Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris

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.

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

“…Alternatively, one can vary the promoter strength driving the expression of these genes. Recent works have identified several constitutive promoters of various strengths that can drive gene expression in P. pastoris [ 36 , 37 , 38 ]. Finally, one can also use metabolic flux analysis to identify bottlenecks in the pathway and engineer the strain further to relieve these bottlenecks [ 39 , 40 ].…”

A regulatory toolkit of arabinose-inducible artificial transcription factors for Gram-negative bacteria

“…In order to establish plant-derived ATFs for bacterial gene expression, full-length plant TFs GRF9, ANAC102 and JUB1-derived ATFs (which function as weak, medium and strong ATFs in yeast 22, 23, 29 ), and the DBD of JUB1, JUB1 DBD (which functions as medium ATF in yeast) were used. The plant TFs were fused to the bacterial SoxS(R93A) activation domain 24, 28 optimized for expression in Salmonella ( Fig.…”

“…Here, we used the same approach as described by Dong et al (2018) to establish ATFs using dCas9 DBD and SoxS AD for tunable gene expression in Salmonella. We further evaluated a novel class of ATFs with heterologous DBDs based on plant-specific transcription factors (TFs) from Arabidopsis thaliana 22,29 , combined with the SoxS AD 23 in order to extend our library of ATFs that allow for inducible gene expression in Gram-negative bacteria. The expression of ATFs is typically controlled by exogenous inducers.…”

“…To address the aforementioned challenges, artificial transcription factors (ATFs) allowing for temporal, tight, and tunable gene expression are favorable. Some ATFs have been developed for synthetic biology applications in both prokaryotic and eukaryotic organisms 22, 23, 24, 25, 26, 27 . However, only a limited number of ATFs are available for Gram-negative bacteria 24, 25, 26, 27 , and to our knowledge, no dedicated ATF to control gene expression in Salmonella has been reported.…”

Advancing Yarrowia lipolytica as a superior biomanufacturing platform by tuning gene expression using promoter engineering

“…The titer of naringenin under xylose-controlled expression reached 715.3 ± 12.8 mg/L in shake-flask cultivation, which was much higher than the 239.1 ± 5.1 mg/L achieved under constitutive expression of UAS1B8TEF. Naseri et al (2017Naseri et al ( , 2021 developed an orthogonal and controllable system in s. cerevisiae and Pichia pastoris, which allows minimal interference with endogenous cellular processes and expression at the desired time. A library of 106 synthetic transcription regulatory units based on plant TFs and their cognate binding sites with up to 2,000-fold IPTG induction level was established.…”