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Title: Geobacillus thermoglucosidans as a thermophile chassis for synthetic biology
Author: Reeve, Benjamin David
ISNI:       0000 0004 7656 8831
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Current reliance on petrochemicals for fuel and chemical production is environmentally damaging and unsustainable. The most promising alternative is bioconversion of lignocellulosic biomass however the organisms commonly used in microbial fermentations for chemical production are not well suited to utilise this feedstock. Alternative microbes for lignocellulose utilisation have been identified and Geobacillus thermoglucosidans is one of the best-adapted organisms currently known. This organism has been used for production of biofuels from lignocellulose but it currently lacks the tools and genetic parts needed to produce a wider variety of products. In this study, novel tools and genetic parts to enable synthetic biology with G. thermoglucosidans were developed. The thermostability of reporter proteins, superfolder GFP, mCherry and flavin-based anaerobic fluorescent proteins was tested and superfolder GFP was shown to be the best reporter protein available for Geobacillus. Two novel constitutive promoter libraries were then generated and characterised. In both G. thermoglucosidans and E. coli, both libraries showed over a 100-fold range of expression strength with the strongest variants comparable in strength to the strongest previously reported Geobacillus promoter pLdh. Predictable tuning of expression strength in G. thermoglucosidans was further demonstrated using translation initiation rate calculator software and the limitations of such tools were reviewed. Finally, a set of seven modular shuttle vectors was developed and characterised. The resulting Geobacillus toolkit allowed for the first time, attempts to produce a more complex biobased product via G. thermoglucosidans genetic engineering. An operon was designed and constructed for biosynthesis of hyaluronic acid, using a newly-discovered hyaluronan synthase from the moderate thermophile Streptococcus thermophilus. The promise of this new enzyme was shown in E. coli where heterologous hyaluronic acid production was demonstrated. The parts and tools developed here for enable more sophisticated genetic engineering with G. thermoglucosidans, making this the first chassis for thermophile synthetic biology.
Supervisor: Ellis, Tom Sponsor: TMO Renewables Ltd ; Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral