Biology Reference
In-Depth Information
determine the enzyme whose low concentration limited the productivity of the
amorphodiene production pathway in
E. coli
. The complete pathway for amorphodiene
production was encoded on a low-copy plasmid and transformed into
E. coli.
Higher-
copy plasmids that encoded each individual pathway enzyme were subsequently
introduced. A plasmid encoding the mevalonate kinase gene (MK) increased
amorphodiene yields, suggesting that concentration of the MK enzyme limited
production.
47
This approach has also been used to improve production of the
amorphodiene precursor mevalonate. Increasing the copy number of rate-limiting enzyme
tHMGR relieved the growth inhibition caused by accumulation of a precursor metabolite,
relieving toxicity and enabling higher mevalonate levels.
41
Additional copies of a gene
may be placed on the same plasmid as the rest of the pathway; however, this may lead
to gene loss through recombination and is best avoided.
48
Plasmid-based expression of pathways may not be well-suited for high-titer production of
biofuels. The metabolic requirements of plasmid replication impose on the host cell a
metabolic burden, which can scale with plasmid copy number.
49
Plasmids are autonomous
genetic elements that may not be distributed evenly upon cell division. If expression of a
plasmid-based biofuel pathway decreases cell growth rate or viability, individuals with fewer
copies of the plasmid will rapidly out-compete the others, leading to a population with low
overall productivity.
50
Techniques that transfer the pathway genes to the chromosome
itself avoid this problem; however, as the chromosome is present in very low copy
numbers, this may place a very low limit on the pathway copy number that can be
introduced. A recently developed innovation, chemically inducible chromosomal
evolution (CIChE), enables the introduction of moderate-copy numbers of pathways onto
the chromosome (
40). Stability of the repeated genes is ensured via knockout of the
native recombinase gene
recA
.
B
214
Biofuel production genes are commonly expressed from well-characterized promoters
induced by the addition of an exogenous chemical, such as anhydrous tetracycline or IPTG.
This provides a simple means with which to modulate the strength of expression, as
increasing inducer concentration should result in higher rates of transcription. While the use
of promoters driven by exogenously added chemical inducers is very useful in the early
stages of pathway demonstration, the price of typical inducers prohibits their use on an
industrial scale. Constitutive promoters with a wide range of promoter strengths have been
developed, enabling finely tuned enzyme levels,
51
without the need for chemical inducers.
Constitutively active promoters can enable an engineer to modulate the transcript levels of
either the whole biofuel pathway,
52
or each individual enzyme can be controlled by a
specific constitutive promoter.
53
However, constitutive expression of pathway enzymes may
present a metabolic burden on the cell, presenting an opportunity for escape mutants to
evolve and contribute to pathway instability.
48
A more sophisticated approach to controlling pathway expression is to use biosensors
(see
Box 11.1
) to couple transcription of pathway enzymes to the concentration of specific
metabolites or other cellular or environmental conditions. This takes advantage of control
mechanisms evolved to dynamically respond to metabolite levels. An early demonstration
of using biosensors to achieve dynamic metabolic control coupled transcription of key
pathway genes of a lycopene biosynthesis pathway with the concentration of acetyl
phosphate resulted in improved titers of the product in
E. coli
.
54
The expression of the rate-
controlling enzymes of lycopene synthesis was placed under control of a promoter native to
E. coli
that responds to acetyl phosphate, a metabolite that increases during periods of excess
glycolytic flux.
55,56
Thus, the lycopene pathway was activated when acetyl phosphate levels
were high, which indicated an imbalance between carbon influx and carbon consumption.
This increased the production of lycopene by 10-fold above what was achieved by
