Abstract:

L-phenylalanine（L-Phe）is an important intermediate in the food and pharmaceutical industries. However, due to the complex regulation mechanisms and the long its biosynthetic pathway, it is difficult to achieve the flux balance between each module by relying only on plasmids or long-cycle iterations of genome editing, thus limiting its production. In this study, seven key genes（ppsA, tktA, aroF^fbr, aroE, aroL, pheA^fbr, and tyrB）carrying the constitutive promoter PF11 and a ribosome binding site（RBS）with the sequence GGGGGGGG in the L-Phe biosynthesis pathway were integrated into the genome of Corynebacterium glutamicum. Using the Base Editor-Targeted and Template-free Expression Regulation（BETTER）technology in our labbased on the CRISPR/Cas system and cytidine deaminase, the RBSs for each key gene were simultaneously edited to generate a RBS mutant library enriched with G/A. The library was subjected to high-throughput screening using a fluorescence protein-based L-Phe biosensor combined with a droplet microfluidics system. Finally, four mutant strains were selected from about 70 000 RBS mutants with varied degrees of L-Phe production improvement. The L-Phe titers in these strains at 72 h during shake-flask fermentation were 2.06, 1.30, 4.42, and 7.44 mmol/L, respectively, which were 2.43, 1.17, 6.37 and 11.40 times higher than the control strain C.g-2（0.6 mmol/L）. The simultaneous regulation of multigene expression levels and screening of combinatorial libraries using base editing techniques may provide a feasible strategy for L-Phe engineering breeding.

Key words: multi-gene simultaneous regulation, base editing technique, L-phenylalanine, Corynebacterium glutamicum, metabolic engineering modification, high-throughput screening, droplet microfluidics, biosensor