Growth-coupled evolution and high-throughput screening assisted rapid enhancement for amylase-producing Bacillus licheniformis.

Bacillus licheniformis α-amylase is a thermostable enzyme used in industrial starch hydrolysis. However, difficulties in the genetic manipulation of B. licheniformis hamper further enhancement of α-amylase production. In this regard, adaptive evolution is a useful strategy for promoting the productivity of microbial hosts, although the success of this approach requires the application of suitable evolutionary stress. In this study, we designed a growth-coupled adaptive evolution model to enrich B. licheniformis strains with enhanced amylase productivity and utilization capacity of starch substrates. Single cells of high α-amylase-producing B. licheniformis were isolated using a droplet-based microfluidic platform. Clones with 67% higher α-amylase yield were obtained and analyzed by genome resequencing. Our findings confirmed that growth-coupled evolution combined with high-throughput screening is an efficient strategy for enhanced α-amylase production. In addition, we identified several potential target genes to guide further modification of the B. licheniformis host for efficient protein expression.