Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain

Abstract

Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y‐11430 (CBS7435), GS115, X‐33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y‐11430 exhibited the highest activity of genes involved in methanol utilization, up to two‐fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X‐33 displayed a six‐fold higher transformation efficiency and up to 1.2‐fold higher titers than Y‐11430. X‐33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X‐33 into Y‐11430 resulted in an optimized base strain that provided up to four‐fold higher transformation efficiency and three‐fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

“…Of all gene sets that were differentially expressed in P[6]-producing cells (Table S2), cytoplasmic translation had the highest enrichment score, followed by gene sets related to cell wall integrity and DNA repair, which are reported signals of stress in K. phaffii (Fig. 4B) [32]. We hypothesized that disrupted translation may preclude both adequate production of P [6] and proper cell growth and maintenance.…”

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

“…Chromosomal ends are not ideal regions for integration of heterologous constructs due to the presence of telomeres, repetitive sequences, and susceptibility to accumulation of polymorphisms. 21 For these reasons, we avoided regions of 5–10 kb at the distal ends of chromosomes where there were no annotated CDSs with high-quality mRNA transcript. We excluded centromeres, which we confirmed to be generally inaccessible as expected ( Figure S2 ).…”

“…We excluded centromeres, which we confirmed to be generally inaccessible as expected ( Figure S2 ). 22 Finally, we excluded regions in the extrachromosomal plasmids maintained in variants of K. phaffii , 21 since the function and stability of these plasmids are not well characterized.…”

“…For hGH- and G-CSF-secreting strains, copy number analysis by qPCR and determination of protein concentration by sandwich ELISA were both performed as described previously. 21 …”

Molecular Engineering Improves Antigen Quality and Enables Integrated Manufacturing of A Trivalent Subunit Vaccine Candidate for Rotavirus

“…Of all gene sets that were differentially expressed in P[6]-producing cells (Table S2), cytoplasmic translation had the highest enrichment score, followed by gene sets related to cell wall integrity and DNA repair, which are reported signals of stress in K. pha i ( Fig. 4B) [32]. We hypothesized that disrupted translation may preclude both adequate production of P [6] and proper cell growth and maintenance.…”