Abstract
The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.
Highlights
Owing to the high yield of recombinant protein production, high similarity of the glycosylation pattern to that in mammalian cells (Balamurugan et al, 2007; Gao et al, 2021), and appropriate folding and secretion of recombinant proteins to the extracellular environment (Yang et al, 2013), yeast Pichia pastoris
A central aim of our work was to establish inducible, heterologous regulators for future use in synthetic biology dealing with P. pastoris
17 artificial transcription factors (ATFs)/binding site (BS) combinations from two Arabidopsis transcription factors (TFs) families, namely: Growth-Regulating Factor 7 (GRF7) (Kim et al, 2012), GRF9 (Kim et al, 2003), the NAC TFs JUNGBRUNNEN1 (JUB1) (Wu et al, 2012), and ANAC102 (Christianson et al, 2009), were selected from our library of plant-derived ATF/BSs developed for S. cerevisiae (Naseri et al, 2017)
Summary
Owing to the high yield of recombinant protein production, high similarity of the glycosylation pattern to that in mammalian cells (Balamurugan et al, 2007; Gao et al, 2021), and appropriate folding and secretion of recombinant proteins to the extracellular environment (Yang et al, 2013), yeast Pichia pastoris Transcriptional regulators play an important role in heterologous protein production (Hartner et al, 2008; Sanjana et al, 2012). Diverse regulators have been developed in recent years for synthetic biology applications in prokaryotic and eukaryotic cells (Hartner et al, 2008; Bruckner et al, 2015; Naseri et al, 2017; Ji et al, 2019), a limited number of artificial regulators are available for P. pastoris. To address the aforementioned challenges, orthogonal transcription factors (TFs) allowing separation of biomass accumulation from the subsequent target molecule production phase, tight control, and tuneable expression of the heterologous gene is desirable (Naseri and Koffas, 2020)
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