Abstract
Multicopy episomal plasmids in yeast, used whenever elevated levels of foreign or homologous gene expression are necessary, are known to be less stable compared to the endogenous 2-μm plasmid they are based on, at least without selective pressure. Considering that rich medium favors growth rate and, simultaneously, is less expensive than selective medium, enhancing stability in non-selective medium is extremely desirable. In this study, we changed the architecture of a multicopy model expression plasmid, creating six isoforms (same size, same DNA content but different positions and orientations of the expression block) and studied mitotic stability, copy number, as well as reporter yEGFP3 expression between isoforms. With one isoform being significantly more stable than the others and another one exhibiting elevated plasmid copy numbers in rich medium, we show that consideration of the arrangement of the plasmid elements might be crucial for productivity employing Saccharomyces cerevisiae as a host. We strongly believe that the ideal architecture has to be assessed for each case and assembly strategy has to begin by evaluating the stability of the vector backbone before insertion of the desired gene. For the plasmid set studied, yEGFP3 reporter production depends more on mitotic stability than on elevated plasmid copy numbers in a small number of cells retaining the plasmid under non-selective conditions.
Highlights
Robustness of an industrial process employing recombinant organisms seems largely dictated by abiotic process parameters
As we did not see a difference in transformation efficiency and segregational stability between the two common laboratory S. cerevisiae strains SY922 and BY4742 (Hohnholz et al 2017), we focused in this study on the widely employed BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0; Brachmann et al 1998; Euroscarf collection, Frankfurt, Germany)
Having observed that the arrangement of the functional segments of a simple multicopy yeast-E. coli shuttle plasmid of the YEp-type can have a significant effect on the segregational stability and plasmid copy numbers (PCNs) (Hohnholz et al 2017), we wanted to go further in investigating the behavior of a set of isomeric expression plasmids
Summary
Robustness of an industrial process employing recombinant organisms seems largely dictated by abiotic process parameters. As a cell-based system, the host inevitably is subject to selection (and counterselection), disfavoring nonphysiological changes of its genetics and metabolism. In order to overproduce proteins or low molecular weight metabolites, homologous or heterologous, almost always genetic information needs to be transformed into the host cell. The necessary sequences in most cases make part of a plasmid vector. The stability of such a vector in the recipient cell is of primordial importance for the robustness and productivity of the production system, instability might render the validation of an industrial process questionable or even impossible (Zhang et al 1996; Delvigne and Goffin 2014; Gustavsson and Lee 2016)
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