Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris

Abstract

Protein production in Pichia pastoris is often based on the methanol‐inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1. Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P AOX1 strain in terms of titer and process performance.

Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

“…The secreted proteins in the sample supernatant of the bioreactor cultivations and small‐scale screenings were analyzed in duplicates with the LabChip® GXII Touch™ (Perkin Elmer, Inc.) according to the manufacturerʹs recommendations (Prielhofer et al, 2018). The specific productivity between two time points t 0 and t 1 was calculated according to Equation (1) and the average overall q P according to Equation (2).…”

Production of protein-based polymers in Pichia pastoris

“…Because cell lysis and accompanying release of proteolytic activity into the medium reportedly occur less on glycerol (Sinha et al, 2005) and glucose (Mattanovich et al, 2009) than on methanol, it would be interesting to investigate the use of methanol-free approaches in situations of persistent degradation. Strong promoters that do not require methanol induction include the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase ( GAP ) gene (Waterham et al, 1997), the glucose-regulated promoter of the GTH1 gene (Prielhofer et al, 2013; Prielhofer et al, 2018), and synthetic derepression variants of the AOX1 promoter (Hartner et al, 2008; Mellitzer et al, 2014). Also recent methanol-free strategies based on overexpression or deletion of regulators of the wild-type AOX1 promoter appear promising (Chang et al, 2018; Shen et al, 2016; Vogl et al, 2018; Wang et al, 2017).…”

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

“…These are mainly related with the increased costs and risk of storing and using methanol, as well as the increased oxygen demand and high heat production derived from methanol metabolization (Heyland et al, 2010 ; Prielhofer et al, 2013 ; Çalik et al, 2015 ). These shortcomings have given ground to dedicate important efforts to identify and to develop methanol-free alternatives, which can be classified in two categories: discovery of alternative efficient promoters for P. pastoris (P GAP , P PGK , P THI11 , P PYK , P SDH ) (Periyasamy et al, 2013 ; Landes et al, 2016 ; Juturu and Wu, 2018 ; Massahi and Çalik, 2018 ; de Macedo Robert et al, 2019 ) and engineering or development of synthetic promoters (Ata et al, 2017 ; Wang et al, 2017 ; Prielhofer et al, 2018 ; Vogl et al, 2018b ).…”