Metabolic engineering of Rhodococcus aromaticivorans RHA1 for muconic acid production from lignin

Lignin has potential as a sustainable feedstock to replace fossil fuels in chemical manufacturing. The coupling of chemical fractionation and biocatalysis has emerged as a promising technology to realize this potential. In this process, chemocatalytic fractionation of biomass or lignin yields heterogeneous mixtures of lignin-derived aromatic compounds (LDACs), which are subsequently funneled to target chemicals by microbial cell factories. The recent expansion of genetic toolkits for non-model bacteria offers burgeoning possibilities for engineering bespoke biocatalysts using natural LDAC degraders such as Rhodococcus aromaticivorans RHA1. Herein, we describe the development of an RHA1 biocatalyst to convert a softwood kraft lignin stream containing vanillin, vanillate and acetovanillone to muconic acid by leveraging Serine integrase-Assisted Genome Engineering (SAGE). We increased vanillin metabolism by co-expressing ligV, encoding a vanillin dehydrogenase, and RHA1's endogenous vanACB, encoding a vanillate O-demethylase, partially overcoming the vanillate bottleneck observed when expressing ligV alone. To funnel aromatics to muconic acid, we tested two aromatic acid decarboxylases, finding that AroY with EcdBD efficiently decarboxylated protocatechuate to catechol. We then integrated the Hpe pathway of Rhodococcus rhodochrous GD02 to enable acetovanillone conversion. Finally, deletion of catB enabled muconic acid accumulation. Our biocatalyst, strain RHAAL14, transformed the LDACs derived from the oxidation of softwood kraft lignin to muconic acid with a 97% molar yield and a titer of 1.4 g/L. The iterative, integrated metabolic engineering strategies described in this work advance the development of rhodococcal strains for microbial cell factories.