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Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion.

ABSTRACT: BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport. RESULTS: The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations. CONCLUSION: This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation.

SUBMITTER: Ruth C

PROVIDER: S-EPMC4161868 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion.

Ruth Claudia C Buchetics Markus M Vidimce Viktorija V Kotz Daniela D Naschberger Stefan S Mattanovich Diethard D Pichler Harald H Gasser Brigitte B

Microbial cell factories 20140903

<h4>Background</h4>The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator ...[more]

PMID: 25205197

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Project description:BACKGROUND: The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. RESULTS: Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. CONCLUSIONS: The current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

Project description:BACKGROUND:The yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases prevented this mis-sorting and resulted in higher levels of secreted heterologous protein. Inspired by the positive results, we now set out to investigate the influence of further parts of the vacuolar pathway, namely the Cvt-pathway and the homotypic fusion and protein sorting (HOPS) complex. RESULTS:Strains impaired in the Cvt pathway (?atg11, ?atg8) had no effect on secretion of the model protein carboxylesterase (CES), but resulted in lower secretion levels of the antibody fragment HyHEL-Fab. Disruption of genes involved in the HOPS complex led to vacuole-like compartments of the B category of vps mutants, which are characteristic for the deleted genes YPT7, VPS41 and VAM6. In particular ?ypt7 and ?vam6 strains showed an improvement in secreting the model proteins HyHEL-Fab and CES. Additional disruption of the vacuolar protease Pep4 and the potential protease Vps70 led to even further enhanced secretion in ?ypt7 and ?vam6 strains. Nevertheless, intracellular product accumulation was still observed. Therefore, the secretory route was strengthened by overexpression of early or late secretory genes in the vacuolar sorting mutants. Thereby, overexpression of Sbh1, a subunit of the ER translocation pore, significantly increased HyHEL-Fab secretion, leading up to fourfold higher extracellular Fab levels in the ?ypt7 strain. The beneficial impact on protein secretion and the suitability of these strains for industrial applicability was confirmed in fed-batch cultivations. CONCLUSIONS:Disruption of genes involved in the HOPS complex, especially YPT7, has a great influence on the secretion of the two different model proteins HyHEL-Fab and CES. Therefore, disruption of HOPS genes shows a high potential to increase secretion of other recombinant proteins as well. Secretion of HyHEL-Fab was further enhanced when overexpressing secretion enhancing factors. As the positive effect was also present in fed-batch cultivations, these modifications likely have promising industrial relevance.

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Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion.

Publications

Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion.

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