Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds

Author:

Clarkson Sonya M.1,Giannone Richard J.23,Kridelbaugh Donna M.1,Elkins James G.1,Guss Adam M.1,Michener Joshua K.13ORCID

Affiliation:

1. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

2. Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

3. BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Abstract

ABSTRACT The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to 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 engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, 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 protocatechuate more than 2-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 conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.

Funder

U.S. Department of Energy

DOE | LDRD | Oak Ridge National Laboratory

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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