A step forward to improve recombinant protein production in Pichia pastoris: From specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy

Abstract

Click here to download Manuscript: 160215_Garcia-Ortega_Adelantado_FINAL.docx Click here to view linked References This is the author's version of a work that was accepted for publication in Process biochemistry (Ed. Elsevier). Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Garcia Ortega, Xavier et al. "A step forward to improve recombinant protein production in Pichia pastoris : $b from specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy" in

Rationale‐based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)

“…Most of the studies involving P GAP have shown q P to increase linearly with increasing µ (Khasa et al , ; Rebnegger et al , ; Garcia‐Ortega et al , ). In the present work, similar behaviour was observed for the SCC.…”

“…Chemostat cultivation. Chemostat cultures of the two strains were prepared in duplicate as described elsewhere (Garcia-Ortega et al, 2016). Different specific growth rates from 0.025 to 0.15 h -1 were evaluated.…”

“…This relationship is crucial to bioprocess development and optimization (Potvin et al, 2012;Looser et al, 2015;C ß alik et al, 2015). Thus, Garcia-Ortega et al (2016) and Maurer et al (2006) characterized P GAP -based strains producing an antibody fragment and obtained robust results with them in chemostat systems; so, they found q P to increase eight times with increasing l. Rebnegger et al (2014) examined the response of this expression system producing human serum albumin (HSA) at different specific growth rates at transcriptomic level and observed marked upregulation of genes involved in translation. However, genes of the glycolytic pathways such as TDH3, which is the endogenous gene regulated by P GAP , were unregulated or weakly regulated, therefore suggesting that effect in the translational machinery played a major role in by causing q P to increase with increasing l. The results obtained with some fed-batch cultures are also consistent with synergism in these two variables (Zhao et al, 2008;Garcia-Ortega et al, 2013).…”

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

“…Over the past decade, experimentalists have sought to elucidate how increases in production in P. pastoris fermentations can be achieved by designing original fed‐batch strategies . Meanwhile, in direct contrast not to the tools used but to the evidence, an operation mode – the so‐called advanced strategy – hypothesized as C‐starving conditions is not an accurate assessment of the process, because this was neither justified in the work nor realistic as P. pastoris can enter into C‐starving conditions during short interruption(s) of the continuous feed‐stream in a fed‐batch operation. To confront the emerging studies on the design of fed‐batch strategies and misevaluations, we recently reported on the parametric continuous feed‐stream design for fed‐batch operations .…”

“…Glucose has been widely used as sole carbon source in fed-batch phase of production under P GAP , [11][12][13][14][15][16][17] and resulted in higher r-protein productivities when fed by a CFS designed with a pre-determined specific growth rate within the range 0.10 h −1 < μ < 0.15 h −1 . In general, one of the economic constraints of r-protein production is the demand for carbon (C) and nitrogen (N) sources as substrates.…”