Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

Abstract

Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.

Keywords: Komagataella phaffii; Pichia pastoris; cell engineering; metabolic engineering; recombinant protein.

Figure 1

Schematic overview of the main pathways, proteins, and transcription factors involved in cellular engineering strategies for the improvement of recombinant protein production in Komagataella phaffii. Abbreviations: Hac1: UPR-regulating transcription factor; Yap1: oxidative stress response transcription factor; Mxr1: methanol expression regulator 1; Pdi1: protein disulphide isomerase; Kar2: immunoglobulin-binding protein; Ero1: endoplasmic reticulum oxidoreductase; Sly1: hydrophilic protein involved in ER/Golgi vesicles trafficking; Sec1: Sm-like protein involved in docking and fusion of exocytic vesicles; Gpx1: glutathione peroxidase; Glr1: glutathione reductase; Pmp20: peroxisome-membrane-associated protein 20; Cat1: catalase; Das1/2: dihydroxyacetone synthase 1 and 2; Fld: formaldehyde dehydrogenase; Fdh: formate dehydrogenase; Zwf1: glucose-6-phosphate dehydrogenase; Sol3: 6-gluconolactonase; GND2: 6-phosphogluconate dehydrogenase.

Figure 2

Diagram illustrating the main steps in the MUT pathway and co-substrate alternatives for enhancement of biomass and energy supply in Komagataella phaffii. AOX: alcohol oxidase; CAT: catalase; DAS: dihydroxyacetone synthase; FLD: formaldehyde dehydrogenase; FGH: S-formylglutathione hydrolase; FDH; formate dehydrogenase; ADH: alcohol dehydrogenase; GS(H): glutathione; CMC: carboxymethyl cellulose.