(15b) Investigating New Pathways and a ‘Funneling’ Strategy to Enhance Production of Chemicals Derived from Shikimic Acid Biosynthesis

The engineering of non-natural pathways is a promising to approach to developing renewable and sustainable alternatives to many conventional petrochemicals. This is true of a multitude of useful chemicals that can be synthesized from precursors of native shikimic acid biosynthesis, including various aromatics and other molecules. For example, several recent studies have demonstrated how Escherichia coli can be engineered for the efficient production of muconic acid which, in turn, can be used as a precursor to adipic acid – a platform chemical used for the synthesis of many different polymers (e.g., nylon 6,6). To date, no fewer than five distinct pathways have been reported for muconic acid production from glucose, each involving different precursors of the shikimic acid pathway and enzyme chemistries. This unique flexibility (i.e., the ability to synthesize the same product via multiple different pathways) arises in part due to the fact that muconic acid is also a naturally-occurring metabolite that arises as a key intermediate in the beta-ketoadipate pathway, used by various microbes to degrade a range of aromatic compounds. Further exploiting this principle, here we demonstrate how three additional routes to muconic acid can be constructed by linking a series of recently-engineering pathways for phenol biosynthesis with its subsequent, partial degradation. The resultant phenol-dependent pathways support muconic acid production at up to 0.5 g/L in shake flasks. We also report a novel, four-step pathway for muconic acid production from endogenous chorismate via p-hydroxybenzoate which supports muconic acid titers of up to about 1 g/L while circumventing auxotrophic limitations experienced via the original, 3-hydroshikimate-derived route. Moreover, as inspired by the mechanisms of ‘biological funneling’ employed by soil microbes for the simultaneous degradation of diverse aromatic mixtures, we investigate the potential of a synthetic ‘funneling’ strategy for enhancing achievable muconic acid titers and yields. More specifically, by co-expressing multiple pathways that stem from different endogenous precursors before then converging on a common intermediate, muconic acid yields have been enhanced by as much as 40% relative to the best performing, single pathway control. In addition to maximizing the availability of precursors, this novel strategy offers a more holistic approach to balancing pathway performance and host fitness. Analogous ‘funneling’ approaches have similarly been extended to investigate the general utility of this strategy for improving the production of aromatic products, including phenol, catechol, and others.