Metabolism in Microbial Communities and the Associated Biochemistry of Polymer Deconstruction
Janine Fu1, Brendan Mahoney1, Allen Takayesu1, Christine Minor1, Giorgia Del Vecchio1, Owen Koetters1, Daniel Ha1, Sergey Knyazev1, Mila Rubbi1, Matteo Pellegrini1, Rachel Ogorzalek Loo1, Katherine Chou2, Robert Clubb1, Joseph A. Loo1 and Robert Gunsalus1* (firstname.lastname@example.org ), and Todd Yeates1
1University of California–Los Angeles DOE Institute for Genomics and Proteomics; and 2National Renewable Energy Laboratory
This team’s microbiology projects within the UCLA DOE Institute for Genomics and Proteomics employ molecular, biochemical, and in silico approaches to examine model microbial communities and their metabolic partners to better understand the processes that drive anaerobic carbon recycling in nature. This information impacts 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 biofuel production. Metabolic pathways for key substrates are being elucidated in model syntrophic communities with focus on their key enzymes and associated oxidation-reduction reactions. Next-generation omics methods are in development to interrogate environmentally relevant pathways and interactions in microbial communities as well as to test newly proposed functions where possible. Using the cellulolytic model microorganism, Clostridium thermocellum, team members are examining how anaerobic microbes synthesize and assemble their extracellular cellulosome structures that degrade lignocellulose.
Major activities within the UCLA-DOE Institute in the past year deal with three core areas of investigation.
Elucidation of syntrophic microbial pathways for metabolism of model substrates. Genomic, proteomic, and informatic studies were performed on defined microbial communities to elucidate how representative fatty acid substrates are metabolized. Core pathway enzymes for short- and branched-chain fatty acids were elucidated and further characterized in Syntrophomonas wolfei and S. wolfei sub sp methylbutyratica cells when grown with Methanospirillum hungatei or Methanobacterium formicicum as the methanogenic partner. Recombinant and structural studies of acyl-CoA reductase enzymes of the beta oxidation pathways were performed to further explore the biochemistry of these thermodynamic limiting steps during syntrophic cell growth. Associated electron transfer pathways leading to hydrogen production were also examined and documented.
To further explore syntrophic microbial diversity, PacBio long read sequencing approaches were used to sequence, assemble, and annotate genomes of previously isolated syntrophic bacterial strains that utilize other model substrates when grown in co-culture with suitable methanogen partners. Team members are extending the gene annotation methods beyond the standard homology-based interferences to those based on co-evolution such as phylogenetic profiling, phenotypic profiling, and operon conservation with the goal of supporting microbial pathway prediction and modeling.
Acyl-lysine modification of syntrophic pathway proteins. Proteomic and mass spectrometry studies were performed to further characterize protein post-translational modifications of carbon and electron transfer pathway enzymes in model syntrophic strains. As protein modification can affect enzyme activity, these data will decipher their relationship with the metabolism of syntrophic microbial communities. Acyl-lysine modifications, which can arise from reactive metabolites, were strikingly found in high abundance in the proteome of model syntrophic bacteria. Acetyl, butyryl, 3-hydroxybutyryl, and crotonyl modifications were observed in both S. wolfei, and S. wolfei sub sp methylbutyratica. Interestingly, the methylbutyratica subspecies, capable of metabolizing longer carbon substrates, also displayed instances of methylbutyrylation, valerylation, and hexanoylation. The type and relative abundance of these modifications do significantly change in response to different carbon sources, correlating with metabolic bottleneck points in the microbes’ degradation pathway.
Cellulosome assembly and display in cellulolytic anaerobic bacteria. In companion microbial studies, the team is investigating how highly cellulolytic anaerobic bacteria synthesize, assemble, and display cellulosomes. Clostridium thermocellum, a model bacterium capable of directly converting cellulosic substrates into ethanol and other biofuels is being used to investigate how the cell fine-tunes the enzyme composition of its cellulosome using anti-σ factors to control gene expression in response to sensing extracellular polymers. Team studies have shown that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit that is likely extended from the cell surface. Current work seeks to elucidate the mechanism of signaling through its Conserved RsgI Extracellular (CRE) domain, which researchers hypothesize is proteolyzed to transduce signals to downstream σ factors that modulate the expression of cellulosome components. Researchers are also employing in silico comparative genomics approaches to identify conserved cellulosome biogenesis pathway components whose functional importance will be assessed in C. thermocellum. The results of these studies will provide new insight into anaerobic carbon recycling by naturally cellulolytic bacteria and could guide rational engineering efforts to create microbes that are capable of converting of plant biomass into biofuels, materials, and chemicals.
Fu, J. Y., et al. 2022. “Dynamic Acylome Reveals Metabolite Driven Modifications in Syntrophomonas wolfei,” Frontiers in Microbiology 13. DOI:10.3389/fmicb.2022.1018220.
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), e2000111.
Muroski, J. M., et al. 2022. “The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationship with Benzoate Degradation,” Molec. and Cellular Proteomics 21(4), 100215.
This research was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-FC-02-02ER63421.