clifton,... 2022Pichia pastoris (syn. Komagataella phaffii) is an industrially relevant recombinant protein platform that has been used to produce over 5000 proteins to date. Cell-free...
Pichia pastoris (syn. Komagataella phaffii) is an industrially relevant recombinant protein platform that has been used to produce over 5000 proteins to date. Cell-free protein synthesis can be used as a screening tool before strain development or for the production of proteins that are difficult or toxic to make in vivo. Here we describe the methods for generating an active cell lysate from P. pastoris using high pressure homogenization and an improved reaction mix which results in high yields of reporter proteins such as luciferase, and complex proteins such as human serum albumin and virus-like particles.
Topics: Humans; Pichia; Protein Biosynthesis; Recombinant Proteins; Saccharomycetales
PubMed: 34985738
DOI: 10.1007/978-1-0716-1998-8_4 -
Methods in Molecular Biology (Clifton,... 2022The knowledge of certain strain-specific parameters of recombinant Pichia pastoris strains is required to be able to set up a feeding regime for fed-batch cultivations....
The knowledge of certain strain-specific parameters of recombinant Pichia pastoris strains is required to be able to set up a feeding regime for fed-batch cultivations. These parameters are commonly determined either by time-consuming and labor-intensive continuous cultivations or by several, consecutive fed-batch cultivations. Here, we describe a fast method based on batch experiments with substrate pulses to extract certain strain characteristic parameters, which are required to set up a dynamic feeding strategy for P. pastoris strains based on the specific substrate uptake rate. We further describe in detail the course of actions, which have to be taken to obtain the desired dynamics during feeding.
Topics: Pichia; Recombinant Proteins; Saccharomycetales
PubMed: 35781209
DOI: 10.1007/978-1-0716-2399-2_14