Efficient genome engineering in Mycolicibacterium neoaurum using Cas9 from Streptococcus thermophilus.

IF 2 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Biotechnology Letters Pub Date : 2024-12-01 Epub Date: 2024-07-31 DOI:10.1007/s10529-024-03519-7
Gedan Xiang, Tao Liu, Lekai Li, Guihong Lin, Ke Liu, Fengqing Wang
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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.

Abstract Image

利用嗜热链球菌的 Cas9 在新牛磺酸霉菌中进行高效基因组工程。
包括新金牛霉菌在内的非致病性分枝杆菌,由于其天然的甾体代谢途径,可以直接利用植物甾醇大规模工业化生产甾体药物中间体。对 M. neoaurum 进行有针对性的基因改造,有利于选育出高产工程菌,并获得高附加值产品,如孕二烯-20-羧酸(PDC),它是合成某些皮质类固醇的重要前体。在异源 II 型 CRISPR/sth1Cas9 系统的基础上,开发了一种简单的策略来对 M. neoaurum 基因组进行遗传工程。在这里,我们从 pMV261 上构建了一个可定制的质粒工具 pMSC9,并整合了 sth1Cas9 蛋白和相应的 sgRNA 支架。随后,在 pMSC9 中插入与不同目的基因相对应的间隔序列,生成编辑质粒,然后将其转化到 M. neoaurum 中。结果,目标基因在 CRISPR/sth1Cas9 系统的作用下被引入 DNA 双站断裂(DSB),然后通过先天性非同源末端连接(NHEJ)机制进行修复。最后,通过无抗性培养将编辑质粒从正确编辑的M. neoaurum突变体中固化,并以由此产生的删除一个目的基因的突变体为宿主,通过相同的过程删除另一个目的基因。这项研究表明,CRISPR/sth1Cas9工具可以快速编辑新毛孢霉菌株。CRISPR/sth1Cas9系统的编辑模式表明,该工具是对M. neoaurum基因编辑工具箱的重要补充。
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来源期刊
Biotechnology Letters
Biotechnology Letters 工程技术-生物工程与应用微生物
CiteScore
5.90
自引率
3.70%
发文量
108
审稿时长
1.2 months
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.
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