Metabolic Engineering of an Aniline Production Pathway in E. coli

• Mekhi Williams, Chemical Engineering, University of Delaware

• Kristala Prather, Chemical Engineering, Massachusetts Institute of Technology

Aniline is a bulk chemical used as an intermediate in the production of dyes, pharmaceuticals, pesticides, and multiple other useful products. Current aniline production requires using the carcinogenic, petroleum-dependent chemical benzene. Using benzene in this chemical process is undesirable due to danger and sustainability concerns, so the development of a better process is in high demand. Metabolic engineering presents a possible solution to this issue. Organisms are capable of facilitating complex chemical reactions on organic matter using a series of enzymes expressed by protein-encoding genes. These enzymatic pathways can be altered by introducing recombinant DNA with genes that overexpress non-native enzymes, allowing for the biosynthesis of novel products. Such a pathway was proposed for the production of aniline in bacteria, but it was not proven to work and lacked optimization for large-scale production. Thus, we sought to introduce the full aniline pathway into E. coli. Four recombinant genes were chemically transformed into E. coli and overexpressed for it to produce aniline from glucose as a feedstock. Expression of non-native enzymes were confirmed via SDS-PAGE analysis of cell lysates, and aniline production was analyzed using HPLC analysis of the titer of engineered cells grown with glucose. Aniline production was optimized by altering plasmid design by substituting homologous genes. By successfully designing a biosynthetic method for producing aniline, this high-use chemical can now be produced with reduced safety and sustainability concerns.