scholarly journals Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Xueyun Zheng ◽  
Yimin Zhang ◽  
Xinying Zhang ◽  
Cheng Li ◽  
Xiaoxiao Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Large Subunit ◽  
Protein A ◽  
Methylotrophic Yeast ◽  
Regulation Mechanism ◽  
Heterologous Proteins ◽  
Foreign Proteins

Abstract Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

scholarly journals Ktr1p is an α-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris

1997 ◽  
Vol 321 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Pedro A. ROMERO ◽  
Marc LUSSIER ◽  
Anne-Marie SDICU ◽  
Howard BUSSEY ◽  
Annette HERSCOVICS
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Methylotrophic Yeast ◽  
Secreted Proteins ◽  
Yeast Genome ◽  
Amino Acid Similarity ◽  
Substrate Specificities ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Absolute Requirement

The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the α1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx. 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit α1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar Km for mannose (280Ő350 mM), methyl-α-mannoside (60Ő90 mM) and GDP-mannose (50Ő90 ƁM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-α-mannoside, α-1,2-mannobiose and methyl-α-1,2-mannobiose, as well as Man15Ő30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize α-1,6-mannobiose, α-1,6-mannotriose, α-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9Ő10GlcNAc. These results indicate that Kre2p/Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.

scholarly journals Evaluación del tiempo de inducción y la concentración de metanol en la expresión de L-asparaginasa II de Saccharomyces cerevisiae usando Pichia pastoris (Muts)

2018 ◽  
pp. 131-134
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Induction Time ◽  
Lymphoblastic Leukemia ◽  
Methylotrophic Yeast ◽  
Culture Conditions ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Low Concentrations ◽  
Eukaryotic Organisms

Evaluación del tiempo de inducción y la concentración de metanol en la expresión de L-asparaginasa II de Saccharomyces cerevisiae usando Pichia pastoris (Muts) Omar Pillaca-Pullo, Karin Torres, Marcela Pimenta, Adalberto Pessoa-Jr & Michele Vitolo Facultad de Ciencias Farmacéuticas – Universidad de São Paulo (Brasil), 0550-8000 DOI: https://doi.org/10.33017/RevECIPeru2015.0020/ Resumen La levadura metilotrófica Pichia pastoris es ampliamente usada como un sistema eucariota para expresar proteínas recombinantes. Más de 500 proteínas recombinantes fueron expresas por P. pastoris con niveles de expresión que alcanzan hasta el 80% de proteínas totales secretadas y hasta 30% de proteínas totales de la célula. Existen tres fenotipos de P. pastoris clasificados de acuerdo con su capacidad de metabolizar metanol, el fenotipo MutS crece lentamente en medios con metanol por lo que generalmente se usan bajas concentraciones de metanol y tiempo de inducción prolongados. Por esta razón, el control de las condiciones de cultura como la concentración del inductor y el tiempo de inducción son factores importantes tanto para el crecimiento de la levadura como para producción de la proteína ya que este sistema es controlado por el promotor AOX inducido con metanol. Por otro lado, L-asparaginase (EC. 3.5.1.1) es un importante biofármaco usado en el tratamiento de leucemia linfoblástica aguda (ALL), la enzima comúnmente utilizada en la terapéutica es procedente de bacterias, estas han demostrado buena actividad, pero causan muchas reacciones inmunológicas severas en los pacientes tratados. La búsqueda de L-asparaginasa procedente y expresada en organismos eucariotas se abre como una posibilidad para reducir las reacciones adversas. En este estudio fueron evaluados el tiempo de inducción (24 - 120 horas) y la concentración de inductor (0.25, 0.5 y 1.0%). Los datos mostraron que la condición de mayor expresión de L-asparaginasa II  de Saccharomyces cerevisiae después de 48 horas de inducción con 1,0% de metanol (~ 25 U.g-1). Finalmente se recomienda evaluar dicha producción en biorreactor donde se lleve un control adecuado de otras variables importantes como el pH del cultivo y la concentración de oxígeno en el medio. Descriptores: Pichia pastoris, Saccharomyces cerevisiae, L-asparaginase, Metanol.  Abstract The methylotrophic yeast Pichia pastoris is widely used as a eukaryotic system for expressing recombinant proteins. Over 500 recombinant proteins were expressed in P. pastoris with expression levels reaching up to 80% of total secreted proteins and up to 30% total cell proteins. There are three phenotypes of P. pastoris classified according to their ability to metabolize methanol, phenotype MutS grows slowly on media containing methanol at generally low concentrations of methanol and longer induction time are used. Therefore, the control of culture conditions such as concentration of the inducer and the induction time are important factors for both yeast growth and for production of the protein since this system is controlled by the AOX promoter induced with methanol. Furthermore, L-asparaginase (EC. 3.5.1.1) is an important biopharmaceutical used to treat acute lymphoblastic leukemia (ALL), the enzyme commonly used in the therapy is from bacteria, these have shown good activity but cause many severe immune reactions in patients. The search for L-asparaginase derived and expressed in eukaryotic organisms opens a possibility to reduce adverse reactions. In this study they were evaluated the induction time (24-120 hours) and inducer concentration (0.25, 0.5 and 1.0% v/v). The data showed that the condition of increased expression of L-asparaginase II from Saccharomyces cerevisiae after 48 hours’ induction with 1.0% methanol (~ 25 U.g-1). Finally, it is recommended to evaluate this production in bioreactor where adequate control of other important variables such as pH of the culture and the concentration of oxygen in the medium is carried. Keywords: Pichia pastoris, Saccharomyces cerevisiae, L-asparaginase, Metanol.

Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

2021 ◽  
Author(s):  
Sonakshi De ◽  
Diethard Mattanovich ◽  
Pau Ferrer ◽  
Brigitte Gasser
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Protein Characterization ◽  
Pathway Engineering ◽  
Small Scale ◽  
Cell Factory ◽  
Scale Production ◽  
Heterologous Proteins ◽  
Metabolic Pathway Engineering

Abstract Besides bakers’ yeast, the methylotrophic yeast Komagataella phaffii (also known as Pichia pastoris) has been developed into the most popular yeast cell factory for the production of heterologous proteins. Strong promoters, stable genetic constructs and a growing collection of freely available strains, tools and protocols have boosted this development equally as thorough genetic and cell biological characterization. This review provides an overview of state-of-the-art tools and techniques for working with P. pastoris, as well as guidelines for the production of recombinant proteins with a focus on small-scale production for biochemical studies and protein characterization. The growing applications of P. pastoris for in vivo biotransformation and metabolic pathway engineering for the production of bulk and specialty chemicals are highlighted as well.

scholarly journals The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris

2018 ◽  
Vol 293 (43) ◽  
pp. 16647-16660 ◽  
Author(s):  
Trishna Dey ◽  
Kamisetty Krishna Rao ◽  
Jesminara Khatun ◽  
Pundi N. Rangarajan
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Signaling Pathway ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Leucine Zipper ◽  
Alcohol Oxidase ◽  
Transcriptional Regulator ◽  
Methylotrophic Yeast ◽  
Methanol Metabolism ◽  
Methylotrophic Yeast Pichia Pastoris

Rtg1p and Rtg3p are two basic helix–loop–helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae. Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris. We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris. We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

scholarly journals Catabolite Repression of Aox in Pichia pastoris Is Dependent on Hexose Transporter PpHxt1 and Pexophagy

2010 ◽  
Vol 76 (18) ◽  
pp. 6108-6118 ◽  
Author(s):  
Ping Zhang ◽  
Wenwen Zhang ◽  
Xiangshan Zhou ◽  
Peng Bai ◽  
James M. Cregg ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Catabolite Repression ◽  
Alcohol Oxidase ◽  
Sugar Metabolism ◽  
Methylotrophic Yeast ◽  
Degradation Pathway ◽  
Hexose Transporter ◽  
Heterologous Proteins ◽  
Induced Expression ◽  
Transcript Levels

ABSTRACT In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an Δhxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris

Gene ◽  
1997 ◽  
Vol 190 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Koti Sreekrishna ◽  
Robert G. Brankamp ◽  
Keith E. Kropp ◽  
Dale T. Blankenship ◽  
Jiu-Tsair Tsay ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Optimal Synthesis ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris
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