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

Applied Microbiology and Biotechnology, May 2017

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.

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

Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris Rochelle Aw 0 1 2 3 Geraint R Barton 0 1 2 3 David J. Leak 0 1 2 3 0 Centre for Synthetic Biology and Innovation, Imperial College London , London SW7 2AZ , UK 1 Department of Life Sciences, Imperial College London , London SW7 2AZ , UK 2 Department of Biology & Biochemistry, University of Bath , Bath BA2 7AY , UK 3 Centre for Integrative Systems Biology and Bioinformatics, Imperial College London , London SW7 2AZ , UK 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. Pichia pastoris/Komagataella phaffii; Transcriptomic analysis/microarray; Clonal variation; Unfolded protein response; ER-associated degradation; Protein expression - The phenomenon of clonal variation has been noted in a variety of organisms, most noticeably Chinese hamster ovary (CHO) cells where it was first described in 1977 (Konrad et al. 1977). Supposedly genetically identical clones plated on agar plates showed variation in colony morphology, as well as variation when used for the production of recombinant proteins. Differences were most noticeable in secreted protein titer and highlighted an underlining heterogeneity in CHO cells that had not previous been identified (Dahodwala et al. 2012; O’Callaghan and James 2008; Pilbrough et al. 2009). Variation has often been attributed to differences in gene integration sites, the use of antibiotics for selection or gene copy number (Kim et al. 2001; Zdzienicka et al. 1985). Variation in plant cells, known as somaclonal variation, is predominantly attributed to stress factors, although epigenetic factors such as copy number variation, gene silencing and gene activation have been noted (Bardini et al. 2003; Kaeppler et al. 2000; Kidwell and Osborn 1993). The methylotrophic yeast Pichia pastoris, reclassified as Komagataella phaffii/pastoris, was described as showing clonal variation in the mid-1980s (Cregg et al. 1989). Indeed, clonal variation is such an integral feature that for commercial production using P. pastoris as an expression host, screening hundreds of clones per phenotype is recommended in order to identify the highest secretor ( Z h en g e t al . 2 01 3 ) . T hi s is a n ex t re m el y ti m e consuming aspect of using P. pastoris, which otherwise has the potential to be a favourable recombinant expression host, particularly after the creation of a strain with a humanized glycosylation pattern (Hamilton and Gerngross 2007). Similarly to mammalian cells, much of the clonal variation has been attributed to the presence of multiple gene copies or varying integration sites (Aw and Polizzi 2013; Clare et al. 1998; Schwarzhans et al. 2016). However, it is clear that other host-related determinants are also operational. To date, two papers have carried out the most comprehensive investigations into clonal variation, the first by Viader-Salvadό et al. (2006) used amplified fragment length polymorphism (ALFP). By analysing 1 4 t r a n s f o r m a n t s a n d t h r e e c o n t r o l s t r a i n s , th e y determined that variation predominantly arose from the transformation process, and that the clones that most closely resembled the host strains gave higher yields. Schwarzhans et al. (2016) also used genomic analysis to investigate clonal variation, yet differences in copy number and integration sites meant that their definition of clonal variation differs from what we have outlined as being genetically identical clones. Their findings indicated that integration loci, copy number and vector orientation were the most fundamental causes of differences to yields. A greater understanding of clonal variation could simplify the process of se (...truncated)


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Rochelle Aw, Geraint R Barton, David J. Leak. Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris, Applied Microbiology and Biotechnology, 2017, pp. 5045-5058, Volume 101, Issue 12, DOI: 10.1007/s00253-017-8317-2