(582bi) High-Throughput Optimization of Genetically Refactored Nitrogenase Gene Clusters

We constructed, sequence-verified, and screened a large library (~3 Mb) comprising diverse permutations of refactored nitrogenase clusters to enable extraction of design rules for tuning refactored gene clusters. Genetic refactoring allows for accessing, transferring, and optimizing highly complex and tightly regulated biological processes, such as gene clusters. By removing known or potentially unidentified regulatory components and constructing a modular genetic system built from parts, genetically refactored systems enable fine-tuning of these large multicomponent processes. Previous work in our group demonstrated refactoring of the nitrogenase cluster from Klebsiella oxytoca and developed a hierarchical cloning strategy to construct refactored clusters from scarlessly assembled transcriptional units. Building upon this work, here we present the construction and screening of 120 whole refactored nitrogenase clusters in an E. coli host. This library was designed to investigate the operon structure, the number of transcriptional units comprised (4-11), and the orientation of genes and operons in the refactored nitrogenase cluster. We verified the sequence of the constructed ~23 kb refactored nitrogenase clusters using next-generation sequencing and sequence assembly tools Velvet and Pilon. Additionally, we are investigating the integration of next-generation sequencing technologies to enable increased throughput for building and optimizing large multipart DNA constructs.