Elucidating and Computing Metabolic Networks for Co-Valorization of Cellulose and Lignin Derivatives

Successful biological valorization of plant matter derivatives relies on optimal carbon balance and redox balance within the metabolic network while minimizing stress on the cellular platforms. However, quantitative and predictive elucidation of this metabolic network, especially within the context of processing monomer mixtures, remains a critical knowledge gap in the bioengineering of lignocellulosic valorization. Members of the Pseudomonas genus, notably strains of Pseudomonas putida, are widely explored for the valorization of lignin and cellulose derivatives. Pseudomonas species, which are metabolically diverse, represent ideal bacterial candidates for engineering valorization objectives due to their innate metabolic capabilities to process both glucose derived from cellulose and the structurally-diverse aromatic monomers derived from lignin. A mechanistic understanding of the responsible metabolic architecture and its regulatory nodes remains largely unknown. This understanding is required to predict the regulatory bottlenecks in substrate valorization and to compute valorization potentials under different substrate stoichiometries, both of which are required to innovate the bioengineering of valorization targets under different feedstock scenarios. The proposed research applies a multi-omics approach that combines isotope-assisted metabolomics, computational fluxomics, comprehensive proteomics, and metabolic engineering to achieve the following three objectives: (1) elucidate the systems-level fluxes through the metabolic network during co-utilization of sugar and aromatic substrates; (2) elucidate the hierarchical metabolism amongst structurally-diverse lignin monomers; (3) engineer cellular metabolic segregation to optimize fluxes to C4 dicarboxylic acids as high-value chemical building blocks. Our intellectual broader impact will involve techno-economic analyses to evaluate economic outcomes of engineered strains and our educational broader impact will consist of outreach programs to engage diverse groups of students at high-school and undergraduate levels.