Development of a Genome-Scale Metabolic Model of Clostridium thermocellum and Its Applications for Integration of Multi-Omics Datasets and Computational Strain Design

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.

Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production

“…Reactions in the GEM-iCbes included 589 cytosolic reactions, 60 transmembrane reactions, and 12 extracellular reactions, all associated with specific gene annotations. A high consistency score (99%) was achieved with GEM-iCbes when analyzed using the Memote consistency check ( 31 ), which is comparable to the consistency score of 98%, for a recently updated model of C. thermocellum , iCBI655 ( 18 ) ( Table 1 ).…”

“…Genome-scale models (GEMs) provide a systems-level view of the metabolism and are frequently applied to optimize the production of bio-based chemicals in engineered microorganisms. This approach has been used to model thermophilic bacteria that are capable of degrading cellulose or hemicellulose, such as Clostridium thermocellum (cellulose; T opt = 55 to 60°C [ 17 , 18 ]) and Thermoanaerobacterium saccharolyticum (hemicellulose; T opt = 70°C [ 19 ]), as well as the mesophile Clostridium cellulolyticum ( 20 ), which degrades both cellulose and hemicellulose. In contrast to these organisms, C. bescii is much more thermophilic, growing up to 90°C ( 3 ) and, more importantly, utilizes C5-based hemicellulose, the second major carbohydrate in plant biomass, in addition to C6-based cellulose ( 21 ).…”

Genome-Wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

“…The bacterium’s unique features and its industrial relevance in the conversion of biomass (lignocellulose) to a chemical or fuel draws increasing interest to understand various aspects of this microorganism. In the past decade, research related to this cellulose-degrading bacterium flourished and there are numerous studies on the bacteria’s physiology and metabolism ( Xiong et al, 2016 ; Olson et al, 2017 ; Dash et al, 2019 ; Jacobson et al, 2020 ), cellulosome genesis ( Yoav et al, 2017 ; Kahn et al, 2020 ), genetic tools development ( Olson and Lynd, 2012 ; Marcano-Velazquez et al, 2019 ; Walker et al, 2020 ) genome engineering for improving the conversion of lignocellulose to a target product ( Hon et al, 2017 ; Mazzoli et al, 2020 ; Tafur Rangel et al, 2020 ), computational modeling related to this microbe ( Dash et al, 2017 ; Garcia et al, 2020 ), and beyond.…”

Computational Design and Analysis of Modular Cells for Large Libraries of Exchangeable Product Synthesis Modules

“…Modular design has gained recent interest as an effective approach to understand and redesign cellular systems. 1 In the fields of metabolic engineering and synthetic biology, various modularization strategies 2–7 have been proposed to address the slow and expensive design-build-test cycles of developing microbial catalysts for renewable chemical synthesis. 8 A promising system-level modularization 9 approach is ModCell, 4 that aims to design a modular (chassis) cell compatible with exchangeable production modules that enable metabolite overproduction.…”