运用CRISPR/Cas系统敲除大肠杆菌磷酸烯醇式丙酮酸羧化酶基因及其对脂肪酸代谢的影响  

Knocking out phosphoenolpyruvate carboxylase gene by CRISPR/Cas and its influence on fatty acid metabolism in Escherichia coli

作　　者：夏军[1] 郑明刚[2] 王玲[3] 孙承君[2] 郑立[2] 祝建波[1] 

机构地区：[1]石河子大学生命科学学院农业生物技术重点实验室,新疆石河子832003 [2]国家海洋局第一海洋研究所海洋生态重点实验室,山东青岛266061 [3]青岛大学化学化工与环境学院,山东青岛266071

出　　处：《微生物学通报》2016年第8期1864-1871,共8页Microbiology China

基　　金：中央级公益性科研院所基本科研业务费专项资金资助项目(No.2015T05);海洋可再生能源专项资金项目(No.GHME2001SW02)~~

摘　　要：【目的】CRISPR/Cas是目前基因编辑的一个重要的新兴技术,拟通过该技术,对大肠杆菌进行快速、高效的基因编辑,获取脂肪酸代谢工程菌。【方法】利用一个二元载体构建出CRISPR/Cas偶联λ-Red重组酶的系统,重叠PCR扩增出敲除的Donor,最后用电转的方式对Escherichia coli MG1655脂肪酸代谢相关基因进行快速编辑。同时,采用气相色谱-质谱(GC-MS)对脂肪酸进行定性及定量分析。【结果】获得了大肠杆菌ΔPEPC(partial)、ΔPEPC、ΔPEPC(partial)-ΔFad D、ΔPEPC-ΔFad D以及ΔFad D突变体菌株。各缺失体菌株脂肪酸含量均比野生型要高,最高的增长了3.7%,但是敲除ppc获得的脂肪酸增长量并没有预期的高。同时在脂肪酸组成上,各菌株均含有11:0、12:0、13:0、14:0、15:0、16:0、17:1、17:0、18:0,并且16:0,17:0,18:0占主要组成部分。【结论】运用该技术可以快速、高效地对大肠杆菌基因进行编辑,是大肠杆菌工程菌株构建的一个新思路,对其他工程菌的构建提供了一定的理论基础。[Objective] To obtain recombinant strain ofEscherichia coli to study fatty acid metabolism, the newly developed CRISPR/Cas system was used to modify the genome of E. coli MG1655. [Methods] A binary vector was constructed with CRISPR/Cas and λ-Red recombinase. Donor genes were then knockout through overlapping PCR. The recombinant vector plasmid was subsequently electro-transformed into E. coli MG1655 to edit the gene associated with fatty acid metabolism. Furthermore, fatty acid contents were measured quantitatively by gas chromatography-mass spectrometry （GC-MS）. [Results] Six recombinant E. coli strains were obtained, including APEPC （partial）, APEPC, APEPC（partial）-AFadD, APEPC-AFadD and AFadD. Compared to the wild one, all reconstructed strains showed higher yields of total fatty acids with maximum increase of 3.7%, whereas the yield of mutant strain APEPC was lower than expected. The fatty acid of 11：0, 12：0, 13：0, 14：0, 15：0, 16：0, 17：1, 17：0, 18：0 were detected in all strains, and 16：0, 17：0, 18：0 were dominant. [Conclusion] The CRISPR/Cas system with Lambda-Red recombinases was an efficient and rapid tool to modify genes ofE. coli, and opened a new way to construct recombinant E. coli strains.

关 键 词：CRISPR/Cas 反向PCR GC-MS λ-Red重组酶 PEPC(磷酸烯醇式丙酮酸羧化酶) 脂肪酸含量 

分 类 号：Q78[生物学—分子生物学]