Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

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Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

Microbial Cell Factories ◽

10.1186/s12934-019-1240-8 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 2

Author(s):

Javier Garrigós-Martínez ◽

Miguel Angel Nieto-Taype ◽

Arnau Gasset-Franch ◽

José Luis Montesinos-Seguí ◽

Xavier Garcia-Ortega ◽

...

Keyword(s):

Gene Expression ◽

Growth Rate ◽

Pichia Pastoris ◽

Specific Growth Rate ◽

Specific Growth ◽

Gene Dosage ◽

Fed Batch ◽

Komagataella Phaffii ◽

Production Kinetics ◽

Kinetics Of

Abstract Background The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. Results The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. Conclusions PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression—heterologous genes included—, thus directly influencing the production kinetics of recombinant protein.

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Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes

Applied Microbiology and Biotechnology ◽

10.1007/s00253-010-2944-1 ◽

2010 ◽

Vol 89 (4) ◽

pp. 1127-1135 ◽

Cited By ~ 36

Author(s):

Taicheng Zhu ◽

Meijin Guo ◽

Yingping Zhuang ◽

Ju Chu ◽

Siliang Zhang

Keyword(s):

Pichia Pastoris ◽

Gene Dosage ◽

Transcriptional Analysis ◽

Foreign Gene ◽

Key Genes

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Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0220273 ◽

1999 ◽

Vol 22 (3) ◽

pp. 273-283 ◽

Cited By ~ 25

Author(s):

C Sen Gupta ◽

RR Dighe

Keyword(s):

Pichia Pastoris ◽

Structure Function ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Recombinant Expression ◽

Expression System ◽

Biologically Active ◽

Maximal Response ◽

Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

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