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

Probing the Mechanisms of Microbial Mediated Polymer Deconstruction on the Molecular- and Systems-Level

Authors:

Robert Clubb1* (rclubb@mbi.ucla.edu), Allen Takayesu1, Christine Minor1, Brendan Mahoney1, Giorgia Del Vecchio1, Ethan Humm1, Daniel Ha1, Rajshree Chettiyar1, Mila Rubbi1, Ann Hirsh2, Rachel R. Ogorzalek Loo1, Joseph A. Loo1, Robert Gunsalus1, Matteo Pellegrini1, Jose Rodriguez1

Institutions:

1UCLA-DOE Institute, University of California–Los Angeles; 2Department of Molecular, Developmental, and Cell Biology, University of California–Los Angeles

URLs:

Goals

The microbiology-based projects within the UCLA-DOE Institute employ molecular, biochemical, genome sequencing, and in silico approaches to better understand biological processes that drive carbon recycling in nature. These findings impact multiple areas of BER interest, including bioconversion of model substrates in natural and manmade environments, the associated biochemistry of key degradative enzymes, and the design of plant-based biomass deconstruction strategies for biobased chemicals production. Genomics programs that instruct metabolic pathways for key substrates are being elucidated in both model and novel microbe systems. Next-generation omics methods are being applied to interrogate environmentally relevant pathways, as well as their interactions with defined microbial communities. In related work, the project seeks to define the pathways used by cellulolytic microbes to degrade lignocellulose and other polymers. Using a combination of experimental and bioinformatics approaches, researchers seek to learn how anaerobic microbes sense environmental changes that induce the synthesis and assembly of extracellular cellulosome-like structures that degrade different types of plant biomass. Collectively, the results of these basic science studies provide fundamental insight into processes that drive carbon recycling and will facilitate the development of new microbial-based methods to produce renewable chemicals and materials from abundant biomass.

Abstract

Elucidation of microbial pathways for metabolism and degradation of model polymeric substrates. Genomic, proteomic, and informatic studies were performed on model and newly identified microbes to elucidate how representative plant-derived substrates are efficiently metabolized. Here, core pathway enzymes for polymers, sugars, and fatty acids are being investigated and further characterized in a spectrum of cellulolytic microbial species. Recombinant, structural, and informatic studies of key enzymes in these pathways were performed to explore the thermodynamic rate-limiting steps during anaerobic cell growth. Associated electron transfer pathways needed for hydrogen and formate production by polymer degrading microbes were also examined.

In complementary studies, proteomic and mass spectrometry methods were performed to further characterize metabolic pathways, protein post-translational modifications (PTMs), and cellular envelope components of model polymer-degrading microbes. Characterizing enzyme-disrupting PTMs will help decipher their relationship with the metabolism of biomass by microbial strains. The team has discovered that acyl-lysine modifications arising from reactive metabolites are strikingly abundant in model beta-oxidation bacteria. Acetyl, butyryl, 3-hydroxybutyryl, and crotonyl modifications were observed in a range of species including Syntrophomonas wolfei and Syntrophus aciditrophicus. Interestingly, the types of modifications that occur are correlated with the complexity of the carbon substrate, and the relative abundance of these modifications significantly change in response to different carbon sources. For example, S. wolfei subsp. methylbutyratica is capable of metabolizing longer carbon substrates displaying diverse methylbutyrylation, valerylation, and hexanoylation modifications.

Probing how bacteria produce cellulosome-like structures for efficient plant polymer degradation. The project’s efforts are focused on elucidating how these bacteria sense different types of biomass to optimize the enzyme composition of the cellulosome and gaining broad insight into how cellulosomes are assembled through comparative genome analyses. Here, the team reports recent results that suggest biomass sensing membrane receptors undergo autoproteolysis via a succinimide intermediate, thereby predisposing them to biomass-induced dissolution triggering gene expression changes in cellulosomal genes. Ongoing efforts are focused on using a transcriptomics-based approach to map the full set of genes controlled by each receptor and to determine the biomass signals which they detect. Finally, the team presents the initial results of a comprehensive analysis of published sequenced genomes from which novel cellulosome displaying bacteria have been identified. The results of this work will shed light onto the diversity of cellulosome architectures present in biology and could facilitate engineering efforts for designer recombinant cellulolytic bacteria.

To explore pathways for plant-derived polymer deconstruction, PacBio long-read sequencing approaches were used to sequence, assemble, and annotate genomes of previously isolated bacterial strains that utilize such substrates when grown in pure or in co-culture with suitable microbial partners. The project is also extending gene annotation methods beyond the standard homology-based interferences of these microbial genomes based on co-evolution such as phylogenetic profiling. To this end, researchers are generating maps of domain interactions by leveraging the data found in UniProt, allowing the mapping of interactions between thousands of domains based on their conservation across 10,000 prokaryotic genomes. The team is developing web-based tools to visualize these interactions and navigate relationships between bacteria.

References

Fu, J. Y., et al. 2022. “Dynamic Acylome Reveals Metabolite Driven Modifications in Syntrophomonas wolfei,” Frontiers in Microbiology 13, 1018220.

Hirsch, A. H., et al. Submitted. “Complete Genomes of Two Variovorax paradoxus Endophytes Isolated from Surface-Sterilized Alfalfa Nodules.”

Mahoney, B. J., et al. 2022. “The Structure of the Clostridium thermocellum RsgI9 Ectodomain Provides Insight into the Mechanism of Biomass Sensing,” Proteins 90, 1457–67.

Muroski, J. M., et al. 2021. “Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets,” Proteomics 21(3-4).

Muroski, J. M., et al. 2022. “The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationship with Benzoate Degradation,” Molecular & Cellular Proteomics 21(4), 100215.

Takayesu, A., et al. Submitted. “Insight into the Autoproteolysis Mechanism of the RsgI9 Anti-σ Factor from Clostridium thermocellum.”

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-FC-02-02ER63421.