(582j) Fitselect: A Synthetic Gene Circuit to Guide Combinatorial Metabolic Engineering Experiments

The synthetic gene circuit “Feedback Inhibition of Transcription for Substrain Selection (FITSelect)” was developed to guide enrichment of a genomic over-expression library in Escherichia coli BL21 for enhanced production of L-arginine. The genomic library was derived from degenerate oligonucleotide priming of environmental samples using a novel procedure that allows for the use of error-prone PCR while producing 1-5Kb reads.  This procedure resulted in significantly enhanced L-arginine concentrations upon library enrichment.  The FITSelect circuit can contain both native and orthogonal parts including the argR promoter, which is responsive to L-arginine concentrations, and a growth inhibitory gene.  Two inhibitory genes were investigated in this research: (i) the Control of Cell Death (ccdb) gene and (ii) a gene encoding a microfiber-forming peptide. Intracellular levels of CCDB are inversely related to the cell growth rate, and microfiber formation has been shown to significantly impact cell physiology and density.  In continuously subcultured mixed cultures, substrains with the highest growth rate will ultimately dominate, allowing for selective isolation of strains with lower CCDB levels and higher L-arginine concentrations.  Likewise, cells producing and incorporating microfibers are easily separated due to their larger size and density.  This research compares these two competing approaches in deriving a culture with superior production of L-arginine.