In planta production of the nylon precursor beta-ketoadipate

Beta-ketoadipate ((3KA) is an intermediate of the (3KA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the in planta synthesis of (3KA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the (3KA pathway. Using Nicotiana benthamiana as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to (3KA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxycis,cis-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (pAt beta KA) with stacked pcaG, pcaH, and pcaB genes into a PCAoverproducing genetic background that expresses AroG and QsuB (referred as QsuB-2). The resulting QsuB-2 x pAt beta KA stable lines displayed (3KA titers as high as 0.25 % on a dry weight basis in stems, along with a drastic reduction in lignin content and improvement of biomass saccharification efficiency compared to wild-type controls, and without any significant reduction in biomass yields. Using biomass sorghum as a potential crop for large-scale (3KA production, techno-economic analysis indicated that (3KA accumulated at titers of 0.25 % and 4 % on a dry weight basis could be competitively priced in the range of $2.04-34.49/kg and $0.47-2.12/kg, respectively, depending on the selling price of the residual biomass recovered after (3KA extraction. This study lays the foundation for a more environmentally-friendly synthesis of (3KA using plants as production hosts.