Improving acid resistance of Escherichia coli base on the CfaS-mediated membrane engineering strategy derived from extreme acidophile

Industrial microorganisms used for the production of organic acids often face challenges such as inhibited cell growth and reduced production efficiency due to the accumulation of acidic metabolites. One promising way for improving the acid resistance of microbial cells is to reconstruct their membranes. Herein, the overexpression of cfa2 from extreme acidophile endowed E. coli with high-performance on resistance to the acid stress. The engineered strain M1-93-Accfa2, constructed by CRISPR/Cas9-mediated chromosome integration, also exhibited a significantly higher resistance to severe acid stress. The analysis of fatty acid profiles indicated that the proportion of Cy-19:0 in the cell membrane of M1-93-Accfa2 increased by 5.26 times compared with the control, while the proportion of C18:1w9c decreased by 5.81 times. Correspondingly, the permeability and fluidity of the membrane decreased significantly. HPLC analysis demonstrated that the contents of intracellular glutamic acid, arginine, methionine and aspartic acid of M1-93-Accfa2 were 2.59, 2.04, 22.07 and 2.65 times that of the control after environmental acidification, respectively. Meanwhile, transmission electron microscopy observation indicated that M1-93-Accfa2 could maintain a plumper cell morphology after acid stimulation. M1-93-Accfa2 also exhibited higher-performance on the resistance to organic acids, especially succinic acid stress. These results together demonstrated the great potential of M1-93-Accfa2 constructed here in the production of organic acids.