Chia-Chuan D. Cho, Waye Michelle Leeuwon, Wenshe Ray Liu
{"title":"嗜甲烷甲酵母菌(Candidatus Methanomethylophilus alvus)吡咯氨酰-tRNA 合成酶的定向进化,在一个蛋白质中遗传性地加入两种不同的非顺式氨基酸","authors":"Chia-Chuan D. Cho, Waye Michelle Leeuwon, Wenshe Ray Liu","doi":"10.1021/acsbiomedchemau.4c00028","DOIUrl":null,"url":null,"abstract":"The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNA<sup>Pyl</sup>, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNA<sup>Pyl</sup> from <i>Candidatus Methanomethylophilus alvus</i> (CmatRNA<sup>Pyl</sup>) is catalytically inert toward PylRS from <i>Methanosarcina mazei</i> (MmPylRS) but has weak activity toward PylRS from <i>Ca. M. alvus</i> (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNA<sup>Pyl</sup>-C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNA<sup>Pyl</sup>-C41AU. This new clone, named R3–14, coupling with CmatRNA<sup>Pyl</sup>-C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant <i>M. mazei</i> tRNA<sup>Pyl</sup> to recognize an ochre codon, to genetically incorporate two different ncAAs, <i>N</i><sup>ε</sup>-(<i>t</i>-butoxycarbonyl)-lysine and <i>N</i><sup>ε</sup>-acetyl-lysine, into one model protein.","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2674 1","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Directed Evolution of Candidatus Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase for the Genetic Incorporation of Two Different Noncanonical Amino Acids in One Protein\",\"authors\":\"Chia-Chuan D. Cho, Waye Michelle Leeuwon, Wenshe Ray Liu\",\"doi\":\"10.1021/acsbiomedchemau.4c00028\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNA<sup>Pyl</sup>, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNA<sup>Pyl</sup> from <i>Candidatus Methanomethylophilus alvus</i> (CmatRNA<sup>Pyl</sup>) is catalytically inert toward PylRS from <i>Methanosarcina mazei</i> (MmPylRS) but has weak activity toward PylRS from <i>Ca. M. alvus</i> (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNA<sup>Pyl</sup>-C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNA<sup>Pyl</sup>-C41AU. This new clone, named R3–14, coupling with CmatRNA<sup>Pyl</sup>-C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant <i>M. mazei</i> tRNA<sup>Pyl</sup> to recognize an ochre codon, to genetically incorporate two different ncAAs, <i>N</i><sup>ε</sup>-(<i>t</i>-butoxycarbonyl)-lysine and <i>N</i><sup>ε</sup>-acetyl-lysine, into one model protein.\",\"PeriodicalId\":29802,\"journal\":{\"name\":\"ACS Bio & Med Chem Au\",\"volume\":\"2674 1\",\"pages\":\"\"},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2024-08-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Bio & Med Chem Au\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1021/acsbiomedchemau.4c00028\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Bio & Med Chem Au","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1021/acsbiomedchemau.4c00028","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Directed Evolution of Candidatus Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase for the Genetic Incorporation of Two Different Noncanonical Amino Acids in One Protein
The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNAPyl, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNAPyl from Candidatus Methanomethylophilus alvus (CmatRNAPyl) is catalytically inert toward PylRS from Methanosarcina mazei (MmPylRS) but has weak activity toward PylRS from Ca. M. alvus (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNAPyl-C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNAPyl-C41AU. This new clone, named R3–14, coupling with CmatRNAPyl-C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant M. mazei tRNAPyl to recognize an ochre codon, to genetically incorporate two different ncAAs, Nε-(t-butoxycarbonyl)-lysine and Nε-acetyl-lysine, into one model protein.
期刊介绍:
ACS Bio & Med Chem Au is a broad scope open access journal which publishes short letters comprehensive articles reviews and perspectives in all aspects of biological and medicinal chemistry. Studies providing fundamental insights or describing novel syntheses as well as clinical or other applications-based work are welcomed.This broad scope includes experimental and theoretical studies on the chemical physical mechanistic and/or structural basis of biological or cell function in all domains of life. It encompasses the fields of chemical biology synthetic biology disease biology cell biology agriculture and food natural products research nucleic acid biology neuroscience structural biology and biophysics.The journal publishes studies that pertain to a broad range of medicinal chemistry including compound design and optimization biological evaluation molecular mechanistic understanding of drug delivery and drug delivery systems imaging agents and pharmacology and translational science of both small and large bioactive molecules. Novel computational cheminformatics and structural studies for the identification (or structure-activity relationship analysis) of bioactive molecules ligands and their targets are also welcome. The journal will consider computational studies applying established computational methods but only in combination with novel and original experimental data (e.g. in cases where new compounds have been designed and tested).Also included in the scope of the journal are articles relating to infectious diseases research on pathogens host-pathogen interactions therapeutics diagnostics vaccines drug-delivery systems and other biomedical technology development pertaining to infectious diseases.