Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Optimizing Biological Funneling of Lignin Streams by Comparison in Several Microbial Platforms


Rebecca A. Wilkes1,2* (, Allison Z. Werner 1,2, Andrew J. Borchert1,2, Richard J. Giannone1,3, Valentina E. Garcia4,5, Gina M. Geiselman4,5, Patrick Suthers1,6, John I. Hendry6, Melanie Callaghan1,7, Eashant Thusoo1,7, Robert L. Hettich1,3, John M. Gladden4,5, Daniel Amador-Noguez1,7, Costas D. Maranas1,6, Gregg T. Beckham1,2, Gerald A. Tuskan1,3


1Center for Bioenergy Innovation; 2National Renewable Energy Laboratory; 3Oak Ridge National Laboratory; 4DOE Joint BioEnergy Institute; 5Biomanufacturing and Biomaterials Department, Sandia National Laboratories; 6Pennsylvania State University; 7University of Wisconsin–Madison



The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, non-model plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) develop sustainable, process-advantaged biomass feedstocks; (2) refine consolidated bioprocessing with cotreatment to create fermentation intermediates; (3) advance lignin valorization for bio-based products and aviation fuel feedstocks; and (4) improve catalytic upgrading for SAF blendstocks certification.


Lignin is a complex aromatic polymer found in plant cell walls and accordingly represents an underutilized, but also recalcitrant, carbon-rich stream in lignocellulosic biorefineries. Processes to valorize lignin to high-value products are therefore of interest. Biological funneling of the heterogeneous lignin-related compounds (LRCs) generated by chemical or enzymatic deconstruction into performance-advantaged products is a promising strategy toward this goal (Rinaldi et al. 2016; Sun et al. 2018). This project compares the catabolic capacities of several microbial platforms for biological funneling and identifies metabolic inefficiencies in the production of muconic acid from LRCs.

To characterize catabolic capabilities of several promising microbial strains as hosts for the valorization of lignin streams, growth, and substrate utilization of six bacterial strains and one yeast were directly compared on representative LRCs. Pseudomonas putida bacteria exhibited the fastest growth rate, highest tolerance, and broadest substrate range when grown on a lignin-rich stream, a model aromatic LRC mixture, guaiacyl (G)-type compounds, p-coumaryl (H)-type compounds, and aliphatic acids. Sphingobium lignivorans bacterial strains utilized the highest concentration of syringyl (S)-type compounds. This work provides a foundational comparison of microbial platforms for LRC catabolism as well as genetic reserves to tap for unique metabolic capabilities.

Systems-level characterization of muconic acid production from aromatic LRCs and biomass production from glucose was conducted in P. putida to identify metabolic inefficiencies and bottlenecks. After rewiring native cellular metabolism of 4-hydroxybenzoate to muconic acid in the production strain P. putida CJ781 (CJ781; Kuatsjah et al. 2022), a bottleneck was identified at the catechol 1,2-dioxygenase. Proteomics, exometabolomics, and fluxomics analyses of glucose conversion to biomass growth and energy revealed that, relative to the wildtype strain, CJ781 exhibited greater secretions of intracellular metabolites, higher periplasmic flux, and increased ATP production. Notably, CJ781 secreted pyruvate and acetate, indicating a potential bottleneck in carbon flux entering the tricarboxylic acid (TCA) cycle. Together, this work improves understanding of divided cellular metabolism between product formation and biomass production and identifies nonintuitive genetic targets for optimization of biological funneling.


Kuatsjah, E., et al. 2022. “Debottlenecking 4-Hydroxybenzoate Hydroxylation in Pseudomonas putida Kt2440 Improves Muconate Productivity from P-Coumarate,” Metabolic Engineering 70, 31–42.

Rinaldi, R., et al. 2016. “Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining, and Catalysis,” Angewandte Chemie International Edition 55, 8164–215.

Sun, Z., et al. 2018. “Bright Side of Lignin Depolymerization: Toward New Platform Chemicals,” Chemical Reviews, 118(2), 614–78.

Funding Information

Funding was provided by the Center for Bioenergy Innovation (CBI) led by Oak Ridge National Laboratory (ORNL). CBI is funded as a DOE Bioenergy Research Center supported by the BER Program in the DOE Office of Science under FWP ERKP886. ORNL is managed by UT-Battelle, LLC, for the DOE under contract no. DE-AC05-00OR22725.