Abstract

Efforts to stimulate economic independence while remaining environmentally conscious have motivated researchers to replace non-renewable resources through the engineering of suitable microbes. The efficient generation of polymer precursors, such as maleic acid, through engineered microbial strains will contribute to this aim. This work proposed an engineered strain of Escherichia coli to produce the non-native dicarboxylic acid, maleic acid. The strategy included the design of a synthetic metabolic pathway to overproduce fumaric acid, which would serve as a substrate for the heterologously expressed maleate isomerase. The modifications to the E. coli strain included 6 knockouts, that resulted in a 10-fold increase in fumaric acid production (7.46mg/l vs. 0.78mg/l). The kinetic characteristics of maleate isomerases had not previously been explored in great detail, which prompted the inclusion of a screening of putative isomerases for optimal activity in the synthetic pathway. A library of 55 maleate isomerase candidates was generated from published literature and bioinformatics databases. Activity of the candidates was screened; the favoured forward reaction (maleic acid to fumaric acid) was observed in 23 variants, and the desired reverse reaction was observed in 13 variants. Maleic acid was successfully produced in vivo at a titre of about 100μg/l, which will require significant improvement before industrial implementation is possible. This is however, to our knowledge, the first time a host has been engineered to produce maleic acid using maleate isomerases. Future optimization of this production strain will contribute to a reduced dependence on petroleum as a resource for the polymer precursor, maleic acid.