Systems Metabolic Engineering of Novosphingobium aromaticivorans for Lignin Valorization

Authors:

Marco N. Allemann, Christopher C. Azubuike, Hannah R. Valentino, Gerald N. Presley, Leah Burdick, Richard J. Giannone, and Joshua K. Michener

Institutions:

Oak Ridge National Laboratory

Goals

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

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, potentially through bacterial conversion of thermochemically depolymerized lignin into valuable bioproducts. Novosphingobium 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 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 (Cecil et al. 2018), the β-1 linked dimer 1,2-diguaiacylpropane-1,3-diol (DGPD; Presley et al. 2021), and, more recently, the monomer guaiacol (Bleem et al. 2022). 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. Researchers have also isolated a Novosphingobium strain that can assimilate the β-β linked dimer pinoresinol and identified the relevant catabolic pathway. Current efforts include detailed characterization of the key pinoresinol pathway enzymes and transfer of the pathway into F199.

In addition to optimizing lignin assimilation, researchers are converting the resulting intermediates into value-added products, such as building blocks for bio-derived polymers, and have demonstrated production from a model lignin-derived aromatic compound, ferulate. 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 intraspecific recombination between strain of N. aromaticivorans and are currently studying and optimizing this process. By combining novel pathway discovery, heterologous expression, and 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 ” Current Opinion in Microbiology 65, 64–72.

Cecil, J. H., et al. 2018. “Rapid, 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.

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.

Funding Information

This work was supported by the U.S. Department of Energy Office of Science, through the Office of Biological and Environmental Research (BER) Early Career Research Program. Preliminary data was collected through the BioEnergy Science Center and Center for Bioenergy Innovation, Bioenergy Research Centers funded by BER.