Engineering Escherichia coli to degrade lignocellulose

UoM administered thesis: Master of Philosophy

  • Authors:
  • Alec Banner

Abstract

The ongoing environmental crisis is affected by our reliance on fossil fuels to fulfil the societal demand for fuels and other petrochemically derived products such as polymers and pharmaceuticals. To fulfil this demand whilst limiting the environmental impact of product generation, pressure is being put on biotechnology to provide ‘green’ solutions to these challenges. Abundant lignocellulose waste is a promising feedstock from which to produce a wide range of compounds. However, high pre-treatment and enzyme saccharification costs limit its commercial application. To reduce these costs, the engineering of a consolidated bioprocessing microorganism capable of degrading lignocellulose and directly producing high-value products, via either native or de novo biosynthetic pathways, is desired. Escherichia coli is an ideal host for development of a consolidated bioprocessing organism as it is easily engineered and already shown to be capable of making a wide range of high value products. In this thesis, E. coli has been shown to be capable of expressing a range of cellulase required for the degradation of lignocellulose. Endoglucanases (CelD, Cel5H and EglZH4), exoglucanase (CbhA) and -glucosidases (Bgl1A and Bgl1B) were assembled into cellulose degrading operons which showed the successful co-expression of cellulases within E. coli. Cell extracts from cells expressing these operons were shown to be capable of releasing free glucose from carboxymethyl cellulose. One of the main challenges associated with the development of consolidated bioprocessing systems is the extracellular localisation of enzymes required to degrade lignocellulose. Free enzyme systems offer the advantage of significantly higher extracellular enzyme loading than surface-display/cellulosome systems. To secrete free enzyme from E. coli, a series of five approaches were tested on -glucosidase, Bgl1A. These approaches included N-terminal signalling tags (N20, HT and OmpA), N-terminal fusion peptide (YebF) and co-expression of the Dickeya dadantii excretory system. Of these approaches, the addition of N-terminal tag, N20, to Bgl1A led to the successful secretion of active hydrolase by E. coli. Further work to secrete members of all enzyme classes could lead to the development of an E. coli strain, capable of directly fermenting lignocellulose to high-value products, for use in industrial consolidated bioprocessing.

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Original languageEnglish
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Award date1 Aug 2019