Conversion of lignin-derived monomers to muconate by engineered pseudomonas
Disclosed herein are engineered Pseudomonas useful to relieve the metabolic bottleneck of 4-hydroxybenzoate transformation in a muconate accumulating strain on an engineered Pseudomonas by swapping its endogenous para-hydroxybenzoate-3-hydroxylase (PHBH), PobA, with a homolog, PraI, that has a broader cofactor preference.
1 . An engineered Pseudomonas bacterium comprising a gene encoding an exogenous PraI, wherein the bacterium is free of a gene encoding an endogenous PobA and wherein the bacterium increases production of muconic acid from p-coumarate when compared to a non-engineered Pseudomonas bacterium that comprises a gene encoding an endogenous PobA and wherein the bacterium produces at least 40 g/L of muconic acid; and wherein the gene encoding for PraI is greater than 85% identical to SEQ ID NO: 3; and wherein the bacterium has increased production of beta-ketoadipic acid when compared to a non-engineered Pseudomonas bacterium that comprises a gene encoding endogenous PobA.
2 . The engineered Pseudomonas bacterium of claim 1 , wherein the Pseudomonas bacterium is Pseudomonas strain CJ781.
3 . An engineered Pseudomonas bacterium comprising a gene encoding an exogenous PraI, wherein the bacterium is free of a gene encoding an endogenous PobA and is free of a gene encoding an endogenous PcaIJ and wherein the bacterium increases production of muconic acid from p-coumarate when compared to a non-engineered Pseudomonas bacterium that comprises a gene encoding an endogenous PobA and a gene encoding an endogenous PcaIJ and wherein the bacterium produces at least 40 g/L of muconic acid; and wherein the gene encoding for PraI is greater than 85% identical to SEQ ID NO: 3; and wherein the bacterium has increased production of beta-ketoadipic acid when compared to a non-engineered Pseudomonas bacterium that comprises a gene encoding endogenous PobA and a gene encoding endogenous PcaIJ and is free of a gene encoding exogenous PraI.
4 . The engineered Pseudomonas bacterium of claim 3 , wherein the Pseudomonas is Pseudomonas strain AW271.