Abstract

Solving environmental and social challenges such as climate change requires a shift from our current non-renewable manufacturing model to a sustainable bioeconomy. To lower carbon emissions in the production of fuels and chemicals, plant biomass feedstocks can replace petroleum using microorganisms as biocatalysts. The anaerobic thermophile Clostridium thermocellum is a promising bacterium for bioconversion due to its capability to efficiently degrade lignocellulosic biomass. However, the complex metabolism of C. thermocellum is not fully understood, hindering metabolic engineering to achieve high titers, rates, and yields of targeted molecules. In this study, we developed an updated genome-scale metabolic model of C. thermocellum that accounts for recent metabolic findings, has improved prediction accuracy, and is standard-conformant to ensure easy reproducibility. We illustrated two applications of the developed model. We first formulated a multi-omics integration protocol and used it to understand redox metabolism and potential bottlenecks in biofuel (e.g., ethanol) production in C. thermocellum. Second, we used the metabolic model to design modular cells for efficient production of alcohols and esters with broad applications as flavors, fragrances, solvents, and fuels. The proposed designs not only feature intuitive push-and-pull metabolic engineering strategies, but also present novel manipulations around important central metabolic branch-points. We anticipate the developed genome-scale metabolic model will provide a useful tool for system analysis of C. thermocellum metabolism to fundamentally understand its physiology and guide metabolic engineering strategies to rapidly generate modular production strains for effective biosynthesis of biofuels and biochemicals from lignocellulosic biomass.

Highlights

  • Global oil reserves will be soon depleted (Shafiee and Topal, 2009), and climate change could become a major driver of civil conflict (Hsiang et al, 2011)

  • We developed a genome-scale metabolic model of the biotechnologically relevant organism C. thermocellum

  • Genome-scale models have a broad range of applications in systems biology, including metabolic engineering, physiological discovery, phenotype interpretation, and studies of evolutionary processes (Feist and Palsson, 2008; Palsson, 2015)

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Summary

Introduction

Global oil reserves will be soon depleted (Shafiee and Topal, 2009), and climate change could become a major driver of civil conflict (Hsiang et al, 2011) These challenges to security and the environment need to be addressed by replacing our current non-renewable production of energy and materials for a renewable and carbon neutral approach (Ragauskas et al, 2006). The metabolic network of C. thermocellum contains various reactions to regulate intracellular concentration levels of NADH, NADPH, and reduced ferredoxin These cofactors are used as electron donors with high specificity throughout metabolism. C. thermocellum possesses several hydrogenases to oxidize these reduced cofactors to molecular hydrogen that is secreted by the cell Removal of these hydrogenases through deletion of ech (encoding the ferredoxindependent hydrogenase, ECH) and hydG (associated with the bifurcating hydrogenase, BIF, and bidirectional hydrogenase, H2ASE_syn) was successfully applied to increase ethanol yield by electron rerouting (Biswas et al, 2015). In a subsequent study, Lo et al (2017) over-expressed rnf (encoding the ferredoxinNAD oxidoreductase, RNF) in the hydG ech strain that is expected to enhance NADH supply, but did not achieve improved ethanol yield

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