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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 -
Applied Microbiology and Biotechnology Jun 2014Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a... (Review)
Review
Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.
Topics: Gene Expression; Industrial Microbiology; Pichia; Recombinant Proteins
PubMed: 24743983
DOI: 10.1007/s00253-014-5732-5 -
Microbial Cell Factories Jul 2023Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of...
BACKGROUND
Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers.
RESULTS
Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system.
CONCLUSION
Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.
Topics: Saccharomycetales; Pichia; Biological Products; Fermentation; Methanol
PubMed: 37481525
DOI: 10.1186/s12934-023-02132-z -
Biological Chemistry Jun 2018Protein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that... (Review)
Review
Protein crystallization in living cells has been observed surprisingly often as a native assembly process during the past decades, and emerging evidence indicates that this phenomenon is also accessible for recombinant proteins. But only recently the advent of high-brilliance synchrotron sources, X-ray free-electron lasers, and improved serial data collection strategies has allowed the use of these micrometer-sized crystals for structural biology. Thus, in cellulo crystallization could offer exciting new possibilities for proteins that do not crystallize applying conventional approaches. In this review, we comprehensively summarize the current knowledge of intracellular protein crystallization. This includes an overview of the cellular functions, the physical properties, and, if known, the mode of regulation of native in cellulo crystal formation, complemented with a discussion of the reported crystallization events of recombinant proteins and the current method developments to successfully collect X-ray diffraction data from in cellulo crystals. Although the intracellular protein self-assembly mechanisms are still poorly understood, regulatory differences between native in cellulo crystallization linked to a specific function and accidently crystallizing proteins, either disease associated or recombinantly introduced, become evident. These insights are important to systematically exploit living cells as protein crystallization chambers in the future.
Topics: Animals; Bacteria; Cell Survival; Crystallography, X-Ray; Humans; Muscle, Skeletal; Oocytes; Particle Size; Pichia; Proteins; Surface Properties
PubMed: 29894295
DOI: 10.1515/hsz-2018-0158 -
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 -
Metabolic Engineering Nov 2018Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a... (Review)
Review
Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a platform to produce metabolites of varying origin. We summarize here the impressive methodological developments of the last years to model and analyze the metabolism of P. pastoris, and to engineer its genome and metabolic pathways. Efficient methods to insert, modify or delete genes via homologous recombination and CRISPR/Cas9, supported by modular cloning techniques, have been reported. An outstanding early example of metabolic engineering in P. pastoris was the humanization of protein glycosylation. More recently the cell metabolism was engineered also to enhance the productivity of heterologous proteins. The last few years have seen an increased number of metabolic pathway design and engineering in P. pastoris, mainly towards the production of complex (secondary) metabolites. In this review, we discuss the potential role of P. pastoris as a platform for metabolic engineering, its strengths, and major requirements for future developments of chassis strains based on synthetic biology principles.
Topics: CRISPR-Cas Systems; Metabolic Engineering; Pichia
PubMed: 29704654
DOI: 10.1016/j.ymben.2018.04.017 -
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 -
Toxins Mar 2017The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of... (Review)
Review
The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-β-d-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing (inhibition of , , etc.).
Topics: Humans; Killer Factors, Yeast; Pichia
PubMed: 28333108
DOI: 10.3390/toxins9040112 -
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 -
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