PhD Thesis Defense: Shu Huang

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"From Enzyme Characterization to Pathway Implementation: Engineering NADPH-Linked Ethanol Production in Clostridium thermocellum"

Abstract

Clostridium thermocellum is a promising organism for consolidated bioprocessing of lignocellulosic biomass because it can both solubilize cellulose and ferment the released sugars. However, achieving high ethanol titers remains challenging. One potential limitation is the native NADH-linked alcohol dehydrogenase (ADH) reaction, which becomes increasingly thermodynamically unfavorable as ethanol accumulates. This thesis investigated an alternative ethanol production strategy in which reduced ferredoxin is used to generate NADPH through a ferredoxin:NADP+ oxidoreductase (Fnor), and ethanol formation is driven by an NADPH-linked ADH reaction.

To establish this pathway, candidate Fnor enzymes were first screened for cofactor specificity, catalytic activity, thermostability, and expression in C. thermocellum. Selected Fnor enzymes were then tested in engineered C. thermocellum strains for their effects on ethanol production. The complete NADPH-linked pathway was subsequently reconstructed in vitro using purified enzymes to determine whether pyruvate could be converted to ethanol through the proposed reaction sequence and whether the pathway remained functional at high ethanol concentrations. Finally, the pathway was incorporated into an engineered C. thermocellum background designed to reduce competing electron-sink reactions. Carbon monoxide (CO) adaptation was used to inhibit hydrogenase activity, increase the availability of reduced ferredoxin for FNOR reaction, and improve strain growth under CO.

Multiple thermostable, NADPH-specific Fnor enzymes were identified and successfully expressed in C. thermocellum, and several increased ethanol production relative to an empty-vector control. The reconstructed enzyme pathway converted pyruvate to ethanol with 100% conversion in vitro and remained active in the presence of up to 1 M ethanol. In vivo, CO-adapted strain expression the Fnor enzyme Hth_1218 and AdhEAP125 showed substantially increased ethanol production, with titers increasing from approximately 60 mM to approximately 200 mM from 50 g/L cellobiose.

Together, these results demonstrate that a NADPH-linked ethanol production pathway can function both in vitro and in C. thermocellum. This work establishes an alternative pathway for ethanol production in C. thermocellum and provides a foundation for further engineering toward higher ethanol titers

Thesis Committee

• Daniel G. Olson (chair)
• Lee R. Lynd
• Margaret E. Ackerman
• Carolyn E. Lubner (National Laboratory of the Rockies)

Contact

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