Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Abstract

Background: The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in highyielding protein production hosts.

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

“…Subsequent overexpression of BMS1, where the endogenous promoter is replaced with a doxycycline-titratable promoter in the genome, revealed that maximal Fps1 yield was significantly correlated with an optimum level of BMS1 transcript, which was further correlated with a changed ribosomal subunit stoichiometry. This observation was also true at different doxycycline concentrations for hA2aR and soluble GFP [8].…”

“…In our own study of the S. cerevisiae response to membrane protein production [7], genes that were down-regulated under low yielding conditions were up-regulated under high-yielding conditions and vice versa [8] (Table 1). This lent confidence to the fact that these genes might have a role in high-yielding protein production, which was subsequently further validated by strain engineering.…”

“…The more recent application of "omics" technologies has been driven by attempts to obtain a global understanding of recombinant membrane protein production in bacteria and yeast [8,15] and to identify the mechanisms associated with high-yielding cells. In the prokaryote host, Escherichia coli, increases in levels of chaperones and proteases were found to be associated with increased membrane protein production [15], which concurred with the SERT experiments in the baculovirus system.…”

“…Specifically, we used this approach to engineer the first yeast production strains (GB0813253.2) by rational design [19]. In particular, the spt3∆, srb5∆ and gcn5∆ mutant strains were effective production hosts not only for the glycerol channel, Fps1, where the yield improvement was 10-to 60-fold higher than a wild-type control, but also the human GPCR, 21 (hA2aR), where we doubled the functional yield compared to wild-type [8].…”

“…In our own work on yeast, we previously identified 39 Saccharomyces cerevisiae genes whose expression was significantly altered when the aquaporin, Fps1, was produced under high-yielding conditions (20°C, pH5) compared to lowyielding standard growth conditions (30°C, pH5) [7]. In particular an essential gene, BMS1, with a role in ribosome biogenesis, was identified as always up-regulated in high-yielding host cells [8].…”