Oligo Pools as an Affordable Source of Synthetic DNA for Cost‐Effective Library Construction in Protein‐ and Metabolic Pathway Engineering

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array‐synthesized oligo pools of thousands of user‐defined sequences (up to ∼350 bases in length) have emerged as a low‐cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next‐generation protein and pathway engineering strategies, such as sequence‐function mapping, enzyme minimization, or de‐novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community.