High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform

[Image: see text] Screening target microorganisms from a mutated recombinant library plays a crucial role in advancing synthetic biology and metabolic engineering. However, conventional screening tools have several limitations regarding throughput, cost, and labor. Here, we used the fluid array platform to conduct high-throughput screening (HTS) that identified Escherichia coli ‘TesA thioesterase mutants producing elevated yields of free fatty acids (FFAs) from a large (10(6)) mutant library. A growth-based screening method using a TetA-RFP fusion sensing mechanism and a reporter-based screening method using high-level FFA producing mutants were employed to identify these mutants via HTS. The platform was able to cover >95% of the mutation library, and it screened target cells from many arrays of the fluid array platform so that a post-analysis could be conducted by gas chromatography. The ‘TesA mutation of each isolated mutant showing improved FFA production in E. coli was characterized, and its enhanced FFA production capability was confirmed.