Construction and optimization of a heterologous pathway for protocatechuate catabolism inEscherichia colienables rapid bioconversion of model lignin monomers

Cellulosic biofuel production yields a substantial lignin byproduct stream that currently has few applications. Biological conversion of lignin compounds into chemicals and fuels has the potential to improve the economics of cellulosic biofuels, but few microbes are able both to catabolize lignin and generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we have engineered E. coli to catabolize a model lignin monomer, protocatechuate, as the sole source of carbon and energy, via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with PCA more than two-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for lignin bioconversions. Highlights A heterologous pathway for PCA catabolism was transferred to Escherichia coli. Evolution identified a mutation that increased growth with PCA by 2.5-fold. Optimization plus further engineering allowed efficient catabolism of 4-HB