The enzymatic deconstruction of lignocellulosic plant biomass is performed by specialist
microbial species. It is a ubiquitous process within nature and central to the global recycling
of carbon and energy. Lignocellulose is a complex heteropolymer, highly recalcitrant and
resistant to hydrolysis due to the major polysaccharide cellulose existing as a crystalline
lattice, intimately associated with a disordered sheath of hemicellulosic polysaccharides and
lignin. In this thesis I aim to transfer the highly efficient cellulolytic mechanism of the
bacterium Cellulomonas fimi, to that of a suitably amenable and genetically tractable
expression host, in the hopes of better understanding the enzymatic hydrolysis of
lignocellulose. Using tools and concepts from molecular biology and synthetic biology, I
constructed a library of standardised genetic parts derived from C. fimi, each encoding a
known enzymatic activity involved in the hydrolysis of cellulose, mannan or xylan; three of
the major polysaccharides present in lignocellulose.
Characterization assays were performed on individual parts to confirm enzymatic activity and
compare efficiencies against a range of substrates. Results then informed the rational design
and construction of parts into modular devices. The resultant genetic devices were introduced
into the expression hosts Escherichia coli and Citrobacter freundii, and transformed strains
were assayed for the ability to utilize various forms of xylan, mannan and cellulose as a sole
carbon source. Results identified devices which when expressed by either host showed
growth on the respective carbon sources. Notably, devices with improved activity against
amorphous cellulose, crystalline cellulose, mannan and xylan were determined. Recombinant
cellulase expressing strains of E. coli and C. freundii were shown capable of both
deconstruction and utilization of pure cellulose paper as a sole carbon source. Moreover, this
capacity was shown to be entirely unhindered when C. freundii strains were cultured in saline
media. These findings show promise in developing C. freundii for bioprocessing of biomass
in sea water, so as to reduce the use of fresh water resources and improve sustainability as
well as process economics. Work presented in this thesis contributes towards understanding
the complementarities and synergies of the enzymes responsible for lignocellulose
hydrolysis. Moreover, the research emphasizes the merits of standardizing genetic parts used
within metabolic engineering projects and how adopting such design principles can expedite
the research process