The development of new genetic tools for strain diversification has, in recent years, greatly increased the potential success of various combinatorial strain improvement strategies.  By coupling these cell engineering techniques with powerful high-throughput screens, strains possessing industrially-relevant phenotypes can be readily identified from large assortments of mutants.  Through an inverse metabolic engineering (IME) approach, such mutants can then be further studied and characterized to help guide future strain improvement endeavors. 

Here, we present an IME framework for optimizing tyrosine production in Escherichia coli.  We first employed genetic techniques such as transposon mutagenesis and global transcription machinery engineering (gTME) to create diverse libraries of mutants derived from a pre-engineered strain.  These populations were subsequently screened for desirable tyrosine-producing candidates using a novel assay based on the synthesis of melanin.  Several improved strains were identified in these searches, some of which exhibited more than two-fold increases in yield and titer.  We are currently conducting an in-depth analysis of these mutants in order to formulate new, directed strategies for optimizing tyrosine production in E. coli.