Enabling growth-decoupled recombinant protein production based on the methanol-free P promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of () can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using has often been driven either by the methanol induced promoter (P ) and/or the constitutive promoter (P ).

Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris.

Background: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P, P) was first conducted in chemostat cultures.

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Cell Factory.

The methylotrophic yeast () is currently considered one of the most promising hosts for recombinant protein production (RPP) and metabolites due to the availability of several tools to efficiently regulate the recombinant expression, its ability to perform eukaryotic post-translational modifications and to secrete the product in the extracellular media. The challenge of improving the bioprocess efficiency can be faced from two main approaches: the strain engineering, which includes enhancements in the recombinant expression regulation as well as overcoming potential cell capacity bottlenecks; and the bioprocess engineering, focused on the development of rational-based efficient operational strategies. Understanding the effect of strain and operational improvements in bioprocess efficiency requires to attain a robust knowledge about the metabolic and physiological changes triggered into the cells.

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) P-driven recombinant producer strains with different target gene dosage.

Background: The P-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear.

Results: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two P-driven Candida rugosa lipase 1 (Crl1) producer clones.

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

Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K.