Abstract
Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed.Key Points• Eight expression systems, producing different reporter proteins were analyzed.• The cells were maintained in steady-state by continuous chemostat culturing.• Protein- and promoter-specific effects were observed.
Highlights
Growing demand for industrial enzymes and biopharmaceutical proteins triggers increasing interest in microbial cell factories that serve as protein production platforms
It was evidenced that Y. lipolytica produces high levels of active enzyme, generates stable isotope-labelled variants of a secretory protein, or that it is suited for the production of complex high molecular weight proteins targeted for secretion (Madzak et al 2005; Boonvitthya et al 2013; Nars et al 2014; Theron et al 2020)
Distribution of C and N was calculated based on molecular weight and elementary composition of the major products synthesized by Y. lipolytica strains in the chemostat cultures
Summary
Growing demand for industrial enzymes and biopharmaceutical proteins triggers increasing interest in microbial cell factories that serve as protein production platforms. Y. lipolytica was found to perform well in bioreactor cultures in terms of cell growth, secretory protein titer, and productivity (Celińska et al 2017a; Theron et al 2020). It was evidenced that Y. lipolytica produces high levels of active enzyme, generates stable isotope-labelled variants of a secretory protein, or that it is suited for the production of complex high molecular weight proteins targeted for secretion (Madzak et al 2005; Boonvitthya et al 2013; Nars et al 2014; Theron et al 2020). Y. lipolytica was shown to manage secretory production of a heterologous protein of molecular weight > 120 [kDa] (Swietalski et al 2020)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
