An improved secretion signal enhances the secretion of model proteins from Pichia pastoris

Abstract

BackgroundProteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal.ResultsWe engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal.ConclusionsThe improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1009-5) contains supplementary material, which is available to authorized users.

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

“…For instance, some authors have used increased gene dosages of target genes involved in product folding and secretion such as HAC1 , PDI1 and/or KAR2 (Bankefa et al , ; Guan et al , ; Liu et al , ; Yang et al , ). Other authors (Barrero et.al., ) have engineered the secretion signal in order to improve translocation into the endoplasmic reticulum (ER).…”

Differential role of segments of α-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene

“…Another study reported enhanced secretion of phytase protein by 7-fold which could be accomplished after codon optimization of the α-MAT secretory signal sequences [ 43 ]. Fusion of the pro-region of the α-MAT with signal peptide of mouse salivary α-amylase [ 68 ] or the Ost1 signal of S. cerevisiae [ 44 ] has been reported to result in increased expression of Rhizopus oryzae glucoamylase by 3.6-fold or of red fluorescent protein E2-Crimson by 20-fold respectively. The fusion with the Ost1 signal peptide, wherein co-translational transport of the cargo protein occurred, also worked favourably (10-fold increase) for lipase BTL2 protein.…”

“…In spite of applying strategies, such as, codon optimization, increasing the copy number, combining both genetic and process conditions [ 37 – 39 ], overexpression of transcription factors [ 40 ], low production has marred the use of P. pastoris as an expression platform. Manipulation of the secretory signal sequences has shown promise for enhanced production of extracellular proteins in this yeast [ 41 – 44 ]. Production of G-CSF has been attempted in several strains of P. pastoris (X-33, SMD1168 and GS115) using the α-MAT secretory signal and reported yield varied from 2 to 20 mg/L of G-CSF at shake flask level [ 45 – 48 ].…”

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

“…This amount limited the number of copies that could be integrated into the genome. The resulting colonies were re-streaked twice in YPD-Zeo plates to avoid population mixing, and 8 colonies were screened, as described elsewhere [45]. This allowed the most representative colony to be selected for further study.…”