Applying a ‘Metabolic Funnel’ for Phenol Production in Escherichia coli

Phenol is an important petrochemical that is conventionally used as a precursor for synthesizing an array of plastics and fine chemicals. As an emerging alternative to its traditional petrochemical production, multiple enzyme pathways have been engineered to date to enable its renewable biosynthesis from biomass feedstocks, each incorporating unique enzyme chemistries and intermediate molecules. Leveraging all three of the unique phenol biosynthesis pathways reported to date, a series of synthetic ‘metabolic funnels’ was engineered, each with the goal of maximizing net precursor assimilation and flux towards phenol via the parallel co-expression of multiple distinct pathways within the same Escherichia coli host. By constructing and evaluating all possible binary and tertiary pathway combinations, one ‘funnel’ was ultimately identified, which supported enhanced phenol production relative to all three individual pathways by 16 to 69%. Further host engineering to increase endogenous precursor availability then allowed for 26% greater phenol production, reaching a final titer of 554 ± 19 mg/L and 28.8 ± 0.34 mg/g yield on glucose. Lastly, using a diphasic culture including dibutyl phthalate for in situ phenol extraction, final titers were further increased to a maximum of 812 ± 145 mg/L at a yield of 40.6 ± 7.2 mg/g. The demonstrated ‘funneling’ pathway holds similar promise in support of phenol production by other, non-E. coli hosts, while this general approach can be readily extended towards a diversity of other value-added bioproducts of interest

Muconic Acid Production <i>via</i> Alternative Pathways and a Synthetic “Metabolic Funnel”

Muconic acid is a promising platform biochemical and precursor to adipic acid, which can be used to synthesize various plastics and polymers. In this study, the systematic construction and comparative evaluation of a modular network of non-natural pathways for muconic acid biosynthesis was investigated in <i>Escherichia coli</i>, including <i>via</i> three distinct and novel pathways proceeding <i>via</i> phenol as a common intermediate. However, poor recombinant activity and high promiscuity of phenol hydroxylase ultimately limited “phenol-dependent” muconic acid production. A fourth pathway proceeding <i>via</i> <i>p</i>-hydroxybenzoate, protocatechuate, and catechol was accordingly developed, though with muconic acid titers by this route reaching just 819 mg/L, its performance lagged behind that of the established, “3-dehydroshikimiate-derived” route. Finally, these two most promising pathways were coexpressed in parallel to create a synthetic “metabolic funnel” that, by enabling maximal net precursor assimilation and flux while preserving native chorismate biosynthesis, nearly doubled muconic acid production to up to >3.1 g/L at a glucose yield of 158 mg/g while introducing only a single auxotrophy. This generalizable, “funneling” strategy is expected to have broad applications in metabolic engineering for further enhancing production of muconic acid, as well as other important bioproducts of interest