(182ab) Challenges in Microbial Production of Aniline

Aniline is a simple organic building block used to produce many critical materials, including polyurethanes, essential medicines, agrochemicals, and colorants. However, the predominant process for manufacturing aniline involves concentrated strong acids and stoichiometric amounts of the petrochemical benzene. With the climate transition motivating the adoption of greener processes, the bioproduction of aniline via microbial fermentation could prove a more sustainable alternative. Previous patents from Genomatica, Inc. and Sumitomo Rubber Industries, Ltd. established metabolic pathway designs involving enzymatic decarboxylation of aminobenzoic acid, citing candidate enzymes from the non-oxidative phenolic acid decarboxylase family. We tested representative decarboxylases in a whole-cell biocatalysis format by overexpressing their corresponding genes in Escherichia coli, and we measured <1% yields on supplemented p-aminobenzoic acid as well as no measurable yield on anthranilic acid. Furthermore, mutants of the Enterobacter cloacae decarboxylase cited in the Sumitomo Rubber Industries, Ltd. patent demonstrated consistently worse aniline yields than that of the wild-type enzyme. These activities underperform the already lackluster yields published in the prior art, and ultimately, these decarboxylases are unviable for the posed biosynthetic pathway for aniline. We also observe that other factors, such as product toxicity and aniline’s innate instability in media, further complicate the outlook on microbial aniline manufacture. Successful realization of aniline’s metabolic pathway requires not only a feat of enzyme engineering for high-conversion decarboxylation of aminobenzoic acid, but also an in situ product removal system that will minimize aniline titer in microbial culture. In light of this, competing approaches for bio-derived aniline manufacture, including a hybrid chemical-biological route developed by Covestro AG, can be considered more attainable. As we seek to transition toward a greener bioeconomy, continued investigation of these options for aniline bioproduction may provide insights into the feasibility of commercializing bio-based monocyclic aromatics.