Curation of the genome annotation ofPichia pastoris(Komagataella phaffii) CBS7435 from gene level to protein function

Abstract

As manually curated and non-automated BLAST analysis of the published Pichia pastoris genome sequences revealed many differences between the gene annotations of the strains GS115 and CBS7435, RNA-Seq analysis, supported by proteomics, was performed to improve the genome annotation. Detailed analysis of sequence alignment and protein domain predictions were made to extend the functional genome annotation to all P. pastoris sequences. This allowed the identification of 492 new ORFs, 4916 hypothetical UTRs and the correction of 341 incorrect ORF predictions, which were mainly due to the presence of upstream ATG or erroneous intron predictions. Moreover, 175 previously erroneously annotated ORFs need to be removed from the annotation. In total, we have annotated 5325 ORFs. Regarding the functionality of those genes, we improved all gene and protein descriptions. Thereby, the percentage of ORFs with functional annotation was increased from 48% to 73%. Furthermore, we defined functional groups, covering 25 biological cellular processes of interest, by grouping all genes that are part of the defined process. All data are presented in the newly launched genome browser and database available at www.pichiagenome.org In summary, we present a wide spectrum of curation of the P. pastoris genome annotation from gene level to protein function.

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

“…We first analyzed the two control strains: (i) the unmodified parental strain and (ii) strain QTV19, which was electroporated without plasmid DNA (to check for detrimental effects of the transformation event itself) ( Table 1). In the mut S control we found 37 changes, including SNPS (12) and indels (25), spread across all four chromosomes and the mitochondrial genomes. Indels were between 1 and 21 bp in length, with insertions being most prevalent (15 insertions between 1 and 10 bp in length and 9 deletions between 11 and 21 bp; see Data Set S8 for the exact changes).…”

“…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.…”

13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched‐chain amino acids

“…The yeast Pichia pastoris, recently classified as the new genus Komagatella phaffii [1,2], is widely used for expression of recombinant proteins that require disulfide bond formation or glycosylation [3]. In spite of its outstanding importance for biotechnology and life sciences in general, however, the incomplete annotation of its genome [4,5] and the lack of a model organism database, as they exist for Escherichia coli and Saccharomyces cerevisiae, impede research progress [6]. Experimental studies of P. pastoris' central carbon metabolism are likewise comparatively rare [7][8][9][10][11][12][13][14][15][16].…”

Transcriptional engineering of the glyceraldehyde‐3‐phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris

“…In S. cerevisiae , there are three MIG genes, which encode the transcriptional regulators Mig1p, Mig2p and Mig3p. In P. pastoris 2 proteins with homology to Mig1 are found, which were designated as Mig1‐1 and Mig1‐2 (Valli et al, ). In their DNA‐binding region, Mig1‐2 has higher similarity to S. cerevisiae Mig1 than Mig1‐1, while similarity outside this region is rather low.…”