Synthetic transcription factors establish the function of nine amino acid transactivation domains of Komagataella phaffii Mxr1

The zinc finger (ZF) transcription factor Mxr1 (methanol expression regulator 1) of the methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) harbors a DNA-binding domain consisting of two C₂H₂ ZFs (Mxr1ZF) between amino acids 36 and 101 and a previously identified nine amino acid transactivation domain (9aaTAD) between residues 365 and 373 (TAD A, QELESSLNA). Beyond this, 21 putative 9aaTADs (designated TAD B-V) located between amino acids 401 and 1155 remain to be characterized. Here, we demonstrate that a compact synthetic transcription factor composed of Mxr1ZF and three tandem copies of TAD A can activate the transcription of Mxr1 target genes for ethanol and methanol metabolism with specificity and efficiency comparable to the full-length protein. Expression of individual synthetic transcription factors containing Mxr1ZF and each of the 20 putative 9aaTADs in K. phaffii Δmxr1 strain revealed that 10 of these putative TADs are functional, capable of reversing the growth defect of the mutant and activating transcription of target genes required for ethanol and methanol metabolism. Functional analysis indicates that Mxr1 9aaTADs rely on General Control Nondepressible 5 (Gcn5), a histone acetyltransferase, for transactivation. These findings suggest that recruitment of Gcn5-mediated histone acetylation at target promoters is a critical step in transcriptional activation by Mxr1 9aaTADs. This study represents the first comprehensive characterization of 9aaTADs in a K. phaffii ZF transcription factor, providing insights into their mechanism and potential applications in synthetic biology.