Strategies for Improving Renewable Phenol Biosynthesis in Engineered Escherichia coli

Phenol is a non-renewable petrochemical building block used widely in the production of fine and commodity chemicals (e.g., bisphenol A, caprolactam, and salicylic acid), as well as for a number of important plastics (e.g., phenolic resins).  Whereas a renewable route to phenol from glucose-derived tyrosine via tyrosine phenol lyase has previously been demonstrated, the pathway and overall strategy suffers from several inherent limitations.  Most notable among these is that the key pathway enzyme, namely tyrosine phenol lyase (tpl), suffers from both equilibrium and feedback inhibition limitations that limit metabolite flux and achievable product titers. With the goal of improving renewable phenol bioproduction, we present here our most recent progress related to understanding and addressing these and other critical factors.  To this end, we have focused on the engineering of feedback-resistant tpl mutants with the aid of a novel, high throughput screening assay that couples apparent enzyme activity with growth rate in minimal media.  Isolated mutants are also being reversed engineered to provide an improved understanding of the evolved phenotypes.  Furthermore, through complementary efforts have also engineered two alternative and non-natural pathways linking phenol biosynthesis with native function of the shikimic acid biosynthesis pathway.  Unlike the tpl-derived pathway, both novel pathways terminate with irreversible, phenylacrylic acid decarboxylase reactions so as to drive flux towards the final product.  Both routes were systematically constructed in Escherichia coli following screening studies to identify the most robust pathway enzymes resulting in initial shake flask titers of 50 mg/L.  Although all three routes possess identical theoretical yields, comparison of the relative pathway performance provides interesting insights into pathway design strategies for non-natural chemical production.  Current strategies to increase final product titers through all three routes include pathway de-bottlenecking by development of optimal expression strategies and re-engineering of the host metabolism to maximize production and availability of respective precursors.