(567bh) FITSelect: Genomic Library Enrichment for Product Production without High-Throughput Screening

Senger, R. S., Virginia Tech
Zhou, R., Virginia Tech

Combinatorial approaches to metabolic engineering in microbes have proven extremely effective for enhancing production of biological chemicals and conferring tolerance to growth inhibitors. One approach is to prepare a plasmid library of genomic DNA (gDNA) fragments and then screen that library to locate gDNA inserts that enhance product production or cellular tolerance. When the objective of the engineering strategy also increases the cellular growth rate, this screening process is easy. Transformants containing plasmids with different gDNA inserts are incubated in the same liquid culture. The transformant with the insert enabling the highest growth rate eventually dominates the culture. This is a growth competition assay and is based on the ?survival-of-the-fittest? concept. This works well when the objective is to increase cellular fitness to a toxic chemical. However, diverting cellular resources to product production generally results in a decreased cellular growth rate, making the growth competition assay ineffective for strain selection. Several high-throughput technologies have developed to select hyper-producing transformants in this case, but this requires screening of individual colonies. This, in turn, demands significant resources and specialized equipment. We have developed a method to tie product production to the cellular growth rate such that a higher product production rate leads to an increased growth rate. This allows for selection of hyper-producing strains using a growth competition assay. Our method is called FITSelect (Feedback Inhibition of Transcription for growth Selection), and here we demonstrate its use for the over-production of L-arginine by Escherichia coli K12.