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Analysis and engineering of central metabolism in Clostridium thermocellum

Time: Fri 2023-06-02 10.00

Location: Lärosal 22, House 4 Albano campus, Albanovägen 12

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Language: English

Subject area: Biotechnology

Doctoral student: Teun Kuil , Industriell bioteknologi

Opponent: Professor Ruud Weusthuis, Wageningen University

Supervisor: Professor Antonius J. A. van Maris, Industriell bioteknologi

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QC 2023-04-25


To mitigate climate change, greenhouse gas emissions must be reduced to net-zero in 2050 requiring a drastic transition in today´s energy sector. To achieve this goal, the use of biofuels produced from lignocellulosic feedstocks, including agricultural and forestry residues, is expected to play an important role. The native ability of the anaerobic thermophile Clostridium thermocellum to efficiently degrade lignocellulose makes this microorganism a promising candidate for consolidated bioprocessing of lignocellulosic feedstocks into the biofuel ethanol. However, improvements in ethanol yield, titre, and tolerance are required for industrial implementation. The aim of this thesis was to increase understanding of the central metabolism of C. thermocellum and thereby aid future metabolic engineering and process optimization efforts focused on improving ethanol production from lignocellulosic material. 

The atypical glycolysis of C. thermocellum uses pyrophosphate (PPi) instead of ATP as phosphoryl donor. This alteration is hypothesized to increase energetic efficiency but simultaneously decrease thermodynamic driving force resulting in lower achievable ethanol titres. As such, improved understanding of the PPi metabolism has both fundamental and applied importance. Knockout studies combined with physiological characterization of four predicted metabolic PPi sources provided valuable insights into the PPi metabolism and demonstrated that the energetic benefits of PPi usage are likely limited. Furthermore, biochemical characterization of the ATP-Pfk from C. thermocellum and other bacteria demonstrated that PPi might be a key allosteric regulator in bacteria with a PPi-dependent glycolysis. 

The low thermodynamic driving force of the ethanol formation pathway combined with a flexible redox network are key factors that impact ethanol titre, yield, and tolerance in C. thermocellum. Apart from dominant thermodynamic limitations, physiological characterization of wild-type and a non-ethanol producing mutant at various exogenous ethanol concentrations and temperatures demonstrated that biophysical limitations also impact ethanol tolerance. Lowering the cultivation temperature decreased chaotropic effects of ethanol and improved ethanol tolerance. 

By-product formation and incomplete substrate utilization decrease obtained ethanol yields. To minimize formation of one specific class of by-products, the mechanism behind amino acid secretion in C. thermocellum was investigated. Cellobiose- or ammonium-limited chemostats of wild-type and knockout strains of NADPH-supplying and NADPH-consuming pathways identified catabolic oversupply of NADPH as the main driver behind amino acid secretion. The malate shunt and the ammonium-regulated shift between nitrogen assimilation pathways with differing cofactor specificities were shown to play key roles in NADPH metabolism and amino acid secretion. 

To improve substrate utilization, laboratory evolution combined with reverse metabolic engineering was used as a tool to provide insights into increased utilization of glucose and fructose. Reproducible and constitutive growth on these hexose sugars was achieved for evolved mutant strains. Additionally, two mutations were identified that are involved in (regulation of) transport or metabolism of these hexose sugars.

Together these findings provide valuable insights into the central metabolism of C. thermocellum and aid future optimizations of this organism for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals.