Expressing Of Shewanella Frigidimarina Fatty Acid Metabolic Genes In E.coli And Effect Of Its Fatty Acid Metabolic By CRISPR/Cas9 System

E. coli is a type strain which has clearly genetic background and mature genetic modification(commonly used to construct it), and so far, engineering E. coli has received more deeply research. Most of the reconstruction of the fatty acid way greatly attract the attention of the researchers. As part of cell membrane, fatty acid has an important physiological significant for organism and correlation with our life. Thus constructed engineering E.coli which can produce high level contents of fatty acid has great significance and a value of applications.CRISPR/Cas is an important and emerging gene editing technology. Many researchers have applied this technology to many species and fields. Our study aimed to take E.coli as target, we constructed CRISPR/Cas system to study some genes which correlation with fatty acid metabolism. Meanwhile we use this system to obtain a recombinant E. coli strain possessing the fatty acid metabolism genes from a lipid-rich marine bacterium(Shewanella frigidimarine). To do this research, we want to clarify the function of CRISPR/Cas in E.coli and evaluate the efficiency of this tool in gene modification, what is more, we expect to increase the contents of fatty acid in E.coli. The experimental results are as follows:(1) Design the spacer which is a significant part of gene editing by the Web http://spot.colorado.edu/~slin/cas9.html, then construct the spacer into the plasmids which function as CRISPR/Cas with high efficiency of gene editing through overlap PCR. For obtain precisely gene editing, we successfully cloned the donor which in correspondence with target gene. Finally, we successfully electroporated the donor and plasmid of CRISPR/Cas system into E.coli.(2) We successfully obtained the ΔPEPC(partly konck-out), ΔPEPC, ΔPEPC(partly konck-out)-ΔFad D, ΔPEPC-ΔFad D and ΔFad D recombinant strains. and the fatty acid regulatory transcription factor(fad R), delta9(Δ9 desaturase) and acetyl-Co A carboxylase(acc) genes were successfully cloned from Shewanella frigidimarina, then we knock this gene into E. coli genome alone or in series. Identified by PCR, we successfully obtained the ΔPEPC::F/D, ΔPEPC::ACC, ΔFad D::F/D and ΔFad D::ACC recombinant strains.(3) Identified by GC-MS, total lipid content of each constructed strain was higher than wild type strain, and the highest nearly 3.7 % higher than wild type strain, but the fatty acid contents of ΔPEPC mutant strain has no obvious change than the wild type strains. For the ΔPEPC::F/D, ΔPEPC::ACC, ΔFad D::F/D and ΔFad D::ACC recombinant strains, the total lipid content of each recombinant strain was also higher than the wild-type strain, with a maximum of 13.1 %, nearly 5.3 % higher than wild-type strain. 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.