Engineering Synthetic Anaerobic Consortia Inspired by the Rumen for Biomass Breakdown and Conversion
Authors:
Bo Zhang1* ([email protected]), Elaina Blair1, Roy Kim2, Joel Howard2, Patrick Leggieri1, Christopher Lawson2, Scott Baker3, Michelle O’Malley1
Institutions:
1University of California–Santa Barbara; 2University of Toronto; 3Pacific Northwest National Laboratory
Goals
This project will leverage a synthetic rumen consortium composed of anaerobic fungi and chain-elongating bacteria to study which metabolites are shared and exchanged between microbes and identify strategies to bolster lignocellulose conversion to value-added products. This approach will develop high-throughput systems and synthetic biology approaches to realize stable synthetic consortia that route lignocellulosic carbon into short- and medium-chain fatty acids (SCFAs/MCFAs) rather than methane. Key research objectives are to: (1) design and predict anaerobic fungal and bacterial consortia that efficiently convert lignocellulosic biomass into MCFAs; (2) understand how fermentation parameters and microbe–microbe interactions regulate and drive microbiome metabolic fluxes; and (3) use genomic editing to alter the fermentation byproducts of anaerobic fungi and bolster MCFA titers and yields.
Abstract
Lignocellulose deconstruction and conversion in nature is driven by mixed microbial partnerships. For example, microbes are particularly well optimized to recycle organic matter in anaerobic habitats, ranging from landfills to intestinal tracts, via interspecies hydrogen transfer and methane release. Compared to aerobic processes, anaerobic digestion can far more efficiently convert substrate to chemical products. This is largely because much less carbon is funneled to cell growth, resulting in higher yields, and far fewer energy inputs are required because pretreatment, aeration, mixing, and heat removal are greatly reduced. Compartmentalizing difficult biomass deconstruction and production steps among specialist anaerobes is an exciting new route to converting biomass into value-added products, especially if consortia can be built predictively and engineered for stability.
Previously, the research team established model bacterial consortia, enriched from the rumen, which convert lignocellulose into high titers of butyrate, a four-carbon (C4) volatile fatty acid (VFA). Metagenomic and metatranscriptomic analyses identified key chain-elongating bacteria in these consortia that maintain high expression of the reverse β-oxidation pathway responsible for production of C4 through C8 VFAs. In parallel, the team demonstrated that anaerobic rumen fungi within the Neocallimastix genus are superior biomass degraders that produce optimal substrates for chain elongators including lactate, acetate, and ethanol. Accordingly, partnering anaerobic fungi and chain-elongating bacteria in synthetic consortia represents a novel strategy for maximizing lignocellulose conversion to C4 through C8 VFAs.
Multiple chain-elongating bacteria were screened, and candidates identified that produce VFAs and grow robustly in culture with known fungal metabolites. The research team paired Pseudoramibacter alactolyticus, a top MCFA producer, with the anaerobic fungus Neocallimastix sp., observing lactate depletion and butyrate and hexanoate production. These strains were paired for several passages and produced consistent metabolic output each time, thus indicating a stable consortium.
Current work involves semi-quantitatively evaluating abundances of consortia members via quantitative polymerase chain reaction (qPCR). The team will also employ RNA sequencing to evaluate differences in gene expression when anaerobic fungi and chain elongators are grown together compared to monoculture, and under different conditions that might increase MCFA production or shift products to longer MCFAs (e.g., such as adding formate into fungal cultures to increase lactate production). These synthetic communities have potential to stably drive conversion of lignocellulose to value-added products.
Anaerobic fungi depend on hydrogenosomes to generate ATP and hydrogen. However, enzymes involved in carbon metabolism and redox balance in hydrogenosomes are not well understood. This accounts for a primary source of uncertainty in genome-scale metabolic models (GSMs) of anaerobic fungi. To address this, the research team isolated hydrogenosomes from Caecomyces churrovis via OptiPrep density gradient centrifugation and confirmed expression of an enzyme complex (i.e., NuoEF and HydA) involved in hydrogen production and redox balance in hydrogenosomes, as well as enzymes for pyruvate metabolism (i.e., PFL and PFOR) using NanoPOTS proteomic analysis and enzyme assays. The function of the heterologous generated NuoEF-HydA complex will be explored with enzyme assays to reveal the role of hydrogenosomal PFL and PFOR by inhibiting PFL with a specific synthesized PFL inhibitor. This approach will enhance understanding of anaerobic fungal metabolism and provide essential data for refining the metabolic modeling of consortia.
Funding Information
This research was supported by the DOE Office of Science, BER Program, grant no. DE-SC0022142.