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 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 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.

中文翻译：

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通过比较转录组学开发一种新型血红素生物传感器，在毕赤酵母中产生高活性血红素蛋白


用于真核生物动态通路调节的血红素响应生物传感器的开发尚未见报道，这对实现多功能血红素蛋白的高效合成和维持细胞内血红素稳态提出了挑战。在此，设计并优化了包含新鉴定的血红素响应启动子 CRISPR/dCas9 和降解标签 N-degron 的生物传感器，以微调高效血红素供应 P1H9 底盘中的血红素生物合成。在鉴定出文献报道的对血红素不敏感的启动子后，通过转录组学挖掘内源性血红素响应启动子，并从不同的调控元件组合中筛选出最佳的生物传感器。通过参与血红素生物合成的基因的转录波动以及随后的细胞内血红素滴度的响应变化，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该调节系统的效率，这些蛋白可用于开发人造肉和人造金属酶。此外，这些发现可以为其他血红蛋白的合成提供有价值的策略。