Innovative Bioprocess Strategies Combining Physiological Control and Strain Engineering of Pichia pastoris to Improve Recombinant Protein Production

Abstract

The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris. Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive PGAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient (RQ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate (qP) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher qP values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy.

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses

“…Glucose accumulation in the culture broth was detected after 10 h of fermentation (data not shown), showing that the μ max of the producer strain under this hypoxic condition is lower than the targeted μ. This fact can be attributed to the negative impact on the maximum cell growth capacity under this oxygen-limiting condition and agrees with previously published results (Gasset et al, 2022;Nieto-Taype et al, 2020).…”

Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter

“…All fed-batch cultivations were conducted in a 5 L Biostat B fermenter (Sartorius Stedim, Goettingen, Germany) starting with an initial volume of 2 L. The media composition was the same as described previosuly ( Gasset et al, 2022 ), but with glycerol instead of glucose in the fed-batch feeding solution. For all cultivations, temperature was controlled at 25°C, pH permanently adjusted to 5 with NH 4 OH 15% v/v and the airflow was 2 L min -1 .…”

The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells

“…There are numerous commercial platforms available with strong and inducible expression promoters for P. pastoris . As a result, this species is also widely used in the pharmaceutical industry [ 35 , 39 , 40 ]. Other yeasts used for recombinant protein production include Hansenula polymorpha , Yarrowia lipolytica , Arxula adeninivorans , Kluyveromyces lactis , and Schizosaccharomyces pombe [ 7 , 36 , 41 , 42 ].…”