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

Abstract

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 (106) 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.

Double-Sided Microwells with a Stepped Through-Hole Membrane for High-Throughput Microbial Assays

“…We demonstrated the potential of the double-sided microwell in a rare cell isolation test. In fact, though we used one device for the proof-of-concept experiment in this study, rare cell isolation at a ratio of 1 to 10 8 or 10 9 could be achieved with the device by parallelized use of multiple devices, which is a 14-fold improvement over recent isolation application …”

“…In fact, though we used one device for the proof-of-concept experiment in this study, rare cell isolation at a ratio of 1 to 10 8 or 10 9 could be achieved with the device by parallelized use of multiple devices, which is a 14-fold improvement over recent isolation application. 47 ■ CONCLUSIONS Microwell arrays have long been a popular analytical tool because of user-friendly simplicity for conducting versatile customized assays, meeting basic requirements for routine analytical processes including the chemical manipulation and extraction of numerous partitioned samples. However, advancements in its performance (e.g., reliability, cost, simplicity, and especially throughput) of microwell arrays have stagnated because further development of sample handling techniques has been strictly constrained by the standardized microwell design.…”