Role of BGS13 in the Secretory Mechanism of Pichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress

“…For the BGS targets no mutation frequency was calculated, as only very few colonies were sequenced. The frameshifts in the targets BGS5 , BGS7 , and BGS12 , as well as the in-frame deletion in BGS13 , however, confirm the results of Cereghino et al who reported insertions of integration cassettes in those open reading frames previously, while still reporting an intact reading frame for BGS13 [ 51 , 52 ].…”

“…In total, 20 genes of interest were identified, of which 5 were chosen for a more detailed analysis of CRISPR/Cas9 on- and off-target effects, based on preliminary results and position in the genome ( Table 1 ). Cereghino et al have identified targets whose disruption might have positive effects on the secretion of the reporter protein beta galactosidase [ 51 , 52 ]. We have chosen to include 4 of those genes as targets and named them BGS5 , BGS7 , BGS12 and BGS13 in accordance with Cereghino et al ( Table 1 ).…”

Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris

“…In contrast to S. cerevisiae, YPS1 gene mutants of P. pastoris grew normally in the presence of CFW or CR (Guan et al, 2012;Naranjo et al, 2019) phenotype of yps1 strains is not affected in the presence of two cell wall-disrupting reagents (Kang et al, 1998;Yao et al, 2009).…”

Komagataella phaffii as Emerging Model Organism in Fundamental Research

“…A BLAST search revealed that Bgs13 is the K. phaffii homolog of S. cerevisiae Pkc1. The role of Bgs13/Pkc1 has been further investigated by analyzing strains expressing the truncated variant of this essential protein ( Naranjo et al, 2019 ). The mutant strain showed an abnormal localization of the Bgs13 variant and its cell wall suffered from inherent structural problems, which most probably promoted improved protein secretion.…”