Biology Reference
In-Depth Information
STRAIN ENGINEERING FOR INCREASED BIOFUEL TOLERANCE
Because many fuel candidates are toxic to microbial strains used in industrial processes,
tolerance of a candidate host organism to the fuel, and to biosynthetic intermediates of
that fuel, must be considered when selecting an appropriate production host. If the
production host cannot tolerate high concentrations of the biofuel it is designed to make,
it might be difficult to produce the compound in high titers. For instance, it makes little
sense to use
E. coli
to produce ethanol, as its tolerance to this compound is limited to
B
4%, whereas
S. cerevesiae
can withstand far higher concentrations. There are many
microbial species known to show high tolerance to hydrophobic solvents, but
unfortunately, the more genetically tractable organisms typically show low tolerance to
many biofuel candidates. Some exceptions include two promising biodiesel compounds,
fatty acid ethyl esters
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and bisabolene,
7
which have been demonstrated to inflict no
toxicity to
E. coli
. Interestingly, there are recent reports of
E. coli
producing butanol above
the concentrations where toxicity is seen.
4,5
We speculate that biofuel tolerance in late
growth phase and early stationary phase, where much of the biofuel is typically produced,
might be higher than in the exponential growth phase, where toxicity studies are typically
conducted. Furthermore, the most commonly used measure of tolerance (inhibition of
growth) may not accurately report on the stability of biochemical pathways within
the cell.
Increased tolerance can also be engineered by modifying the host organism, or equipping it
with mechanisms to resist toxicity. An excellent review of the mechanisms of biofuel toxicity
and techniques for engineering-increased tolerance has been written,
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but will be briefly
sketched out here. Microbial species possess efflux pumps that can expel hydrophobic
compounds and enable the cells to thrive in the presence of low levels of the toxic
compounds. For instance, in
E. coli,
the AcrAB-TolC efflux pump provides some tolerance to
biofuel compounds. More effective efflux pumps from hardier microbial species (e.g. species
isolated from oil fields) can be harnessed to increase tolerance in the host organism. This
approach was used to demonstrate that expression of solvent-tolerance pumps can result in
increased biofuel production.
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A library of efflux pumps was cloned from a variety of
microbial species and expressed in
E. coli
in the presence of several fuel candidates.
A competition experiment among strains expressing the various pumps revealed the pumps
that bestowed the most tolerance to the fuels. While this study demonstrated the potential
of using heterologous efflux pumps for increasing tolerance to biofuels, compatibility of the
foreign pumps with the host may need to be adjusted through additional engineering
efforts.
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A metagenomic library of heterologous genomic DNA was used to increase the tolerance
of
E. coli
to various toxins produced in biomass pretreatment.
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Microbial genomic DNA
(gDNA) was isolated from peat bog soil and other environments with high concentrations
of toxic inhibitors, and a phage library was made from the gDNA and infected into
E. coli
.
Transfected cells that exhibited increased tolerance were obtained by imposing a selection
for tolerance to inhibitory concentrations of the toxins. An investigation into the
heterologous genes that bestowed higher tolerance suggested that increased tolerance may
have been achieved by complementation of an
E. coli
enzyme affected by the toxins with a
foreign enzyme that could tolerate the toxin. This study is particularly attractive as it
provided a strong clue to the mechanism of toxicity of those particular poisons. While this
method was demonstrated to engineer increased tolerance to biomass pretreatment toxins,
there seems no obvious reason why it could not also be applied to engineering increased
biofuel toxicity as well. It might also provide information as to the specific enzymes that
are affected the most in the presence of biofuels, which are often assumed to be
systemic.
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