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

The zinc finger transcription factor Mxr1 (Methanol expression regulator 1) of the methylotropic yeast Komagataella phaffii, aka, Pichia pastoris, harbors a DNA binding domain (DBD) consisting of two C2h2 zinc fingers (Mxr1ZF) between amino acids 36-101 and previously identified nine amino acid activation domain (9aaTAD) between residues 365-373(TAD A). Beyond this 21 putative 9aaTADs (designated TAD B-V) located between amino acids 400-1155 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 Dmxr1 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 Non-derepressible 5 (Gcn5), a hsitone 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 zinc finger transcription factor, providing insights into their mechanism and potential applications in synthetic biology.

甲醇营养型酵母巴斯德毕赤酵母（Komagataella phaffii，旧称Pichia pastoris）的锌指转录因子Mxr1（甲醇表达调控因子1，Methanol expression regulator 1），其氨基酸残基36-101位包含由两个C2H2型锌指（C2h2 zinc fingers，Mxr1ZF）构成的DNA结合结构域（DNA binding domain，DBD），且此前已鉴定出位于残基365-373位的九氨基酸激活结构域（nine amino acid activation domain，9aaTAD，命名为TAD A）。除此之外，位于氨基酸残基400-1155区间内的21个推定九氨基酸激活结构域（命名为TAD B至V）仍有待表征。本研究证实，由Mxr1ZF与3个串联拷贝的TAD A组成的紧凑型人工合成转录因子，可特异性且高效地激活Mxr1靶基因的转录，这些靶基因参与乙醇和甲醇代谢，其激活效果与全长Mxr1蛋白相当。在敲除MXR1的巴斯德毕赤酵母（K. phaffii Δmxr1）菌株中，分别表达携带Mxr1ZF与各推定九氨基酸激活结构域的人工合成转录因子后发现，其中10个推定TAD具有功能活性，能够恢复该突变株的生长缺陷，并激活参与乙醇和甲醇代谢的靶基因的转录。功能分析表明，Mxr1的九氨基酸激活结构域的转录激活依赖于General Control Non-derepressible 5（Gcn5）——一种组蛋白乙酰转移酶。上述研究结果表明，在靶基因启动子区域募集Gcn5介导的组蛋白乙酰化，是Mxr1九氨基酸激活结构域实现转录激活的关键步骤。本研究首次对巴斯德毕赤酵母锌指转录因子中的九氨基酸激活结构域进行了系统性表征，为阐明其作用机制以及在合成生物学中的潜在应用提供了新见解。