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Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

While many heterologous molecules can be produced at trace concentrations via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth couplingpara-coumarate to indigoidine production and prototype two cut sets inPseudomonas putidaKT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth onp-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY inpara-coumarate minimal media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.