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Journal of Cellular Physiology Sep 2020One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression... (Review)
Review
One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.
Topics: Fungal Proteins; Proprotein Convertases; Protein Biosynthesis; Proteomics; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Saccharomycetales
PubMed: 32057111
DOI: 10.1002/jcp.29583 -
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 -
Bioresources and Bioprocessing Jun 2023Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino...
Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K and Mg) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.
PubMed: 38647944
DOI: 10.1186/s40643-023-00653-4 -
Biotechnology For Biofuels and... Dec 2022Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia... (Review)
Review
Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia pastoris has become the leading eukaryotic host organism for bioproduct generation. Most of the P. pastoris bioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeast P. pastoris bioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential of P. pastoris for the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generation P. pastoris platforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated for P. pastoris cell growth promoting effects and recombinant protein production. Further metabolic pathway engineering of P. pastoris to construct renewable and low cost substrate utilization pathways was discussed. Although, second generation P. pastoris bioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering of P. pastoris will pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns. P. pastoris high tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generation P. pastoris strain design and bioprocess engineering, in the years to come.
PubMed: 36581872
DOI: 10.1186/s13068-022-02234-7 -
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 -
Frontiers in Microbiology 2022(syn. spp.) has attracted extensive attention as an efficient platform for recombinant protein (RP) production. For obtaining a higher protein titer, many researchers... (Review)
Review
(syn. spp.) has attracted extensive attention as an efficient platform for recombinant protein (RP) production. For obtaining a higher protein titer, many researchers have put lots of effort into different areas and made some progress. Here, we summarized the most recent advances of the last 5 years to get a better understanding of its future direction of development. The appearance of innovative genetic tools and methodologies like the CRISPR/Cas9 gene-editing system eases the manipulation of gene expression systems and greatly improves the efficiency of exploring gene functions. The integration of novel pathways in microorganisms has raised more ideas of metabolic engineering for enhancing RP production. In addition, some new opportunities for the manufacture of proteins have been created by the application of novel mathematical models coupled with high-throughput screening to have a better overview of bottlenecks in the biosynthetic process.
PubMed: 36504810
DOI: 10.3389/fmicb.2022.1059777 -
Journal of Fungi (Basel, Switzerland) Oct 2023is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell... (Review)
Review
is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell factory for the production of chemicals and natural products. In this review, the general physiological properties of are summarized and the readily available genetic tools and elements are described, including strains, expression vectors, promoters, gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and adaptive laboratory evolution. Moreover, the recent achievements in -based biosynthesis of proteins, natural products, and other compounds are highlighted. The existing issues and possible solutions are also discussed for the construction of efficient cell factories.
PubMed: 37888283
DOI: 10.3390/jof9101027 -
International Journal of Environmental... Mar 2022A variety of flocculants have been used to aggregate colloidal substances. However, recently, owing to the adverse effects and high costs of conventional flocculants,...
A variety of flocculants have been used to aggregate colloidal substances. However, recently, owing to the adverse effects and high costs of conventional flocculants, natural flocculants such as microbial flocculants are gaining attention. The aim of the study was to produce and characterize a bioflocculant from MH545928.1 and apply it in wastewater treatment. A mixture of butanol and chloroform (5:2 /) was used to extract the bioflocculant. Phenol-sulphuric acid, Bradford and Carbazole assays were utilized for the identification of carbohydrates, proteins and uronic acid, respectively. Scanning electron microscopy (SEM) and elemental detector were employed to determine the surface morphology and elemental compositions. The removal efficiencies were 73%, 49% and 47% for BOD, COD and P, respectively. The bioflocculant (2.836 g/L) obtained showed the presence of carbohydrates (69%), protein (11%) and uronic acid (16%). The bioflocculant displayed a cumulus-like structure and the elemental composition of C (16.92%), N (1.03%), O (43:76%), Na (0.18%), Mg (0.40%), Al (0.80%), P (14.44%), S (1.48%), Cl (0.31%), K (0.34%) and Ca (20.35). It showed the removal efficiencies of 43% (COD), 64% (BOD), 73% (P) and 50% (N) in coal mine wastewater. This bioflocculant is potentially viable to be used in wastewater treatment.
Topics: Flocculation; Hydrogen-Ion Concentration; Pichia; Uronic Acids; Wastewater; Water Purification
PubMed: 35328836
DOI: 10.3390/ijerph19063148 -
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 -
Endogenous Ethanol and Triglyceride Production by Gut , and Yeasts in Non-Alcoholic Steatohepatitis.Cells Oct 2022Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably...
Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing (HiAlc Kpn). yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05-1.43] vs. 0.034 [0.008-0.57], = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, = 0.0001). was the most frequent (four patients), while , and were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49-5.60] vs. 0.32 [0.009-0.43], = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to , gut and yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production.
Topics: Humans; Pichia; Ethanol; Non-alcoholic Fatty Liver Disease; Candida albicans; Candida glabrata; Triglycerides; Candida; Fructose
PubMed: 36359786
DOI: 10.3390/cells11213390