Bioethanol, produced from organic waste as a
second-generation biofuel, is an important renewable energy source. Here,
recalcitrant carbohydrate sources, such as municipal and agricultural waste,
and plants grown on land not suitable for food crops, are exploited.
The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is
naturally very flexible in its growth substrates and produces a variety of fermentation
products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd.
used metabolic engineering to enhance ethanol production, creating the
production strain TM242 (NCIMB 11955 ∆ldh,
∆pfl, pdhup). Ethanol yield has been increased to 82% of the
theoretical maximum on glucose and up to 92% of the theoretical maximum on
cellobiose. However, this strain still produces acetate, presumably derived
from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate
An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a
homoethanologenic pathway. Yeast and a very limited range of mesophilic
bacteria use the homoethanol fermentation pathway, which employs pyruvate
decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert
pyruvate to ethanol. Despite extensive screening, no PDC has yet been
identified in a thermophilic organism.
Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop
a thermophilic version of the homoethanol pathway for use in Geobacillus spp.
This Thesis reports the in
vitro characterization and crystal structure of one of the most
thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to
improve expression of active PDC at high growth temperatures. This
includes codon harmonization and the successful development of a PET (producer
of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored
as an alternative engineering approach, but did not yield a PDC more
thermostable than ZpPDC.
vitro ZpPDC is
most active at 65°C with a denaturation temperature of 70°C, when sourced from
a recombinant mesophilic host. Codon harmonization improved detectable PDC
activity in G. thermoglucosidasius cultures
grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to
65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of
up to 15°C higher than previously reported for any PDC expressed
|Date of Award||1 Mar 2017|
|Sponsors||Biotechnology and Biological Sciences Research Council & TMO Renewables Ltd|
|Supervisor||Michael Danson (Supervisor) & David Leak (Supervisor)|
- pyruvate decarboxylase
- Geobacillus thermoglucosidasius