Gedan Xiang, Tao Liu, Lekai Li, Guihong Lin, Ke Liu, Fengqing Wang
{"title":"Efficient genome engineering in Mycolicibacterium neoaurum using Cas9 from Streptococcus thermophilus.","authors":"Gedan Xiang, Tao Liu, Lekai Li, Guihong Lin, Ke Liu, Fengqing Wang","doi":"10.1007/s10529-024-03519-7","DOIUrl":null,"url":null,"abstract":"<p><p>Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":" ","pages":"1319-1332"},"PeriodicalIF":2.0000,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Letters","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s10529-024-03519-7","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/31 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.
期刊介绍:
Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them.
All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included.
Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields.
The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories.
Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.