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

Abstract

Background The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. Results Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. Conclusions Among the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

“…When the flexible GS linker was inserted into the pro-peptide, the expression of extracellular mature rhG-CSF reached a maximum of 54.1 mg/L, which was 1.9-fold greater than the initial pro-peptide "B" (Table 1). This value is higher than that reported in other studies (yields up to approximately 39 mg/L) in which rhG-CSF was produced using the α-MAT secretion signal in another yeast species, Pichia pastoris (Aggarwal & Mishra, 2020;Lasnik et al, 2001).…”

“…Using yeast as a host to produce hG‐CSF can increase the rate of production, while simultaneously producing the glycosylated form. Thus, attempts to produce hG‐CSF using yeast are still in progress, but the production level has not yet reached a commercial scale (Aggarwal & Mishra, 2020 ; Lasnik et al, 2001 ; Wittman et al, 2004 ).…”

Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach

“…In the reference strain (Sb-NPA), we utilized Sb’s endogenous αMF-prepro secretion signal. This signal has been extensively used in other yeasts to secrete a range of proteins (Aggarwal & Mishra, 2020; Brake et al, 1984; Møller et al, 2017; Wang et al, 2021). Using the αMF-prepro signal, we achieved 250 mg/L NPA after 36 hours of cultivation.…”

The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii

“…The pre-peptide helps the nascent protein translocate to the ER. The pro-peptide is believed to play a significant role in secretion efficiency [26,27]. The mutation or deletion on pro-peptide of S. cerevisiae α-MF changed secretion efficiency of reporter proteins [26,28].…”

“…Amino acids 50-56 and 60-67 constitute two β-sheet while amino acids 68-78 are present in an α-helix. Studies showed that deletion of amino acids 57-70 located within the secondary structure of S. cerevisiae α-MF propeptide increased secretion of recombinant protein [27,28]. Most of α-MF pro-peptides from various yeasts have the same secondary structure like S. cerevisiae α-MF propeptide, indicating the structure possibly plays a functional role in expression regulation of α-MF pheromone in yeasts.…”