(213a) An Inverse Metabolic Engineering Approach For Tyrosine Production In Escherichia Coli

The development of new tools for introducing random genetic perturbations has, in recent years, allowed for the construction of large combinatorial libraries of strains exhibiting a wide range of cellular phenotypes. This ability to generate such rich phenotypic diversity has significantly expanded the potential in identifying strains with a particular desired property. Through an inverse metabolic engineering approach, these strains can then be further characterized in order to gain a better understanding of how the imposed genetic changes lead to its phenotype and, more importantly, how such information can be used to design subsequent strain optimization strategies. Despite these advances in library construction, however, we have found that for most systems of interest, a severe rate-limiting step occurs in strain identification. Therefore, the successful application of the inverse metabolic engineering approach must first begin with the development of a high throughput screening tool to facilitate target identification from these large combinatorial libraries. Here, we present an inverse metabolic engineering approach for the production of tyrosine in Escherichia coli. We begin with the development of a novel screening tool that allows for the high throughput identification and isolation of recombinant Escherichia coli optimized for tyrosine production. This is accomplished through the introduction of a heterologous gene encoding for a bacterial tyrosinase, which allows us to identify desirable mutants by visual detection of the dark and diffusible pigment melanin. We will discuss the application of this assay in screening a variety of combinatorial libraries generated by transposon mutagenesis and global transcription machinery engineering (gTME). The best strains obtained from these searches will be evaluated and compared in order to formulate new, directed strategies for enhancing tyrosine production in Escherichia coli.