Molecular optimization of rabies virus glycoprotein expression in <i>Pichia pastoris</i>

Azoun, Safa Ben; Belhaj, Aicha Eya; Göngrich, Rebecca; Gasser, Brigitte; Kallel, Héla

doi:10.1111/1751-7915.12350

Cited by 34 publications

(22 citation statements)

References 63 publications

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“…These results are in agreement with several previously reported cases where such a plateau-like correlation was observed22454950. Nevertheless, this pattern does not seem to be a general rule for P AOX1 -based systems, as other studies pointed at a positive correlation between gene copy number and mRNA level when increasing gene dosage2051. Strikingly, normalization of mRNA levels with the number of DNA copies of ROL (i.e.…”

Section: Resultssupporting

confidence: 92%

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

Cámara

Landes

Albiol

et al. 2017

Sci Rep

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The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.

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Section: Resultssupporting

confidence: 92%

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

Cámara

Landes

Albiol

et al. 2017

Sci Rep

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“…(), they showed that the genes involved in dissimilatory pathway were upregulated in high copy clones expressing porcine insulin precursor (PIP) under the control of AOX1 promoter. The significant activation of methanol assimilation pathway compared to central carbon metabolism pathway could be the reason for reduced RABV‐G protein production in the aox7 clone compared to gap7 clone, particularly when we take into account that RABV‐G transcript level was similar for both clones (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ). These data indicate that methanol does not offer enough carbon and energy to strongly sustain cell growth and RABV‐G production.…”

Section: Discussionmentioning

confidence: 99%

“…Expression levels of RABV‐G obtained by these clones were compared and a significant difference was seen in extracellular and intracellular compartments. We showed that this difference was obviously substrate and not promoter‐dependent as mRNA levels of RABV‐G gene were comparable (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ).…”

Section: Introductionmentioning

confidence: 87%

“…Optimized RABV‐G gene (Genbank accession number KT878717 ) was used for the construction of the expression cassette. The generation of multi‐copy clones used in this work (gap7, aox7) was previously described in details (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ); these clones were constructed using two different recombinant vectors pPICZαA or pGAPZαB. The expression was controlled by AOX1 or GAP as a promoter, α‐mating factor of S. cerevisiae was used to direct the recombinant protein to extracellular medium.…”

Section: Methodsmentioning

confidence: 99%

“…In previous studies, we generated two recombinant clones of P. pastoris KM71H Mut S harboring seven copies of the rabies virus glycoprotein (RABV‐G) gene (Ben Azoun, Belhaj, Göngrich, Gasser, & Kallel, ; Ben Azoun, Belhaj, & Kallel, ). The expression of the target protein was driven either by AOX1 promoter (aox7) or GAP promoter (gap7) and directed in both clones to secretion by the alpha mating factor of Saccharomyces cerevisiae .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Investigating the effect of carbon source on rabies virus glycoprotein production inPichia pastorisby a transcriptomic approach

Azoun

Kallel

2017

MicrobiologyOpen

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Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV‐G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 ( AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV‐G in P. pastoris.

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High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

Li,

Shao,

Xiang

et al. 2024

Biotechnology Journal

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Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis‐sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty‐two single‐mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer‐aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1‐P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1‐P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1‐P6 was produced by the secretion‐enhanced P. pastoris chassis to 199.6 ± 20 mg L−1 with a specific activity of 96 ± 0.32 U mg−1, resulting in a total enzyme activity of 19137 ± 1131 U L−1, demonstrating significant potential for industrial applications.

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Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

Cited by 34 publications

References 63 publications

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

Investigating the effect of carbon source on rabies virus glycoprotein production inPichia pastorisby a transcriptomic approach

High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

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