Comparative genomics and transcriptomics of Pichia pastoris

Abstract

BackgroundPichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species.ResultsHere, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts.ConclusionsOur studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2876-y) contains supplementary material, which is available to authorized users.

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

“…We hypothesized that conserved genetic polymorphisms among strains may cause phenotypic differences, so we sequenced the genome of each strain and performed variant calling based on alignment to our reference genome for Y‐11430 (Love et al, ). We analyzed the genomic sequences for the presence of functional and nonfunctional SNPs (Figure ) and larger structural variants such as insertions or deletions relative to Y‐11430.…”

“…We previously determined that GS115 possesses 74 SNPs relative to Y‐11430, 35 of which are potentially functional (Love et al, ). Several of the functional SNPs occur in DNA repair ( RAD5, EXO1 , and DNL4 ) or cell wall genes ( CWH43 and GQ67_02041 ).…”

“…Several of the functional SNPs occur in DNA repair ( RAD5, EXO1 , and DNL4 ) or cell wall genes ( CWH43 and GQ67_02041 ). All 74 SNPs present in GS115 were also present in Y‐48124 (an NRRL banked strain of X‐33) and X‐33, with the exception of an amino acid variant at the highly conserved position 557 of the essential His4p protein (Love et al, ). Both Y‐48124 and X‐33 possess a p.C557G substitution in HIS4 , rather than the p.C557R substitution found in GS115 (Figure b).…”

“…Approximately one‐third of these SNPs are potentially functional and many are concentrated at the ends of chromosomes or in repetitive sequence regions, consistent with strain or organismal divergence (Figure c; Louis, Naumova, Lee, Naumov, & Haber, ). Despite these high SNP counts, these strains likely belong to K. phaffii , and not K. pastoris , since the latter species has on the order of one million SNPs relative to K. phaffii Y‐11430 (Love et al, ). Of note, YB‐378, YB‐4289, and Y‐17741 all possess the larger variant of Rsf2p present in Y‐7556 and S. cerevisiae .…”

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

“…With the advent of next-generation whole-genome sequencing (WGS), reference genomes (20)(21)(22) and refined sequencing and annotations (23)(24)(25) have become available for different P. pastoris wild-type strains. With further decreases in the cost of these technologies, systematic studies of the underlying mechanisms for clonal variation in P. pastoris transformants have become feasible.…”