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地衣芽孢杆菌是具有广泛应用的重要工业微生物,迄今为止这一菌株的基因编辑工具仍十分有限。CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9系统已在许多物种中成功应用于基因编辑,然而地衣芽孢杆菌极低的转化和重组效率为CRISPR/Cas9系统在这一宿主中的应用带来障碍。本研究构建了诱导型表达Cas9蛋白的重组质粒,成功实现了CRISPR/Cas9系统介导的地衣芽孢杆菌淀粉酶编码基因amy L的敲除。结果显示,所设计的3个sg RNA均能实现目的基因的有效编辑,基因敲除的成功率分别为58%、39%和37%。淀粉酶基因敲除的重组大肠杆菌生长无显著影响,淀粉酶活力为原始菌的0.86%。以上成果为研究地衣芽孢杆菌的基因功能及通过菌种改造提升这一工业微生物的发酵性能提供了新型、有效的基因编辑工具。
Abstract：Bacillus licheniformis is an important industrial microorganism with wide applications. Presently, the genomic editing tools for this species are still limited. CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9 system has been successfully used in many species for genomic editing, while its application in Bacillus licheniformis is seriously hampered by extremely low efficiency in transformation and recombination. In this study, Cas9-inducible-expression recombinant plasmids were constructed, and the knockout of amy L gene,which was a amylase encoding gene from B. licheniformis mediated by CRISPR/Cas9 system, was successfully realized. The results showed that all the three designed sg RNA all can achieve effective editing of target genes,with success rates of 58%, 39% and 37%, respectively. Amylase gene knockout of recombinant Escherichia coli had no significant effect on growth, while its residual activity of α-amylase was only 0.86% compared with that of the parent one. The results brought a new, effective genome editing tool for investigating gene functions in B.licheniformis, as well as engineering the host for improved ferment performance in industry.
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基本信息:
DOI：10.13417/j.gab.036.004188
中图分类号:Q78
引用信息:
[1]李由然,顾正华,张梁,等.CRISPR/Cas9系统介导的地衣芽孢杆菌基因敲除[J].基因组学与应用生物学,2017,36(10):4188-4196.DOI:10.13417/j.gab.036.004188.
基金信息:
国家自然科学基金青年基金项目(31401674);; 十三五国家重点研发计划(2016YFD0401404);; 江南大学自主重点项目(JUSRP51503)共同资助
2017-10-25
2017-10-25