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.