title: Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: College of Life Sciences, Shihezi University, Shihezi, 832000, People's Republic of China. triangle_gz@163.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Xia
authors.affiliation: College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. soa_zmg@163.com.
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Shihezi University, Shihezi, 832000, People's Republic of China. 274831213@qq.com.
authors.forename: Jian-bo
authors.initials: JB
authors.lastname: Zhu
authors.affiliation: The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. csun@fio.org.cn.
authors.forename: Cheng-jun
authors.initials: CJ
authors.lastname: Sun
authors.affiliation: The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. zmg@fio.org.cn.
authors.forename: Ming-gang
authors.initials: MG
authors.lastname: Zheng
authors.affiliation: The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People's Republic of China. zhengli@fio.org.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.affiliation: College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. 86944535@qq.com.
authors.forename: Ying-hua
authors.initials: YH
authors.lastname: Lou
authors.affiliation: College of Chemical Engineering, Qingdao University, Qingdao, 266071, People's Republic of China. 66574667@qq.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005227:Fatty Acids / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D045522:Recombinases / Q000378:metabolism*; D020592:Shewanella / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D045522:Recombinases
keywords: Acetyl-CoA carboxylase; CRISPR/cas9; Fatty acid regulatory transcription factor; Lipid production; Shewanella frigidimarina; Δ9 Desaturase; λ-Red recombinases