Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here,we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114 increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cellwide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.
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