Post by: Celine Bien-Aime
It is no secret
that our environment is being destroyed, but what are we doing about it? Well there
are extensive efforts to produce more eco-friendly transportation fuels and
n-butanol production has become an area of active research in regards to this. Researchers
are very eager to reduce our dependence on imported oils, as well as lower
greenhouse gas emission due to the use of fossil fuels. This is why the search
for biofuels is so important. Currently, ethanol is the major biofuel used for
transportation; however, butanol has several advantages over ethanol, such as a
higher energy density and the ability to be transported via pipelines as
opposed to barges and trucks. The N-butanol pathway has been well studied in a
bacterium called Clostridium
acetobutylicum. This bacterium is a mesophile, which means that it grows
best under moderate temperatures of 20-45 ºC. The genes in N-butanol pathway of
C. acetobutylicum have been
introduced into other mesophiles, like Escherichia
coli and Bacillus subtilis, among
others, in order to induce n-butanol production. The genes used are thl, hbd,
crt, etfA, etfB, adhE2 and adhE1, which encode the enzymes thiolase, β-hydroxybutyryl
CoA dehydrogenase, crotonase, butyryl CoA dehydrogenase, electron transfer
flavoproteins subunit A & B and the bifunctional enzyme aldehyde-alcohol
dehydrogenase respectively.
 |
| The N-butanol Pathway (Steen et .al. 2008) |
Previous
studies have found that using thermophilic anaerobic
bacteria, which are
bacteria that grow best at higher temperatures and in environments with little
oxygen, is more advantageous than using mesophiles. This is because there is a
reduced risk of contamination, higher reaction rates and lower differential
costs. For this reason, researchers in this paper looked at the ability and
efficiency of n-butanol production in Thermoanaerobacterium
saccharolyticum. T. saccharolyticum
is a gram positive, thermophilic, anaerobic bacterium found in Yellowstone
National Park. It grows between temperatures of 45 and 65 ºC and a pH between 4.0
and 6.8. T. saccharolyticum is an
attractive bacterium for genetic engineering of butanol production because it
is naturally competent, meaning it easily takes up and incorporates foreign DNA
either from it’s environment or from other bacteria. It can use a variety of
sugars found in biomass, which is just plant based materials, like cellobiose,
glucose, xylose, mannose, galactose and arabinose. It can also hydrolyze xylan,
mannan, starch and pectin. T.
saccharolyticum has been engineered to produce higher yields of ethanol,
and the goal is to do the same for butanol production. Essentially they want to
know “Can T. saccharolyticum produce more
butanol for your buck?”
Several
different strains of T. saccharolyticum
were made by introducing genes from a closely related thermophile, Thermoanaerobacterium thermosaccharolyticum, which produces
n-butanol via an n-butanol pathway that is not well characterized, and C. acetobutylicum. Genes from T. thermosaccharolyticum were used
because certain enzymes encoded by C.
acetobutylicum aren’t stable at higher temperatures. First, the researchers
introduced plasmids that only contained single genes from the n-butanol pathway
into the T. saccharolyticum. Then the
researchers introduced non-replicative plasmids that contained all the genes in
the n-butanol pathway.
T. saccharolyticum was able to express
all the individual genes from the n-butanol pathway using the single gene
plasmids. Despite being able to grow on a variety of sugars, the engineered T. saccharolyticum with the entire
n-butanol pathway was only able to produce butanol when grown on xylose. This
was because the operon that was added to the bacterium was under a tightly
regulated promoter. Researchers also found that butanol was not produced in
strains that could not produce acetate, because the butanol became lethal to
the cell. Overall, researchers were successful in engineering T. saccharolyticum to produce n-butanol.
Engineered T. saccharolyticum showed
an 8-10 fold increase in butanol production as compared to T. thermosaccharolyticum.
Since the
n-butanol pathway was successfully incorporated into T. saccharolyticum, researchers believed that this study
demonstrated the “portability” of the N-butanol pathway. Further studies should
include the incorporation of this pathway in other thermophilic bacteria. For future engineering efforts, researchers also
suggested that native ethanol producing genes in T. saccharolyticum should be replaced proteins that are
specifically used for butanol production. Now that there are viable and cost-efficient
ways to produce butanol over ethanol, more efforts can be made to divert our
fuel dependency to biofuels and away from fossil fuels. Studies like this can
help us ensure that future generations will have a safe, clean environment to
live in.
Literature Cited
Emerging
Technologies: Biofuels. (n.d.). Retrieved December 22, 2015, from
http://needtoknow.nas.edu/energy/energy-sources/emerging-technologies/biofuels/
Steen, E., Chan, R., Prasad, N., Myers, S., Petzold, C.,
Redding, A., Ouellet, M., Keasling, JD. (2008). Metabolic engineering of
Saccharomyces cerevisiae for the production of n-butanol. Microbial Cell
Factories, 7(36).
doi:10.1186/1475-2859-7-36
No comments:
Post a Comment