Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells
Rana Azeem Ashraf, Matthias Bureik, Mario Andrea Marchisio
{"title":"Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells","authors":"Rana Azeem Ashraf, Matthias Bureik, Mario Andrea Marchisio","doi":"10.1016/j.synbio.2024.03.013","DOIUrl":null,"url":null,"abstract":"<div><p>Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes. In this study, we combined, in the yeast <em>S. cerevisiae</em>, genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac. <em>S. cerevisiae</em> cells were permeabilized and used as enzyme bags in order to host these metabolic reactions. We engineered three different (genetic)-enzymatic basic Boolean gates (YES, NOT, and N-IMPLY). In the YES and N-IMPLY gates, human CYP2C9 was expressed under the galactose-inducible <em>GAL1</em> promoter. The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe<sup>+2</sup>- heme prosthetic group in the active site of the human enzyme. Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts. This new approach will help study precise metabolic attributes of human P450s.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":"9 3","pages":"Pages 406-415"},"PeriodicalIF":4.4000,"publicationDate":"2024-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24000474/pdfft?md5=1b0e86900ca2e71dc42b1d5d44a1c2c4&pid=1-s2.0-S2405805X24000474-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Synthetic and Systems Biotechnology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405805X24000474","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes. In this study, we combined, in the yeast S. cerevisiae, genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac. S. cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions. We engineered three different (genetic)-enzymatic basic Boolean gates (YES, NOT, and N-IMPLY). In the YES and N-IMPLY gates, human CYP2C9 was expressed under the galactose-inducible GAL1 promoter. The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2- heme prosthetic group in the active site of the human enzyme. Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts. This new approach will help study precise metabolic attributes of human P450s.
期刊介绍:
Synthetic and Systems Biotechnology aims to promote the communication of original research in synthetic and systems biology, with strong emphasis on applications towards biotechnology. This journal is a quarterly peer-reviewed journal led by Editor-in-Chief Lixin Zhang. The journal publishes high-quality research; focusing on integrative approaches to enable the understanding and design of biological systems, and research to develop the application of systems and synthetic biology to natural systems. This journal will publish Articles, Short notes, Methods, Mini Reviews, Commentary and Conference reviews.