scholarly journals Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System

2021 ◽  
Vol 43 (3) ◽  
pp. 2289-2304
Author(s):  
Wei Xia ◽  
Mengkai Hu ◽  
Yang Pan ◽  
Dan Wu ◽  
Jing Wu
Keyword(s):  
Pichia Pastoris ◽  
Copy Number ◽  
Kluyveromyces Lactis ◽  
Expression System ◽  
Constitutive Expression ◽  
Potential Hazard ◽  
Autonomously Replicating Sequence ◽  
Streptomyces Sp ◽  
Episomal Vectors ◽  
Pharmaceutical Industries

Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL−1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.

A constitutive expression system for Pichia pastoris based on the PGK1 promoter

2015 ◽  
Vol 38 (3) ◽  
pp. 509-517 ◽  
Author(s):  
Andrelisse Arruda ◽  
Viviane Castelo Branco Reis ◽  
Vinícius Daniel Ferreira Batista ◽  
Bruno Sahim Daher ◽  
Luiza Cesca Piva ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Constitutive Expression ◽  
Pgk1 Promoter

An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris

2019 ◽  
Vol 155 ◽  
pp. 43-47 ◽  
Author(s):  
Quan Chen ◽  
Yuhang Zhou ◽  
Jianli Yu ◽  
Wenshuai Liu ◽  
Fei Li ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Constitutive Expression ◽  
Yeast Pichia Pastoris

scholarly journals Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system

2019 ◽  
Vol 19 (6) ◽  
Author(s):  
Shinobu Takagi ◽  
Noriko Tsutsumi ◽  
Yuji Terui ◽  
XiangYu Kong ◽  
Hiroya Yurimoto ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Constitutive Expression ◽  
Free Expression ◽  
Aox1 Promoter ◽  
Phytase Production ◽  
Model Case ◽  
Komagataella Phaffii ◽  
Gap Promoter ◽  
Important Region

ABSTRACT The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals Modeling of copy number variability in Pichia pastoris

2021 ◽  
Vol 118 (5) ◽  
pp. 1832-1839
Author(s):  
Amos E. Lu ◽  
Andrew J. Maloney ◽  
Neil C. Dalvie ◽  
Joseph R. Brady ◽  
Kerry R. Love ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Copy Number ◽  
Copy Number Variability

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 A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
Jeffrey G Linger ◽  
Larry E Taylor ◽  
John O Baker ◽  
Todd Vander Wall ◽  
Sarah E Hobdey ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Expression System ◽  
Constitutive Expression ◽  
Hypocrea Jecorina ◽  
Glycosyl Hydrolase Family ◽  
Hydrolase Family

scholarly journals rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

2006 ◽  
Vol 395 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Chiara Ciaccio ◽  
Alessandra Gambacurta ◽  
Giampiero DE Sanctis ◽  
Domenico Spagnolo ◽  
Christina Sakarikou ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Expression System ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Kinetic Properties ◽  
Specific Substrate ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Pichia Pastoris Expression System

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

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