Biology Reference
In-Depth Information
binding site (RBS) sequestering sequences. The library of mutated intergenic regions was
constructed and applied to the mevalonate pathway. To select, the library was transformed into
a melavonate auxotroph strain and then screened by a mevalonate biosensor. The product
concentration of the final mutant pathway was increased seven-fold compared to that of the
original wild-type pathway. It was interesting to note that the reason for improved production
was actually a down-regulation of two genes within the operon, which was counterintuitive.
Thus, the ability to tune multiple regulatory mechanisms simultaneously without specific
rational design is a major strength of the TIGR approach.
PROMOTER STRENGTH
More than increasing mRNA stability, the promoter strength can be tuned to increase
the gene expression by producing more quantities of the mRNA. The strength is based on
efficient promoter recognition and rapid binding of the DNA polymerase.
56
However,
multiple genes in a pathway do not all need to be expressed at the same level.
Promoters can be mutagenized to achieve precise strength and regulation. These promoters
of varying strengths have been applied to exhibit a broad range of genetic control, which
can be selected to construct an optimal metabolic pathway. Alper et al.
57
used a library of
mutant promoters with varying strengths to regulate the expression of the
dxs
gene, which
led to the improvement of the volumetric productivity of lycopene. It was found that
optimal gene expression did not involve the strongest promoter. This library method allows
for the precise quantitative control of gene expression in vivo. A more broadly applicable
method was recently established by Du et al.,
58
which relies on the DNA assembler method
to automatically combine multiple libraries of mutant promoters of different strengths in
a multigene pathway. Through a rigorous screening and selection process, the optimal
combination of mutant promoters in the pathway can be identified (
Fig. 3.1B
).
This technique is especially powerful because it involves multiple promoters in a multigene
pathway, optimizing the expression of all genes simultaneously. By balancing the flux
through the pathway, no bottlenecks were observed. Additionally, it was shown that the
technique allowed for identification of customized pathways for specific
Saccharomyces
cerevisiae
strains.
51
DYNAMIC TRANSCRIPTIONAL REGULATION
Genetic circuits have great potential for dynamic transcriptional regulation through inducible
transcription factors and
cis
-regulatory elements. They can be used to reduce metabolic
burden and toxic byproduct formation. For example, when protein translation was coupled
to glucose availability and other metabolic byproducts of the system, a dynamic control was
introduced to regulate flux through the desired pathway.
59,60
The circuit was designed to
detect the concentration of acetyl phosphate (ACP), a byproduct of the lycopene pathway.
If ACP was detected, two enzymes were turned
by the genetic circuit, and high expression
of those enzymes rerouted the carbon flux from the byproduct back to the product. Thus,
these significant enzymes would only be expressed when needed, and did not overburden the
cell too significantly.
'
on
'
COMPUTATIONAL METHOD FOR RBS ENGINEERING
Similar to transcriptional engineering, such as promoter engineering and combinatorial
engineering of the tunable intergenic regions of operons, translational engineering can also
be used to effectively regulate gene expression. RBS and other regulatory RNA sequences
are the major control elements for translation initiation. It is possible to mutate the RBS
sequence and create a library with varying RBS strengths,
55,61,62
and it was shown that an
optimal RBS can improve product titer. However, the library size that must be screened
increases combinatorially with the number of RBS sites to engineer. Thus, attempts to
optimize gene expression within a system, without specific rational design, can be
