Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

Abstract

cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroF FBR , aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

“…cerevisiae (Weber et al, 2012, Curran et al, 2013 and E. coli (Sun et al, 2013, Yu et al, 2014, Babu et al, 2015, Sengupta et al, 2015. Exceptions are the early work of the Frost lab on muconic acid production from glucose in E. coli (Draths & Frost, 1994, Frost & Draths, 1997, Niu et al, 2002 and routes via salicylate and from lignin-derived species (Deng et al, 2016) as well as the production of adipic acid with Thermobifida fusca (Deng & Mao, 2015).…”

“…After early work on muconic acid production in the mid 1990s (Frost & Draths, 1996, Frost & Draths, 1997) the target adipic acid has recently moved back into the spotlight with several companies (Genomatica, Verdezyne) filing patents (Boussie et al, 2010, Raemakers-Franken et al, 2012 and the metabolic engineering research community re-focussing on this compound (Niu et al, 2002, IHS Chemical, 2012, Weber et al, 2012, Curran et al, 2013, Sun et al, 2013, Yu et al, 2014, Babu et al, 2015, Clomburg et al, 2015, Deng & Mao, 2015, Horwitz et al, 2015, Reizman et al, 2015, Sengupta et al, 2015, Vardon et al, 2015. Considering the extensive work that has been done it is not surprising that there is a wide variety of proclaimed metabolic routes to produce adipic acid or the precursors muconic or glucaric acid (Figure 4).…”

“…Another shikimate pathway based route, which is a hybrid of the other pathways, producing ccMA from glucose via pHBA (Figure 5d) in E. coli (Sengupta et al, 2015) achieved only a titer that was 216-fold below the benchmark, at a yield of 2.155%.…”

“…To further increase flux to the target compounds the chorismate mutase/prephenate dehydratase (pheA) was disrupted. Recently the complete pathway outgoing from glucose until ccMA was established (Sengupta et al, 2015). A different approach to sustainable ccMA production proceeds outgoing from ligninderived species (Deng et al, 2016).…”

“…Six fundamentally different pathways featuring unique intermediates and enzymatic conversions were compiled from patents (Frost & Draths, 1996, Frost & Draths, 1997, Boussie et al, 2010, Raemakers-Franken et al, 2012 and scientific publications (Draths & Frost, 1994, Weber et al, 2012, Curran et al, 2013, Sun et al, 2013, Yu et al, 2014, Babu et al, 2015, Clomburg et al, 2015, Deng & Mao, 2015, Sengupta et al, 2015, Vardon et al, 2015. In total 16 different main routes for carbon-flow were considered.…”