Can too many copies spoil the broth?

Abstract

The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potential success of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increase recombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the first reported use of multi-copy clones and it is still an active area of research to find the fastest and most efficient method for generating these strains. It has also become apparent that there is not always a linear correlation between copy number and protein titer, leading to in-depth investigations into how to minimize the negative impact of secretory stress and achieve clonal stability.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

“…Since it was determined here that the titre of terpene synthase was not yet sufficient to exhaust the freely available FPP pool within the cytoplasm of C. reinhardtii, we sought other methods to increase the titre of the PcPs. One strategy in over expression of target proteins is gene-loading, involving either high copy plasmids or multiple copies of a transgene expression cassette introduced into the genome (Aw and Polizzi, 2013). The only examples of multiple transgene expression from the nuclear genome of C. reinhardtii have been through double transformations with selectable markers (Lauersen et al, 2015b), mating of two separately expressing strains (Rasala et al, 2014), or using viral 2A peptides for cleavage (Rasala et al, 2014).…”

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

“…P. pastoris has several beneficial traits, such as suitability for high-cell-density bioreactor cultivation (5), the availability of exceptionally strong, tightly regulated promoters (6), and the capacity for secreting large amounts of heterologous proteins (1)(2)(3). Furthermore, P. pastoris is readily amenable to genetic modification, and it is relatively easy to integrate multiple copies of an expression cassette, thereby further boosting yields (7)(8)(9). Expression cassettes containing the gene of interest are typically integrated into the genome, resulting in stable expression strains (despite recent advances [10], plasmids have been used infrequently).…”

Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

“…3,21 In several cases this has led to a significant improvement in heterologous protein production, most notably when considering intracellular expression. 22 However, in some cases involving secreted proteins, a higher gene copy number resulted in reduced heterologous protein production.…”

“…This is usually accomplished by driving the expression of the heterologous gene with a suitable promoter or by simply increasing the copy number of the target gene. 3 One should consider that gene overexpression may pose a metabolic burden to the physiology of the cell thus resulting in disappointing outputs. Nonetheless, most researchers still consider these approaches (summarized in Fig.…”

“…3,22 Moreover, there are concerns that the accidental released of genetically modified organisms bearing drug-resistance markers may be horizontally transferred to environmental organisms. 31 Marker-removal has been successfully accomplished in K. phaffii with the use of the Cre/loxP 32 and Flp/FRT 27 site-specific recombination systems.…”