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

Global Proteomics and Resource Allocation Modeling Reveals Thermodynamic Bottleneck and Highlights Effective Genetic and Metabolic Interventions for C. thermocellum

Authors:

Wheaton L. Schroeder1,3* (wls5190@psu.edu), Tommy Willis1,3, Daven Khana2, Daniel Amador-Noguez2,3, Costas Maranas1,3, Gerald A. Tuskan3

Institutions:

1The Pennsylvania State University; 2University of Wisconsin–Madison; 3Center for Bioenergy Innovation, Oak Ridge National Laboratory

URLs:

Goals

The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, non-model plants and microbes to enable high impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) develop sustainable, process-advantaged biomass feedstocks; (2) refine consolidated bioprocessing with cotreatment to create fermentation intermediates; (3) advance lignin valorization for bio-based products and aviation fuel feedstocks; and (4) improve catalytic upgrading for SAF blendstocks certification.

Abstract

Consolidated bioprocessing (CBP) with Clostridium thermocellum is a promising route to produce renewable biochemicals (such as ethanol) from lignocellulosic biomass. A disadvantage of the organism is the relatively low thermodynamic driving force (TDF) through its glycolysis, which limits ethanol titer, as less favorable energy substrates (e.g., pyrophosphate instead of adenosine triphosphate) or metabolic pathways (such as the malate shunt) are used. Recently an improved genome scale metabolic model (GSMM) was developed to help highlight potential energetic solutions in C. thermocellum (Schroeder et al. 2023). This presentation highlights recent efforts to quantify the proteomic and metabolic cost of low TDF and proposes interventions minimizing proteomic cost while maximizing TDF. Absolute concentration of 18 proteins and relative protein concentration (iBAQ) were measured under cellobiose-limed chemostat growth conditions. The correlation between absolute and iBAQ values were used to estimate absolute concentration for each measured protein. Enolase was discovered to be the enzyme with the greatest number of copies per cell, with more than 3.3 times that of the next most abundant enzyme accounting for more than 14% of estimated total protein mass per cell. Enolase has been previously shown to occur at the end of a series of four near-equilibrium reactions, the enzyme of which accounts for nearly 20% of total protein mass per cell, suggesting C. thermocellum uses a “pull” approach through these steps and is a key target for both improving TDF and reducing protein burden. To evaluate potential interventions, a resource allocation model (RAM) of C. thermocellum was reconstructed from the proteomics data and the recent stoichiometric GSMM model. The RAM was used to quantify proteomic and metabolomic effects of several intervention strategies including improving TDF through these steps, creating a greater “push” effect, and creating a greater “pull” effect. This allows researchers to test possible metabolic solutions in silico, prior to experimental validation via genetic engineering.

References

Schroeder, W. L., et al. 2023. “A Detailed Genome-Scale Metabolic Model of Clostridium thermocellum Investigates Sources of Pyrophosphate for Driving Glycolysis,” Metabolic Engineering 77, 306–22. DOI:10.1016/j.ymben.2023.04.003.

Funding Information

Funding was provided by the Center for Bioenergy Innovation (CBI) led by Oak Ridge National Laboratory. CBI is funded as a U.S. DOE Bioenergy Research Center supported by the BER program in the DOE Office of Science under FWP ERKP886. ORNL is managed by UT-Battelle, LLC for the U.S. DOE under contract no. DE-AC05-00OR22725.