DEVELOPMENT OF TRANSCRIPTION FACTOR-BASED WHOLE CELL BIOSENSORS FOR LIGNIN VALORIZATION

  • Leopoldo Ferreira Marques Machado

Student thesis: Phd

Abstract

Valorization of lignin biomass for the sustainable production of high-value chemicals and materials is a key aspect of a nascent global bioeconomy. Challenges in the whole cell bioprocess of this abundant feedstock include its depolymerisation associated with the uptake of the monomeric substrates and biotransformation into valuable products. Whole cell biosensors permit real time intracellular detection and measurement of metabolites allowing for high-throughput screening, evolution and metabolic pathway optimisation applications. Therefore, they are potential biotechnological tools to enable the biosynthetic conversion of lignin biomass feedstocks into value-added products. This thesis presents the development, characterisation and optimisation of transcription factor-based whole cell biosensors for key aromatic substrates and products in the lignin valorization bioprocessing. Firstly, the ferulic acid (FA) biosensor system was developed for the detection of major aromatic chemicals derived from lignin biomass, including ferulic acid. Then, it was employed for the screening of enzymatic degradation of complex biomass sources. Secondly, the protocatechuic acid (PCA) biosensor was developed for detection of protocatechuic acid, a central intermediate in the microorganism-based biocatalysis of lignin. This biosensor was employed for the directed evolution of its transcription factor component PcaV aiming to alter its substrate specificity for sensing of new effectors. The evolution method led to the generation of the Van2 biosensor for detection of aromatic aldehydes, including vanillin. This biosensor was characterised biochemically and structurally. Moreover, the role of the amino acid substitutions that led to vanillin sensing were assessed in vivo. Furthermore, the FA and PCA biosensors were optimised employing a Design of Experiment methodology for dose response curve tuning. Finally, the FA biosensor was applied to demonstrate two biosensor-based strategies to overcome membrane transportation challenges in a lignin whole cell bioprocessing concept. Overall, biosensor systems were developed to permit the detection of key molecules in different stages of the biological valorization of lignin, including the degradation, biocatalysis and final product. They will provide new tools and applications to expedite the biological lignin valorization.
Date of Award1 Aug 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorNicholas Turner (Supervisor) & Neil Dixon (Supervisor)

Keywords

  • Protocatechuic acid
  • Biosensors
  • Synthetic Biology
  • Biotechnology
  • Directed Evolution
  • Lignin valorization
  • Ferulic acid

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