新金色分枝杆菌CRISPR基因编辑技术的构建及其初步应用
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Construction and Initial Application of Gene Editing Using Type II CRISPR System in Mycobacterium neoaurum JC-12
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    摘要:

    新金色分枝杆菌Mycobacterium neoaurum JC-12是一株用于转化植物甾醇合成甾体激素类药物中间体的重要菌株,传统的基因编辑方法效率低、周期长、操作复杂并且需带入抗性标签。近期CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9系统在许多非模式菌株中成功应用为本研究提供了参考。作者结合非同源黏性末端连接(NHEJ)构建了应用于新金色分枝杆菌基因组编辑CRISPR-Cas系统,对Mycobacterium neoaurum JC-12基因组上催化雄甾-4-烯-3,17-二酮(AD)合成雄甾-1,4-二烯-3,17-二酮(ADD)的3-甾酮-脱氢酶(KSDD)分别设计了敲除和转录激活的靶向sgRNA。结果显示成功敲除了ksdd基因并且突变菌株KSDD的酶活下降了80%,突变株发酵96 h的AD产量比原始菌株提高了30%,并且突变株未代谢产生ADD。转录调控结果显示,靶向-29位点相比于无靶向对照ksdd的转录水平下降了38%,而靶向-83位点和-141位点则对ksdd的转录水平分别提升了2.24倍和3.23倍,同时对应的AD和ADD的产量在对-29位点激活时没有显著变化,而AD和ADD的产量在-83位点分别提升了26%和25%,在-141位点分别提升了29.5%和36%。以上研究为进一步对新金色分枝杆菌基因组水平的代谢工程改造提供了更便捷的方式。

    Abstract:

    Mycobacterium neoaurum JC-12 is an important strain to transform phytosterolsto steroid hormones. Traditional gene editing methods are inefficient, long cycles, complicated to manipulate and resistance labels required. The successful application of CRISPR(clustered regular interspaced short palindromic repeats)/Cas9 system in many non-model strains provides a referencefor this study. A CRISPR-Cas system for genome editing of Mycobacterium neoaurum JC-12 was constructed by combining non-homologous end joining(NHEJ). Targeted sgRNAs were respectively designed for the knockout and transcriptional activation of anthrone-dehydrogenase(KSDD) synthesizing androst-1,4-diene-3,17-dione(ADD) by catalyzing 4-androstene-3,17-dione(AD)on the Mycobacterium neoaurum JC-12 genome. The results showed that the ksdd gene was successfully knocked out and the enzyme activity of KSDD decreased by 80%. The AD yield of the mutant strain after 96 h was 30% higher than that of the wild-type strains, and no ADD was produced. Furthermore, the transcriptional regulation results indicated that the transcriptional level of ksdd at -29 target site decreased by 38% compared to that of the control, while the transcriptional level of target-83 and-141 increased 2.24 and 3.23 times, respectively. However, the corresponding AD and ADD yields did not change significantly when the -29 site was activated. Notably, there was 26% and 25% increase in the yields of AD and ADD respectively at the -83 site, and 29.5% and 36% increase at the -141 site. This study provides amore extensive and convenient way to further implement genome level metabolic engineering in Mycobacterium neoaurum.

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林 春,童丽珍,杨套伟,邵明龙,张 显,徐美娟,廖祥儒,饶志明.新金色分枝杆菌CRISPR基因编辑技术的构建及其初步应用[J].食品与生物技术学报,2021,40(7):50-58.

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  • 在线发布日期: 2021-07-26
  • 出版日期: 2021-07-25

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