Engineering E. coli for Consolidated Bioprocessing

  • Alec Banner

Student thesis: Phd

Abstract

The ongoing environmental crisis is in-part, driven by the overuse and reliance on fossil fuels as the carbon feedstock for both fuels and as synthons for the manufacture of chemicals in industries such as pharmaceuticals, materials and flavour/fragrance compounds. Alternative “green” routes to these compounds are being developed using microorganisms for their biosynthesis. Currently, the production of high-value compounds using microorganisms relies on the fermentation of highly-processed, free sugars obtained from crops in direct competition with those used for food and farming. Lignocellulose is a waste feedstock of which billions of tonnes are produced annually. Currently, the release of glucose for fermentation from lignocellulose requires large amounts of energy intensive pre-processing and expensive, commercial cellulase cocktails. The engineering of an organism for CBP would allow the saccharification and fermentation steps to be carried out within a single organism. This could greatly reduce the amount of pre-treatment required and reduce our reliance on commercial enzyme cocktails. The development of an organism for CBP could quickly allow the commercialisation of many biosynthetic processes as many products can already be made biosynthetically. The reduced cost of feedstock could help develop these into green alternatives to traditional petrochemical production. This thesis describes attempts to engineer the model organism E. coli for use in CBP. A range of endo-/ exo-glucanases and b-glucosidases were expressed and characterised in order to select candidates which can then be targeted for secretion. Due to the poorly understood secretory mechanisms across the outer membrane of E. coli, a screening approach was used, whereby each of the selected cellulases was targeted for secretion by a range of signalling tags. This screen led to the identification of two successfully exported cellulases, the processive endoglucanase Cel5H_PelB and the bi-functional endo-/exoglucanase CelD04_AspT. Unfortunately, no exported b-glucosidase candidate was identified and hence, the engineering of a cellulolytic system in E. coli was not possible. Instead, the effect of glucose and glycerol, another alternative feedstock, on the fermentation to linalool were assessed. Challenges with plasmid stability made drawing conclusions difficult but it was clear that both feedstocks were able to produce high titres of linalool. During these fermentations, interesting observations were made relating to the MVA pathway and its effects on acetate overflow metabolism.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorDavid Leys (Supervisor) & Nigel Scrutton (Supervisor)

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