(63c) Application of Enzyme Promiscuity to Establish Non-Natural Biosynthetic Pathways for the Production of Phenolic Compounds

Phenolic compounds have extensive applications in solvent, flavor, fragrance, cosmetics and pharmaceutical industries. Due to the lack of natural biosynthetic pathways or the absence of the responsible pathway enzymes, biosynthesis of a large proportion of phenolic compounds have not yet achieved. Here, we report utilization of the enzyme promiscuity to construct novel biosynthetic pathways for the synthesis of several high value chemicals. First, we explored the promiscuity of phenol hydroxylase (PH) from Pseudomonas stutzeri OX1, which enable us to construct a new pathway for the production of pyrogallol. Linking the catechol biosynthetic pathway with the hydroxylation reaction catalyzed by PH enabled 76.7 mg/L pyrogallol in shake flask experiments. To further improve the titer, we constructed an alternative pathway for pyrogallol by identifying and characterizing a highly efficient 2,3-DHBA monooxygenase based on the structural similarity of substrates and products. Over-expression of pathway enzymes, enhancement of the carbon flux into the shikimate pathway, modular optimization of the pathway enzymes and alleviation of the product autoxidation enabled the production of 1035.8 mg/L pyrogallol, which is the highest titer reported so far. Finally, we achieved biosynthesis of salicyl alcohol and gentisyl alcohol for the first time by applying the promiscuity of a carboxylic acid reductase (CAR). Optimization of the carbon flux through the shikimate pathway enabled titer of 594.4 mg/L salicyl alcohol and 30.1 mg/L gentisyl alcohol. Our work not only achieved biosynthesis of several phenolic compounds for the first time, but also demonstrated utilization of enzyme promiscuity to construct non-natural biosynthetic pathways to produce value-added products.