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

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0120-5) contains supplementary material, which is available to authorized users.

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris

“…Heterologous expression of CES secreted from P. pastoris turned out as good alternative to extraction from natural sources, however, further improvement of production levels was necessary. Previously in our group, we achieved enhancement of the production of CES by overexpressing a novel transcription factor Aft1 in P. pastoris . During further studies attempting to enhance CES secretion, we found that significant amounts of CES were localized to the vacuole in immunofluorescence microscopy, indicating that vacuolar mis‐sorting is indeed a bottleneck in P. pastoris .…”

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

“…The homologue of S. cerevisiae Activator of Ferrous Transport , AFT1, was found to have induced expression levels in excess glycerol, methanol and limiting glucose conditions and has been reported to play a role in the regulation of carbon repressed genes in P. pastoris recently [ 42 ]. The transcription factors PAS_chr4_0324, CTH1, PAS_chr1-1_0422, PAS_chr3_1209, PAS_chr1-1_0122 were related to excess conditions .…”

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…Yuan et al [ 81 ] produced the type 1 ribosome inactivating protein Saporin L3 from Saponaria officinalis (soapwort) leaves which is toxic for E. coli successfully in P. pastoris using the EnBase technology. Ruth et al [ 82 ] successfully applied the Feed Bead ® Technology to study transcription factor of P. pastoris and Ashoor et al [ 83 ] to produce human immunoglobulin receptors with EnPresso in P. pastoris . In the approach by Panula-Perälä et al [ 84 ] the slow glucose release of the EnBase technology was used as a continuous background energy and carbon supply bridging the gaps between methanol pulses needed for protein expression under the AOX1 promotor.…”