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利用 CRISPR/Cas9 基因组编辑技术敲除胆固醇诺卡氏菌 NRRL 5767 中的次级醇脱氢酶。

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.

机构信息

Department of Chemistry, Western Illinois University, Macomb, IL, United States of America.

Department of Biological Sciences, Western Illinois University, Macomb, IL, United States of America.

出版信息

PLoS One. 2020 Mar 27;15(3):e0230915. doi: 10.1371/journal.pone.0230915. eCollection 2020.

DOI:10.1371/journal.pone.0230915
PMID:32218601
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7101164/
Abstract

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (88%, w/w) and 10-ketostearic acid (11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

摘要

胆固醇诺卡氏菌 NRRL5767 以将油酸转化为 10-羟基硬脂酸（88%，w/w）和 10-酮硬脂酸（11%，w/w）的能力而闻名。油酸转化为 10-羟基硬脂酸，然后转化为 10-酮硬脂酸，分别被认为是由油酸盐水合酶和仲醇脱氢酶催化的。羟基脂肪酸具有许多工业应用的附加值。本研究的目的是通过 CRISPR/Cas9 基因组编辑技术对胆固醇诺卡氏菌 NRRL5767 菌株进行改良，敲除仲醇脱氢酶基因，从而阻止 10-羟基硬脂酸转化为 10-酮硬脂酸。改良后的菌株将仅从油酸中产生 10-羟基硬脂酸。这种改进将通过消除 10-羟基硬脂酸与 10-酮硬脂酸的下游分离来提高 10-羟基硬脂酸的产量。在这里，我们报告：（1）胆固醇诺卡氏菌 NRRL5767 中两种功能重组油酸盐水合酶同工酶和一种功能重组仲醇脱氢酶的分子克隆和特性。两种油酸盐水合酶同工酶的存在可能解释了油酸转化为 10-羟基硬脂酸的高转化率。（2）通过 Golden Gate 组装构建了一种特异性靶向仲醇脱氢酶基因的 CRISPR/Cas9/sgRNA 嵌合质粒。（3）通过电穿孔将嵌合质粒转化为胆固醇诺卡氏菌 NRRL5767，并筛选出仲醇脱氢酶缺失突变体。两种突变体在蛋白质水平上缺乏仲醇脱氢酶活性以及 DNA 水平上靶向 5'编码区和 5'上游突变得到验证。缺失突变体通过将添加的油酸转化为仅 10-羟基硬脂酸提供了改进，从而消除了 10-羟基硬脂酸与 10-酮硬脂酸的下游分离。据我们所知，我们首次使用 CRISPR/Cas9/sgRNA 介导的基因组编辑技术成功敲除了诺卡氏菌属中的目标基因。

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