Abstract：
The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins.
摘要：
在真核生物中用于动态通路调节的血红素响应性生物传感器的开发从未被报道,对实现多功能血液蛋白的有效合成和维持细胞内血红素稳态构成挑战。在这里，含有新鉴定的血红素反应启动子的生物传感器，设计并优化了CRISPR/dCas9和降解标签N-degron，以微调高效供应血红素的毕赤酵母P1H9底盘中的血红素生物合成。在鉴定了文献报道的对血红素不敏感的启动子后，内源性血红素反应启动子通过转录组学挖掘，从不同的调控元件组合中筛选出最优的生物传感器。通过参与血红素生物合成的HEM2基因的转录波动以及随后的细胞内血红素滴度的响应性变化，验证了生物传感器的动态调节模式。我们通过提高高活性猪肌红蛋白和大豆血红蛋白的产量来证明这种监管系统的效率，可用于开发人造肉和人工金属酶。此外，这些发现可以为其他血液蛋白的合成提供有价值的策略。
