{"title":"How to engineer giant enzymes: A methodology for mutagenesis of polyketide synthases in native hosts.","authors":"Susanna Kushnir, Uschi Hübner, Frank Schulz","doi":"10.1016/bs.mie.2025.02.007","DOIUrl":null,"url":null,"abstract":"<p><p>Natural products are a fascinating source of chemical diversity and their biosynthetic pathways of biological complexity. The investigation and engineering of biosynthetic pathways towards polyketides in Actinomycetes provides challenges across all steps of the mutagenesis procedure. The typically GC-rich and long genes require robust PCR protocols. The resulting amplicons, often exceeding 10 kbp in length, require equally robust cloning procedures. Finally, the genetic manipulation of Actinomycetes, especially Streptomyces spp., calls for specialized procedures, in particular when the construction of several hundred variants is needed. This chapter will detail methods for all three steps of the process and have been previously used to generate numerous polyketide synthase variants in several Actinomycete species.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"239-267"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2025.02.007","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/3/7 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Natural products are a fascinating source of chemical diversity and their biosynthetic pathways of biological complexity. The investigation and engineering of biosynthetic pathways towards polyketides in Actinomycetes provides challenges across all steps of the mutagenesis procedure. The typically GC-rich and long genes require robust PCR protocols. The resulting amplicons, often exceeding 10 kbp in length, require equally robust cloning procedures. Finally, the genetic manipulation of Actinomycetes, especially Streptomyces spp., calls for specialized procedures, in particular when the construction of several hundred variants is needed. This chapter will detail methods for all three steps of the process and have been previously used to generate numerous polyketide synthase variants in several Actinomycete species.
期刊介绍：
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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