Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris

Abstract

Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8317-2) contains supplementary material, which is available to authorized users.

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

“…Commonly, large numbers of transformant strains are screened using high-throughout (HTP) systems (13) resulting in a few highly producing "jackpot clones" (14). Although the term jackpot clone is sometimes used to refer to high-copynumber strains (9), other effects, such as the integration site and genome rearrangements, also may influence expression (15)(16)(17)(18)(19). The transcriptomes of P. pastoris strains secreting different amounts of human serum albumin were compared using microarrays, but the authors did not find clear regulatory patterns which could mechanistically explain the clonal variation observed (17).…”

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members

“…phaffii (35). Moreover, this set of common "DE up" genes showed a high degree of overlap with "DE up" genes in the de novo methylating conditions, demonstrating the generality of the ribosome-related transcriptional response across conditions ( Figure S8B,C).…”

Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform

“…The coding regions were fused to the constitutive GAP promoter in the pKANB vector and transformed into the YTM1 biosensor strain for initial screening as this biosensor showed a relatively high fluorescence (Figure b) and low population variability in previous experiments (Supporting Information Figure 2). To control for differences in behavior due to clonal variation, three independent colonies of each strain were selected for the analysis (Aw et al, ; Schwarzhans et al, ). The cells were grown in YPD medium for 18 hr from a starting OD 600 of 0.1, and the fluorescence of the YTM1 biosensor measured using flow cytometry (Figure ).…”