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
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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)