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

Abstract:

[Objective] To obtain recombinant strain of Escherichia 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 ΔPEPC (partly knock-out), ΔPEPC, ΔPEPC(partly knock-out)-ΔFadD, ΔPEPC-ΔFadD and ΔFadD. 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 ΔPEPC 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 of E. coli, and opened a new way to construct recombinant E. coli strains.

Key words:CRISPR/Cas, Overlapping PCR, GC-MS, λ-Red recombinases, PEPC (Phosphoenolpyruvate carboxylase), Fatty acid