There is increasing concern regarding alternative, sustainable energy sources, such
as biofuels, to replace declining oil reserves. The abundance of lignocellulosic
biomass makes it the only imaginable resource that can potentially substitute a
substantial portion of the fossil fuels we use today, but current methods for producing
biofuels from non-food crops are cost intensive and not economically viable. Synthetic
biology provides several potential approaches for developing biologically mediated
processes for the conversion of lignocellulosic biomass into biofuels. Such systems
are based on engineered microbes that produce enzymes for catalysing the
conversion of cellulose into fermentable sugars and subsequently into high value
products. Effective degradation of cellulose requires multiple classes of enzyme
working together. In naturally occurring cellulose degrading microbes, bioconversion
is catalysed by a battery of enzymes with different catalytic properties. However,
naturally occurring cellulases with multiple catalytic domains seem to be rather rare
in known cellulose-degrading organisms.
Using synthetic biology approaches, seven cellulases with multiple catalytic domains
were engineered and tested to determine the usefulness of such chimeric enzymes
to replace cloning of multiple enzymes for biomass conversion. Catalytic domains
were taken from Cellulomonas fimi endoglucanases CenA, CenB and CenD,
exoglucanase Cex, and β-glucosidase, Cfbglu as well as Cytophaga hutchinsonii
cellodextrinase CHU2268. All fusions retained both catalytic activities of the parental
enzymes. To investigate the benefits of fusion, Citrobacter freundii NCIMB11490 was
transformed with either fused or non-fused enzymes and cultured with cellulose
blotting papers as main carbon source.
Cells expressing fusions of Cex with CenA or CenD reproducibly showed higher
growth than cells expressing non-fused versions, as well as more rapid physical
destruction of paper. The opposite was observed for the other combinations.
Comparing two different Cex and CenA fusions, CxnA2, which contains two
carbohydrate binding modules (CBMs), degraded filter paper faster and led to better
growth than CxnA1, which contains only one CBM. It was observed that CxnA1 was
exported to the supernatant of E. coli and C. freundii cultures, as also seen for Cex
and CenA, although there is no clear biological mechanism for this.
Monitoring of growth using colony counts is laborious, but the use of optical density is
not possible for cellulose-based cultures as it is affected by the insoluble cellulose
particles. The SYBR Green I/propidium iodide live/dead staining protocol was
therefore evaluated for growth measurements and was found to allow rapid
measurements of large numbers of samples.
In conclusion, these studies have demonstrated a simple and useful method for
making chimeric proteins from libraries of multiple parts. The results demonstrate that
use of fusion proteins can improve biomass conversion in vivo, and could potentially
reduce the necessity for cloning of multiple enzymes and improve product yields. A
simple and effective method for monitoring growth of bacteria in turbid cultures using
a fluorimeter has also been developed
