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Biotechnol Lett. 2016 Jan;38(1):117-22. doi: 10.1007/s10529-015-1956-4. Epub 2015 Sep 10.

Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering.

Author information

1
College of Life Sciences, Shihezi University, Shihezi, 832000, People's Republic of China. triangle_gz@163.com.
2
College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. soa_zmg@163.com.
3
College of Life Sciences, Shihezi University, Shihezi, 832000, People's Republic of China. 274831213@qq.com.
4
The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. csun@fio.org.cn.
5
The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. zmg@fio.org.cn.
6
The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. zhengli@fio.org.cn.
7
College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. 86944535@qq.com.
8
College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. 66574667@qq.com.

Abstract

OBJECTIVE:

To construct a clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system and use this system to obtain a recombinant Escherichia coli strain possessing the fatty acid metabolism genes from a lipid-rich marine bacterium.

RESULTS:

The fatty acid regulatory transcription factor (fadR), delta9 (Δ(9) desaturase) and acetyl-CoA carboxylase (acc) genes were cloned from Shewanella frigidimarina. The fatty acid regulatory transcription factor (fadD) and phosphoenolpyruvate carboxylase inactivated strains were used to construct the fadR/delta9 and acc knock-in strains, which are both markerless and "scar"-less, and identified the change in fatty acid composition in the recombinant strains. There was no change in fatty acid composition between the wild-type strain and recombinant strains. All strains had 11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:1, 17:0 and 18:0 fatty acids, with 16:0 and 18:0 fatty acids being dominant. The total lipid content of each recombinant strain was higher than the wild-type strain, with a maximum of 13.1 %, nearly 5.3 % higher than wild-type strain.

CONCLUSION:

The CRISPR/cas9 system, in conjunction with λ-Red recombinases, can rapidly and efficiently edit the E. coli genome. The CRISPR/cas9 recombineering machinery can be modified to select biotechnologically-relevant bacteria other than E. coli.

KEYWORDS:

Acetyl-CoA carboxylase; CRISPR/cas9; Fatty acid regulatory transcription factor; Lipid production; Shewanella frigidimarina; Δ9 Desaturase; λ-Red recombinases

PMID:
26358622
DOI:
10.1007/s10529-015-1956-4
[Indexed for MEDLINE]

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