Abstract: In order to obtain an engineered strain with good genetic stability and high ability to produce L-phenylalanine without plasmid, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to introduce the deinhibited bifunctional enzyme (PheA) of chorisate mutase and prephenate dehydratase into E. coli W3110, and then used the promoters with different strengths to regulate the enzymes involved in the shikimate pathway. The optimal expression intensity of the enzymes in the shikimic acid pathway was determined by shaking flask fermentation. Finally, strain PHE12 with the best performance of L-phenylalanine production was obtained by increasing the supply of the precursor phosphoenolpyruvate. The L-phenylalanine titer of PHE12 was 20.5 g/L after 24 h shaking flask fermentation. And after fed-batch fermentation for 48 h, L-phenylalanine production reached 81.8 g/L, which increased by 12.2% as compared with the highest yield reported in the literature. The production intensity and the conversion rate of sugar and acid were 1.7 g/(L·h) and 0.24 g/g glucose, respectively, indicating that this strain is promising for industrial application.

Key words: L-phenylalanine; Escherichia coli; clustered regularly interspaced short palindromic repeats/Cas9; fermentative production