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

Systems Metabolic Engineering of Novosphingobium aromaticivorans for Lignin Valorization

Authors:

Joshua K. Michener1* (michenerjk@ornl.gov), Marco N. Allemann1, Christopher C. Azubuike1, Ryo Kato2, Hannah R. Valentino1, Fachuang Lu3, Gerald N. Presley1, Leah H. Burdick1, Delyana P. Vasileva1, Dawn M. Klingeman1, Alexander R. Fisch1, Brian C. Sanders1, Lindsay D. Eltis4, James G. Elkins1, Richard J. Giannone1, John Ralph3, Eiji Masai2

Institutions:

1Oak Ridge National Laboratory; 2Department of Materials Science and Bioengineering, Nagaoka University of Technology, Japan; 3Department of Biochemistry, University of Wisconsin–Madison; 4Department of Microbiology and Immunology, University of British Columbia–Canada

Goals

The goal of this project is to engineer a non-model bacterium, Novosphingobium aromaticivorans, for valorization of depolymerized lignin to value-added bioproducts. The project involves (1) discovery and optimization of pathways for assimilation of lignin-derived aromatic compounds; (2) engineering conversion pathways that match the stoichiometry of aromatic catabolism; and (3) development of genome-scale mapping techniques to identify new engineering targets in non-model bacteria.

Abstract

The goal of this project is to engineer a non-model bacterium, Novosphingobium aromaticivorans, for valorization of depolymerized lignin to value-added bioproducts. The project involves (1) discovery and optimization of pathways for assimilation of lignin-derived aromatic compounds; (2) engineering conversion pathways that match the stoichiometry of aromatic catabolism; and (3) development of genome-scale mapping techniques to identify new engineering targets in non-model bacteria.

Lignin is one of the abundant renewable materials found in nature. This heterogeneous aromatic polymer is composed of a variety of p-hydroxyphenyl (H), guaiacyl (G), and syringyl

(S) monomers that are connected by diverse chemical linkages. Lignin valorization would improve biofuel economics, for example, through bacterial conversion of thermochemically depolymerized lignin into valuable bioproducts. N. aromaticivorans F199 is an Alphaproteobacterium capable of degrading G, S, and H monomers and, due to its genetic tractability and broad catabolic capabilities, is an emerging model organism for conversion of lignin-derived aromatic compounds. However, F199 cannot natively catabolize every component of depolymerized lignin, which limits conversion yields (Azubuike et al. 2022).

Researchers are identifying new aromatic degradation pathways to further increase the catabolic potential of F199 using a combination of barcoded transposon insertion sequencing, proteomics, experimental evolution, and in vitro biochemistry. The team demonstrated this approach with the aromatic monomer syringate, the β-1 linked dimer 1,2-diguaiacylpropane-1,3-diol (DGPD), and, more recently, the monomer guaiacol (Bleem et al. 2022; Cecil et al. 2018; Presley et al. 2021). However, there are multiple lignin-derived aromatic compounds that F199 catabolizes poorly or not at all. Researchers have evolved F199 to rapidly and completely catabolize the common β-O-4 dimer guaiacylglycerol-β-guaiacyl ether (GGE) and, in the process, identified an uncharacterized native catabolic pathway for the monomeric intermediate β-hydroxypropiovanillone. Researchers have also isolated a Novosphingobium strain that can assimilate the β-β linked dimer pinoresinol and fully characterized the pinoresinol catabolic pathway. Current efforts focus on transfer of heterologous catabolic pathways into F199.

In addition to optimizing lignin assimilation, researchers are converting the resulting intermediates into value-added products, such as building blocks for bioderived polymers. Using a combination of heterologous pathway expression, experimental evolution, and targeted chromosomal modification, researchers have enabled and improved conversion in F199 of the model lignin-derived aromatic substrate ferulate into 5-aminovaleric acid (5-AVA). Degradation pathways for 5-AVA have also been identified in F199 and are being removed. Additional optimization targets have been identified through metabolomic analysis of wild-type and engineered strains.

Finally, to better understand the effect of host genetic variation on pathway function, researchers are adapting a novel technique, bacterial quantitative trait locus (QTL) mapping, to F199. Researchers have demonstrated directional intraspecific recombination between strains of N. aromaticivorans driven by an Integrative and Conjugative Element (ICE) in the donor strain. The team is currently identifying the origin of transfer of this ICE to improve transfer. By combining novel pathway discovery, heterologous expression, and genome-scale optimization, researchers are engineering N. aromaticivorans F199 to efficiently valorize lignin-derived compounds.

References

Azubuike, C. C., et al. 2022. “Microbial Assimilation of Lignin-Derived Aromatic Compounds and Conversion to Value-Added Products,” Current Opinion in Microbiology 65, 64–72.

Bleem, A. et al. 2022. “Discovery, Characterization, and Metabolic Engineering of Rieske Non-Heme Iron Monooxygenases for Guaiacol O-Demethylation,” Chem Catalysis 2, 1989–2011.

Cecil, J. H., et al. 2018. “Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans,” Applied and Environmental Microbiology 84, AEM.01185-18.

Presley, G. N. et al. 2021. “Pathway Discovery and Engineering for Cleavage of A Β-1 Lignin-Derived Biaryl Compound,” Metabolic Engineering 65, 1–10.

Funding Information

This work was primarily supported by the DOE, Office of Science, BER program, though an Early Career Award to JKM. Additional funding was provided by the Center for Bioenergy Innovation and Great Lakes Bioenergy Research Center, DOE Bioenergy Research Centers supported by the BER program in the DOE Office of Science.