Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Abstract

Pichia pastoris is a well‐known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent‐free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site‐specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α‐factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co‐expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high‐quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol‐II, and hyaluronic acid. The cell‐surface display of enzymes has obviously increased enzyme stability, and high‐level intracellular expression of acyl‐CoA and ethanol O‐acyltransferase, lipase and d‐amino acid oxidase has opened up applications in whole‐cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High‐level expression of various food‐grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening.

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

“…Therefore, important efforts are being dedicated to increase bioprocess efficiency and profitability. Two widely reviewed complementary approaches are currently being developed to reach these goals, namely strain engineering (Zahrl et al , ; Juturu and Wu, ; Vogl et al , ) and bioprocess optimization (Theron et al , ; Yang and Zhang, ).…”

Expression of multidrug transporter P‐glycoprotein in Pichia pastoris affects the host's methanol metabolism

“…Over one thousand proteins have been successfully expressed in P. pastoris with yields as high as 22 g l −1 for intracellular production of recombinant hydroxynitrile lyase and as high as 14.8 g l −1 for extracellular production of recombinant gelatines (Werten et al ., ; Ren and Yuan, ). Three phenotypes of P. pastoris are being used: Mut + , where both AOX1 (encodes alcohol oxidase I) and AOX2 (encodes alcohol oxidase II) genes are intact; Mut S , a mutant with an inactivated AOX1 gene; and Mut − , where both AOX1 and AOX2 genes are disrupted (Juturu and Wu, ). The Mut + and Mut S strain, able to metabolize methanol as the only carbon source, are the most commonly used strains (Looser et al ., ).…”

Comparative study of μ‐stat methanol feeding control in fed‐batch fermentation of Pichia pastoris producing HBsAg: an open‐loop control versus recurrent artificial neural network‐based feedback control

“…In many previous studies, the classic AOX1 promoter (P AOX1 ), including P AOX1 ‐derived variants, was mostly used for recombinant protein expression in P. pastoris owing to its strong and tightly regulated features . From the industrial perspective, however, the main drawbacks of this type of strain are the high amount of heat production and oxygen requirement as well as the presence of methanol as a toxic and flammable substance in the upstream and sometimes downstream stages . Glyceraldehyde 3‐phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol‐free protein expression without any requirement for induction by shifting the culture from one carbon source to another .…”

“…[11][12][13][14] Glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter is one of the alternative natural promoters for methanol-free protein expression without any requirement for induction by shifting the culture from one carbon source to another. 14 Apart from natural promoters, since the last decade, there has also been growing interest in using other synthetic-engineered promoters to increase the production rate without the major challenges related to P AOX1. 15,16 PFLD1, ICL1 PEX8, YPT1, PCAT1, PPMP20, PDAS2 and NPS are some of the highly regulated promoters with a high production level of heterologous proteins which were extensively described by Vogl et al [15][16][17] Despite the technical and safety constraints related to methanol, P. pastoris with P AOX1 is still the most well-characterized and widely studied strain; [18][19][20][21] it has already been used as an industrial strain for commercial-scale production of recombinant hepatitis B vaccine (HBV) for more than 20 years.…”