Tian-Qiong Shi , Cai-Ling Yang , Dong-Xun Li , Yue-Tong Wang , Zhi-Kui Nie
{"title":"建立可选择标记循环系统,用于对镰刀菌进行迭代基因编辑","authors":"Tian-Qiong Shi , Cai-Ling Yang , Dong-Xun Li , Yue-Tong Wang , Zhi-Kui Nie","doi":"10.1016/j.synbio.2024.01.010","DOIUrl":null,"url":null,"abstract":"<div><p>Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus <em>Fusarium fujikuroi</em>. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in <em>F. fujikuroi</em>, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5′-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.</p></div>","PeriodicalId":22148,"journal":{"name":"Synthetic and Systems Biotechnology","volume":null,"pages":null},"PeriodicalIF":4.4000,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405805X24000103/pdfft?md5=263ce68a4e92b98b20bf2ba0ea5b0a9b&pid=1-s2.0-S2405805X24000103-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi\",\"authors\":\"Tian-Qiong Shi , Cai-Ling Yang , Dong-Xun Li , Yue-Tong Wang , Zhi-Kui Nie\",\"doi\":\"10.1016/j.synbio.2024.01.010\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus <em>Fusarium fujikuroi</em>. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in <em>F. fujikuroi</em>, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5′-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.</p></div>\",\"PeriodicalId\":22148,\"journal\":{\"name\":\"Synthetic and Systems Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.4000,\"publicationDate\":\"2024-02-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2405805X24000103/pdfft?md5=263ce68a4e92b98b20bf2ba0ea5b0a9b&pid=1-s2.0-S2405805X24000103-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/S2405805X24000103\",\"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}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Synthetic and Systems Biotechnology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405805X24000103","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}
Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi
Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5′-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.
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.