CALDICELLULOSIRUPTOR BESCII GENOME-SCALE METABOLIC MODEL SERVES AS A PLATFORM TO REVEAL THE STRATEGIES TO INCREASE ETHANOL AND 2,3-BUTANEDIOL PRODUCTION

Author

Ke Zhang, University of Rhode IslandFollow

Date of Award

2023

Degree Type

Thesis

Degree Name

Master of Science in Biological and Environmental Sciences (MSBES)

Specialization

Cell and Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Ying Zhang

Abstract

The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor bescii (C. bescii), degrades plant biomass at high temperature without any pretreatments and can serve as a strategic platform for industrial applications. C. bescii can utilize various carbohydrates, and its high growth temperature offers benefits such as reduced risk of phage infection and contamination, increased solubility of plant biomass polysaccharides, and the potential to distill volatile products directly from fermentation broths. Therefore, it has become an interest of research and has been metabolically engineered to produce desired bioproducts. The metabolic engineering of C. bescii, however, faces potential bottlenecks in bio-based chemical productions. Genome-scale metabolic modeling (GEM) is a common approach used to examine potential bottlenecks that may be encountered in metabolic engineering for optimizing the production of certain substances. In this work, we reconstructed the GEM for C. bescii based on its genome and other related information from reliable sources (e.g., peer-reviewed literature). The model utilizes subsystems-based genome annotation, targeted reconstruction of carbohydrate utilization pathways, and biochemical and physiological based experimental validations. Specifically, carbohydrate transport and utilization pathways involving 160 genes and their corresponding functions were incorporated, representing the utilization of C5/C6 monosaccharides, disaccharides, and polysaccharides such as cellulose and xylan. To illustrate its utility, the model predicted that optimal production from biomass-based sugars of the model product, ethanol, was driven by adenosine triphosphate (ATP) production, redox balancing, and proton translocation, mediated through the interplay of an ATP synthase, a membrane-bound hydrogenase, a bifurcating hydrogenase and a bifurcating NAD- and NADP-dependent oxidoreductase. Also, it revealed that ATP is a limiting factor for producing another valuable chemical, 2,3-Butanediol (2,3-BDO), via metabolic engineered C. bescii. These mechanistic insights guided the design and optimization of new engineering strategies for product optimization, which were subsequently tested in the C. bescii model, showing a near two-fold increase in ethanol yields. Overall, the C. bescii model provides a useful platform for investigating the potential redox controls that mediate the carbon and energy flows in metabolism and sets the stage for future design of engineering strategies aiming at optimizing the production of ethanol and other bio-based chemicals.

Recommended Citation

Zhang, Ke, "CALDICELLULOSIRUPTOR BESCII GENOME-SCALE METABOLIC MODEL SERVES AS A PLATFORM TO REVEAL THE STRATEGIES TO INCREASE ETHANOL AND 2,3-BUTANEDIOL PRODUCTION" (2023). Open Access Master's Theses. Paper 2331.
https://digitalcommons.uri.edu/theses/2331