scholarly journals A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Yasuyuki Nakamura ◽  
Teruyuki Nishi ◽  
Risa Noguchi ◽  
Yoichiro Ito ◽  
Toru Watanabe ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Dna Sequences ◽  
Inverted Repeat ◽  
Genetic Manipulation ◽  
Plasmid Vector ◽  
Methylotrophic Yeast ◽  
Centromeric Dna ◽  
Content Type ◽  
Repeat Sequences

ABSTRACT The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris. Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability. IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

scholarly journals Role of BGS13 in the Secretory Mechanism of Pichia pastoris

2019 ◽  
Vol 85 (24) ◽  
Author(s):  
Christopher A. Naranjo ◽  
Anita D. Jivan ◽  
Maria N. Vo ◽  
Katherine H. de Sa Campos ◽  
Jared S. Deyarmin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pichia Pastoris ◽  
Mutant Strain ◽  
Recombinant Proteins ◽  
Economic Benefits ◽  
Heterologous Protein Expression ◽  
Content Type ◽  
Kinase C

ABSTRACT The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

scholarly journals Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

2015 ◽  
Vol 112 (11) ◽  
pp. E1263-E1271 ◽  
Author(s):  
Yalin Liu ◽  
Handong Su ◽  
Junling Pang ◽  
Zhi Gao ◽  
Xiu-Jie Wang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
B Chromosome ◽  
Chromosome 9 ◽  
Centromeric Dna ◽  
Repeat Sequences ◽  
Centromeric Repeat ◽  
And Function ◽  
Centromere Assembly ◽  
Derivatives Of

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

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 The Curious Case of Nonrepetitive Centromeric DNA Sequences in Candida auris and Related Species

mBio ◽  
2021 ◽  
Author(s):  
Alexander Lorenz ◽  
Nicolas Papon
Keyword(s):  
Intensive Care ◽  
Dna Sequences ◽  
Chromosome Structure ◽  
Whole Genome ◽  
Health Issues ◽  
Candida Auris ◽  
Centromeric Dna ◽  
Content Type ◽  
Genomic Divergence ◽  
Systemic Infections

2009 saw the first description of Candida auris , a yeast pathogen of humans. C. auris has since grown into a global problem in intensive care settings, where it causes systemic infections in patients with underlying health issues. Recent whole-genome sequencing has discerned five C. auris clades with distinct phenotypic features which display genomic divergence on a DNA sequence and a chromosome structure level.

scholarly journals A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

2015 ◽  
Vol 81 (9) ◽  
pp. 3176-3181 ◽  
Author(s):  
Christopher J. Pappas ◽  
Nadia Benaroudj ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Shuttle Vector ◽  
Plasmid Vector ◽  
Wild Type ◽  
Pathogenic Leptospira ◽  
Content Type ◽  
Large Panel ◽  
Stable Plasmid ◽  
The Stability ◽  
Functional Copy

ABSTRACTLeptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including theparA,parB, andrepgenes into pMAT, a plasmid that cannot replicate inLeptospiraspp. and contains a backbone consisting of anaadAcassette,oriR6K, andoriTRK2/RP4. The inserted DNA segment was isolated from a 52-kb region withinLeptospiramayottensisstrain 200901116 that is not found in the closely related strainL. mayottensis200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use oftranscomplementation in the pathogenLeptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of theperRgene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel ofLeptospirastrains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation ofLeptospiraspp.

scholarly journals Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an ArtificialattBSite for Specific Recombination

2011 ◽  
Vol 77 (21) ◽  
pp. 7508-7516 ◽  
Author(s):  
Jiequn Wu ◽  
Qinglin Zhang ◽  
Wei Deng ◽  
Jiangchao Qian ◽  
Siliang Zhang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Saccharopolyspora Erythraea ◽  
Scale Production ◽  
Content Type ◽  
Large Scale Production ◽  
Endogenous Genes ◽  
Erythromycin A ◽  
Adenosylmethionine Synthetase

ABSTRACTLarge-scale production of erythromycin A (Er-A) relies on the organismSaccharopolyspora erythraea, in which lack of a typicalattBsite largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificialattBsite in an industrialS. erythraeastrain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene,nrps1-1, with a cassette containing eightattBDNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including theS-adenosylmethionine synthetase gene for positive regulation,vhbfor increasing the oxygen supply, and two tailoring genes,eryKanderyG, for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack theattBsite.

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.

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.

scholarly journals Centromeres of the yeast Komagataella phaffii (Pichia pastoris) have a simple inverted-repeat structure

2016 ◽  
Author(s):  
Aisling Y. Coughlan ◽  
Sara J. Hanson ◽  
Kevin P. Byrne ◽  
Kenneth H. Wolfe
Keyword(s):  
Candida Albicans ◽  
Pichia Pastoris ◽  
Inverted Repeat ◽  
Methylotrophic Yeast ◽  
H3k9 Methylation ◽  
Komagataella Phaffii ◽  
Repeat Structure ◽  
Different Types ◽  
Mating Type Switching ◽  
The Relationship

AbstractCentromere organization has evolved dramatically in one clade of fungi, the Saccharomycotina. These yeasts have lost the ability to make normal eukaryotic heterochromatin with histone H3K9 methylation, which is a major component of pericentromeric regions in other eukaryotes. Following this loss, several different types of centromere emerged, including two types of sequence-defined ("point") centromeres, and the epigenetically-defined "small regional" centromeres of Candida albicans. Here we report that centromeres of the methylotrophic yeast Komagataella phaffii (formerly called Pichia pastoris) are structurally-defined. Each of its four centromeres consists of a 2-kb inverted repeat (IR) flanking a 1-kb central core (mid) region. The four centromeres are unrelated in sequence. CenH3 (Cse4) binds strongly to the cores, with a decreasing gradient along the IRs. This mode of organization resembles Schizosaccharomyces pombe centromeres but is much more compact and lacks the extensive flanking heterochromatic otr repeats. Different isolates of K. phaffii show polymorphism for the orientation of the mid regions, due to recombination in the IRs. CEN4 is located within a 138-kb region that changes orientation during mating-type switching, but switching does not induce recombination of centromeric IRs. The existing genetic toolbox for K. phaffii should facilitate analysis of the relationship between the IRs and the establishment and maintenance of centromeres in this species.

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