Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.600
Filtrar
1.
Methods Mol Biol ; 2489: 115-127, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524048

RESUMO

Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an irreplaceable technique to functionalize fungal biosynthetic gene clusters and synthesize fungal natural products in various chassis organisms. This chapter describes the general method of using Pichia pastoris as a chassis host to investigate fungal biosynthetic pathways.


Assuntos
Produtos Biológicos , Saccharomycetales , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Proteínas Fúngicas/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
2.
Microb Cell Fact ; 21(1): 70, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468837

RESUMO

BACKGROUND: The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus. RESULTS: Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and 13C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83-99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species. CONCLUSIONS: By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.


Assuntos
Saccharomycetales , Xilose , Carbono/metabolismo , Fenótipo , Pichia/metabolismo , Saccharomycetales/genética , Xilose/metabolismo , Leveduras
3.
Food Microbiol ; 105: 104012, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35473973

RESUMO

Specialty coffee can be developed by the application of explicit microorganisms or starters to obtain desired fermentation. In the present study, natural fermentation (NF) of Arabica coffee was carried out spontaneously, the other set was inoculated with Pichia kudriavzevii (Y) starter culture (isolated, identified and mass cultured). The effect of microbial fermentation, metagenomics, production of functional metabolites, volatiles and their sensorial aspects were studied. The bioprocess illustrated cohesive interface of coffee nutrients and microbial communities like Mycobacterium, Acinetobacter, Gordonia, etc., in NF, Lactobacillus and Leuconostoc were prevailing in Y. The Pichia and Rhodotorula dominated in both the groups. The bioactivity of bacteria and fungi induced complex changes in physicochemical features like pH (4.2-5.2), Brix° (9.5-3.0), and metabolic transition in sugar (3.0-0.7%), alcohol (1.4-2.7%), organic acids modulating flavour precursors and organoleptics in the final brew. In the roasted bean, Y exhibited higher sugar (42%), protein (25%), polyphenol (3.5%), CGA (2.5%), caffeine (17.2%), and trigonelline (2.8%) than NF. The volatile profile exhibited increased flavour molecules like furans, ketones, and pyrazines in Y, besides lactone complexes. The organoleptics in Y were highlighted with honey, malt and berry notes. P. kudriavzevii coffee fermentation could be beneficial in specialty coffee production and enhancement of distinct characteristic flavours.


Assuntos
Café , Pichia , Café/química , Fermentação , Aromatizantes/metabolismo , Pichia/metabolismo , Açúcares
4.
Biotechnol Lett ; 44(4): 561-570, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35243590

RESUMO

With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide. The current PPT products in the market are usually a racemic mixture with two configurations, the D-type and L-type, of which only the L-PPT has the herbicidal activity. The racemic product is not atom economic, more toxic and may cause soil damage. Asymmetric synthesis of L-PPT has become a research focus in recent years, while biological synthesis methods are preferred for its character of environmental friendly and requiring less reaction steps when being compared to the chemical methods. We have developed a biological synthesis route to produce optically pure L-PPT from D,L-PPT in two steps using 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as the intermediate. In this study, we expressed the glutamate dehydrogenase and glucose dehydrogenase using Pichia pastoris as the first time. After a series of optimization, the total L-PPT yield reached 84%. The developed synthesis system showed a high potential for future industrial application. Compare to the previous plasmid-carrying-E. coli expression system, the established method may avoid antibiotic usage and provided an alternative way for industrial synthesis of optically pure L-PPT.


Assuntos
Herbicidas , Saccharomycetales , Aminobutiratos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/metabolismo
5.
PLoS One ; 17(3): e0265647, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35298551

RESUMO

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.


Assuntos
Endo-1,4-beta-Xilanases , Pichia , Bacillus , Clonagem Molecular , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Temperatura
6.
Protein Expr Purif ; 194: 106076, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35240278

RESUMO

Producing recombinant proteins with incorporated selenomethionine (SeMet) facilitates solving X-ray crystallographic structures of novel proteins. Production of SeMet labeled proteins in the yeast Pichia pastoris (syn. Komagataella phaffii) is difficult because SeMet is mildly toxic, reducing protein expression levels. To counteract this yield loss for a novel protease, Epicoccum sorghi chitinase modifying protein (Es-cmp), a novel disease promoting protease secreted by these plant pathogenic fungi, we isolated a yeast strain that secreted more protein. By comparing the expression level of 48 strains we isolated one that produced significantly more protein. This strain was found to be gene dosed, having four copies of the expression cassette. After optimization the strain produced Es-cmp in defined media with SeMet at levels nearly equal to that of the original strain in complex media. Also, we produced SeMet labeled protein for a homologous protease from the fungus Fusarium vanettenii, Fvan-cmp, by directly selecting a gene dosed strain on agar plates with increased zeocin. Linearization of plasmid with PmeI before electroporation led to high numbers of 1 mg/mL zeocin resistant clones with significantly increased expression compared to those selected on 0.1 mg/mL. The gene dosed strains expressing Es-cmp and Fvan-cmp allowed production of 8.5 and 16.8 mg of SeMet labeled protein from 500 mL shake flask cultures. The results demonstrate that selection of P. pastoris expression strains by plating after transformation on agar with 1 mg/mL zeocin rather than the standard 0.1 mg/mL directly selects gene dosed strains that can facilitate production of selenomethionine labeled proteins.


Assuntos
Quitinases , Selenometionina , Ágar/metabolismo , Ascomicetos , Quitinases/metabolismo , Citidina Monofosfato/metabolismo , Endopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Saccharomycetales , Selenometionina/metabolismo
7.
Molecules ; 27(6)2022 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-35335392

RESUMO

Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.


Assuntos
Galinhas , Transcriptoma , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Galinhas/metabolismo , Peptídeos/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Saccharomycetales
8.
J Biotechnol ; 348: 55-63, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35304164

RESUMO

Chitosanase was widely used in the production of bioactive chitooligosacchride (CHOS) due to their safety, controllability, environmental protection, and biodegradability. Studies showed that the bioactivity of CHOS is closely related to its degree of polymerization. Therefore, the production of ideal polymerized CHOS becomes our primary goal. In this study, the glycosyl hydrolase (GH) family 5 chitosanase was successfully expressed heterologously in Pichia pastoris. After 96 h of high-density fermentation, the chitosanase activity reached 90.62 U·mL-1, the protein content reached 9.76 mg·mL-1. When 2% chitosan was hydrolyzed by crude enzyme (20 U/mL), the hydrolysis rate reached 91.2% after 8 h, producing a mixture of CHOS with 2-4 desirable degrees of polymerization (DP). Then, the antioxidant activity of CHOS mixture was investigated, and the results showed that the antioxidant effect was concentration-dependent and had great application potential in the field of nutrition.


Assuntos
Quitosana , Saccharomycetales , Antioxidantes , Quitosana/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Pichia/genética , Pichia/metabolismo , Saccharomycetales/metabolismo
9.
J Agric Food Chem ; 70(8): 2664-2672, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35148078

RESUMO

Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, Komagataella phaffii (K. phaffii) was selected as the production host. We found that the K. phaffii host X33 was suitable for expressing the target protein, yielding 5.2 mg·L-1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters AOX1, GAP, and GAP(m) were compared and applied. The strain with the promoter AOX1 produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (hPDIA3) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L-1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in K. phaffii and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.


Assuntos
Lactalbumina , Saccharomycetales , Criança , Humanos , Lactalbumina/genética , Lactalbumina/metabolismo , Leite Humano , Pichia/metabolismo , Saccharomycetales/metabolismo
10.
Int J Mol Sci ; 23(3)2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35163307

RESUMO

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 ß-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)-beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize ß-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.


Assuntos
Glucuronatos/metabolismo , Glicosídeos/metabolismo , Oligossacarídeos/metabolismo , Fenóis/metabolismo , Talaromyces/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Cinética , Pichia/metabolismo , Prebióticos/microbiologia , Especificidade por Substrato , Xilanos/metabolismo , Xilose/metabolismo
11.
Methods Mol Biol ; 2446: 181-203, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35157274

RESUMO

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.


Assuntos
Saccharomycetales , Anticorpos de Domínio Único , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Saccharomycetales/metabolismo , Anticorpos de Domínio Único/metabolismo
12.
J Biotechnol ; 346: 47-51, 2022 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-35122934

RESUMO

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H2O2-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H2O2 by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H2O2-producing oxidases in P. pastoris.


Assuntos
Oxirredutases do Álcool , Peróxido de Hidrogênio , Ágar , Oxirredutases do Álcool/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales
13.
Molecules ; 27(3)2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35164064

RESUMO

Chitosanase hydrolyzes ß-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.


Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Glicosídeo Hidrolases/genética , Pichia/genética , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Estabilidade Enzimática , Fermentação , Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Appl Microbiol Biotechnol ; 106(5-6): 1905-1917, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35218387

RESUMO

Glutathione (GSH) is a metabolite that plays an important role in the fields of pharmacy, food, and cosmetics. Thus, it is necessary to increase its production to meet the demands. In this study, ScGSH1, ScGSH2, and StGshF were heterologously expressed in Pichia pastoris GS115 to realize the dual-path synthesis of GSH in yeast. To explore the effects of ATP metabolism on the synthesis of GSH, enzymes (ScADK1, PpADK1, VsVHB) of the ATP-related metabolic pathway and the energy co-substrate sodium citrate were taken into account. We found that both ScADK1 and sodium citrate had a positive influence on the synthesis of GSH. Then, a fermentation experiment in Erlenmeyer flasks was performed using the G3-SF strain (containing ScGSH1, ScGSH2, StGshF, and ScADK1), with the highest GSH titer and yield of 999.33 ± 47.26 mg/L and 91.53 ± 4.70 mg/g, respectively. Finally, the fermentation was scaled up in a 5-L fermentor, and the highest titer and yield were improved to 5680 mg/L and 45.13 mg/g, respectively, by optimizing the addition conditions of amino acids (40 mM added after 40 h). Our work provides an alternative strategy by combining dual-path synthesis with energy metabolism regulation and precursor feeding to improve GSH production. Key Points • ScGSH1, ScGSH2, and StGshF were overexpressed to achieve dual-path synthesis of GSH in yeast. • ScADK1 was overexpressed, and sodium citrate was added to increase the energy supply for GSH synthesis. • The addition conditions of amino acids were optimized to realize the efficient synthesis of GSH.


Assuntos
Reatores Biológicos , Pichia , Fermentação , Glutationa , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales
15.
Bioprocess Biosyst Eng ; 45(2): 409-424, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34999948

RESUMO

Human serum albumin (HSA) is an important therapeutic used in clinical settings for restoration of blood volume and treatment of chemotherapy induced neutropenia. Currently sourced from human serum, it carries the risk of contamination with viruses. The production of stable extracellular recombinant (r)HSA was achieved at nearly 1 g/L at shake-flask level in Pichia pastoris (syn. Komagataella phaffii) containing a three-copy containing HSA expression cassette, prepared in vitro. The HSA specific transcripts were increased by 1.82- to 2.46-fold in the three-copy containing clones indicating increased transcript levels to result in enhanced production of extracellular rHSA. The purified rHSA displayed secondary structure, zeta potential, size distribution and biological efficacy that matched with that of the commercial HSA. Cultivation strategy was developed at bioreactor level for the single HSA expression cassette containing recombinant which led to productivity of 300 mg/L/d of rHSA with minimum proteolytic cleavage.


Assuntos
Pichia , Albumina Sérica Humana , Reatores Biológicos , Humanos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Saccharomycetales , Albumina Sérica Humana/química , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismo
16.
J Microbiol Biotechnol ; 32(4): 464-472, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35001012

RESUMO

An endo-polygalacturonase (endo-PGase) exhibiting excellent performance during acidic fruit juice production would be highly attractive to the fruit juice industry. However, candidate endo-PGases for this purpose have rarely been reported. In this study, we expressed a gene from Penicillium oxalicum in Pichia pastoris. The recombinant enzyme PoxaEnPG28C had an optimal enzyme activity at pH 4.5 and 45°C and was stable at pH 3.0-6.5 and < 45°C. The enzyme had a specific activity of 4,377.65 ± 55.37 U/mg towards polygalacturonic acid, and the Km and Vmax values of PoxaEnPG28C were calculated as 1.64 g/l and 6127.45 U/mg, respectively. PoxaEnPG28C increased the light transmittance of orange, lemon, strawberry and hawthorn juice by 13.9 ± 0.3%, 29.4 ± 3.8%, 95.7 ± 10.2% and 79.8 ± 1.7%, respectively; it reduced the viscosity of the same juices by 25.7 ± 1.6%, 52.0 ± 4.5%, 48.2 ± 0.7% and 80.5 ± 2.3%, respectively, and it increased the yield of the juices by 24.5 ± 0.7%, 12.7 ± 2.2%, 48.5 ± 4.2% and 104.5 ± 6.4%, respectively. Thus, PoxaEnPG28C could be considered an excellent candidate enzyme for acidic fruit juice production. Remarkably, fruit juice production using hawthorn as an material was reported for the first time.


Assuntos
Sucos de Frutas e Vegetais , Poligalacturonase , Clonagem Molecular , Estabilidade Enzimática , Frutas/metabolismo , Concentração de Íons de Hidrogênio , Penicillium , Pichia/genética , Pichia/metabolismo , Poligalacturonase/genética , Poligalacturonase/metabolismo
17.
ACS Synth Biol ; 11(1): 297-307, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-34994189

RESUMO

Pichia pastoris (P. pastoris) is the workhorse in the commercial production of many valuable proteins. Traditionally, the regulation of gene expression in P. pastoris is achieved through induction by methanol which is toxic and flammable. The emerging optogenetic technology provides an alternative and cleaner gene regulation method. Based on the photosensitive protein EL222, we designed a novel "one-component" optogenetic system. The highest induction ratio was 79.7-fold under blue light compared to the group under darkness. After switching cells from dark to blue illumination, the system induced expression in just 1 h. Only 2 h after the system was switched back to the darkness from blue illumination, the target gene expression was inactivated 5-fold. The induction intensity of the optogenetic system is positively correlated with the dose and periodicity of blue illumination, and it has good spatial control. These results provide the first credible case of optogenetically induced protein expression in P. pastoris.


Assuntos
Regulação Fúngica da Expressão Gênica , Pichia , Metanol/metabolismo , Optogenética , Pichia/genética , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales
18.
Metab Eng ; 70: 181-195, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35091068

RESUMO

Yeasts are widely used cell factories for commercial heterologous protein production, however, specific productivities are usually tightly coupled to biomass formation. This greatly impacts production processes, which are commonly not run at the maximum growth rate, thereby resulting in suboptimal productivities. To tackle this issue, we evaluated transcriptomics datasets of the yeast Pichia pastoris (syn. Komagataella phaffii), which is known for its high secretory efficiency and biomass yield. These showed a clear downregulation of genes related to protein translation with decreasing growth rates, thus revealing the yeast translation machinery as cellular engineering target. By overexpressing selected differentially expressed translation factors, translation initiation was identified to be the main rate-limiting step. Specifically, overexpression of factors associated with the closed-loop conformation, a structure that increases stability and rates of translation initiation before start codon scanning is initiated, showed the strongest effects. Overexpression of closed-loop factors alone or in combination increased titers of different heterologous proteins by up to 3-fold in fed-batch processes. Furthermore, translation activity, correlating to the obtained secreted recombinant protein yields, selected transcript levels and total protein content were higher in the engineered cells. Hence, translation factor overexpression, globally affects the cell. Together with the observed impact on the transcriptome and total protein content, our results indicate that the capacity of P. pastoris for protein production is not at its limit yet.


Assuntos
Pichia , Biomassa , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales
19.
Methods Mol Biol ; 2433: 75-88, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34985738

RESUMO

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.


Assuntos
Pichia , Biossíntese de Proteínas , Humanos , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales
20.
Methods Mol Biol ; 2406: 401-416, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35089571

RESUMO

Purification of inclusion bodies (IBs) is gaining importance due to the raising of novel applications for these submicron particulate protein clusters, with potential uses in the biomedical and biotechnological fields among others. Here, we present five optimized methods to purify IBs adapting classical procedures to the material nature, as well as the requirements of the producer cell (Gram-negative bacteria, Gram-positive bacteria, or yeast) and the IB final application.


Assuntos
Corpos de Inclusão , Saccharomyces cerevisiae , Bactérias/metabolismo , Biotecnologia , Corpos de Inclusão/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...