5 research outputs found
Applying a âMetabolic Funnelâ 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 âmetabolic funnelsâ 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 âfunnelâ 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 âfunnelingâ 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 âMetabolic Funnelâ
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 âphenol-dependentâ 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, â3-dehydroshikimiate-derivedâ
route. Finally, these two most promising pathways were coexpressed
in parallel to create a synthetic âmetabolic funnelâ
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, âfunnelingâ
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