Degradation of cellulosic material by cellulomonas fimi
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Date
29/06/2015Author
Kane, Steven Daniel
Metadata
Abstract
The world stocks of fossil fuels are dwindling and may be all but out before the end of
the century. Despite this there is increasing demand for them to be used for transport,
and the ever increasing green house gases which their use produces. Renewable and less
environmentally damaging forms of fuel are needed. Biofuels, particularly bioethanol,
are a possibility to subsidise or replace fossil fuels altogether. Ethanol produced from
fermentation of starch sugars from corn are already in wide use. As this bioethanol is
currently produced from crops such as corn and sugar cane, that puts fuel crops in direct
competition for space and resources with food crops. This has led to increases in food
prices and the search for more arable land. Hydrolysis of lignocellulosic biomass, a
waste by-product of many industries, to produce the sugars necessary for ethanol
production would ease many of the problems with current biofuels. Degradation of
lignocellulose is not simple and requires expensive chemical pre-treatments and large
quantities of enzymes usually from fungal species making it about 10 times more
expensive to produce than corn starch bioethanol. The production of a consolidated
bioprocessor, an organism able to degrade, metabolise and ferment cellulosic material to
produce ethanol or other useful products would greatly reduce the cost currently
associated with lignocellulosic biofuel.
Cellulomonas fimi ATCC 484 is an actinomycete soil bacterium able to degrade
efficiently cellulosic material. The US Department of Energy (DOE) released the
genome sequence at the start of 2012. In this thesis the released genome has been
searched, for genes annotated as encoding polysaccharide degrading enzymes as well as
for metabolic pathways. Over 100 genes predicted to code for polysaccharide
hydrolysing enzymes were identified. Fifteen of these genes have been cloned as
BioBricks, the standard synthetic biology functional unit, expressed in E. coli and C.
freundii and assayed for endo β-1,4-glucanase activity using RBB-CMC, endo
β-1,4-xylanase activity using RBB-xylan, β-D-xylosidase activity using ONPX,
β-D-cellobiohydrolase activity using ONPC and α-L-arabinofuranosidase activity using
PNPA. Eleven enzymes not previously reported from C. fimi were identified as active
on a substrate with the strongest activities being for 2 arabinofuranosidases (AfsA+B), 4
β-xylosidases (BxyC, BxyF, CelE and XynH), an endoglucanase (CelA), and 2
multifunctional enzymes CelD and XynF, active as cellobiohydrolases, xylosidases and
endoxylanases. Four enzymes were purified from E. coli cell lysates and characterised.
It was found that AfsB has an optimum activity at pH 6.5 and 45ºC, BxyF has optimum
activity at pH 6.0 and 45ºC and XynH has optimum activity at pH 9.0 and 80ºC. XynF
exhibited different optima for the 3 substrates with pH 6.0 and 60ºC for ONPC, pH 4.5
and 50ºC for ONPX and pH 5.5 and 40ºC for RBB-xylan.
Searching the genome and screening genes for activities will help genome annotation in
the future by increasing the number of positively annotated genes in the databases. The
BioBrick format is well suited for rapid cloning and expression of genes to be classified.
Searching and screening the genome has also given insights into the complex and large
network of enzymes required to fully hydrolyse and metabolise the sugars released from
lignocellulose. These enzymes are spread across many different glycosyl hydrolase
families conferring different catalytic activities. The characterisation of these novel
enzymes points towards a system adapted to not only a broad specificity of substrate but
also environmental factors such as high temperature and pH. Genomic analysis revealed
gene clusters and traits which could be used in the design of a synthetic cellulolytic
network, or for the conversion of C. fimi into a consolidated bioprocessor itself.