AUTHOR=Naseri Gita , Prause Kevin , Hamdo Housam Haj , Arenz Christoph 

TITLE=Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.676900

DOI=10.3389/fbioe.2021.676900

ISSN=2296-4185

ABSTRACT=<p>The non-conventional yeast <italic>Pichia pastoris</italic> (syn. <italic>Komagataella phaffii</italic>) 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 <italic>P. pastoris</italic>. To establish orthogonal regulators for use in <italic>P. pastoris</italic>, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant <italic>Arabidopsis thaliana</italic>, in combination with the activation domains of yeast <italic>GAL4</italic> and plant <italic>EDLL</italic> and a synthetic promoter harboring the cognate <italic>cis</italic>-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in <italic>P. pastoris</italic>, 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 <italic>P. pastoris</italic> cells that produce β-carotene. Notably, the productivity of β-carotene in <italic>P. pastoris</italic> was ∼4.8-fold higher than that in <italic>S. cerevisiae</italic>, reaching ∼59% of the β-carotene productivity obtained in a <italic>S. cerevisiae</italic> 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 <italic>S. cerevisiae</italic> to <italic>P. pastoris</italic>. 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 <italic>P. pastoris</italic>.</p>