Developing a novel heme biosensor to produce high-active hemoproteins in Pichia pastoris through comparative transcriptomics

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Fei Yu , Chenyang Li , Tao Zhang , Jingwen Zhou , Jianghua Li , Jian Chen , Guocheng Du , Xinrui Zhao
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引用次数: 0

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.

通过比较转录组学,开发一种新型血红素生物传感器,在 Pichia pastoris 中生产高活性血蛋白。
在真核生物中开发用于动态途径调控的血红素响应型生物传感器的研究从未有过报道,这对实现多功能血红蛋白的高效合成和维持细胞内血红素平衡构成了挑战。在此,我们设计并优化了一种生物传感器,该传感器包含新发现的血红素响应启动子、CRISPR/dCas9 和降解标签 N-degron,用于微调高效血红素供应 Pichia pastoris P1H9 底盘中的血红素生物合成。在确定了文献报道的对血红素不敏感的启动子后,通过转录组学挖掘出了内源血红素响应启动子,并从不同的调控元件组合中筛选出了最佳生物传感器。参与血红素生物合成的 HEM2 基因的转录波动以及随后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控模式。我们通过改进高活性猪肌红蛋白和大豆血红蛋白的生产,证明了这一调控系统的效率,可用于开发人造肉和人造金属酶。此外,这些发现还能为其他血红蛋白的合成提供有价值的策略。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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