Synthetic Biology Parts, Devices and Applications

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254 12 Metabolic Channeling Using DNA as a Scaffold


in a consecutive manner. The arrangement [1 : 1 : 2] with an 8‐bp spacer pro­
duced the best results, followed closely by [1 : 1 : 3], [1 : 1 : 4]. They found that the
production rate was threefold higher than that of [1 : 1 : 1] (Figure 12.2a).
For the 1,2‐propanediol and mevalonate synthesis, the scaffolds were designed
bidirectionally in the way that the first enzyme was flanked on both sites by the
second, followed by the third enzyme [1 : 2 : 2]. In addition, a consecutive
arrangement of the second enzyme [1 : 2 : 1] and [1 : 4 : 1] for both 1,2‐ propanediol
and mevalonate biosynthesis, the DkgA and HMGS, respectively, was tested.
The DNA scaffold arrangement [1 : 2 : 1] 4 12 bp spacer gave the best yield of
1,2‐ propanediol, closely followed with [1 : 2 : 2] 4 12 bp and [1 : 4 : 2] 2 12 bp
(Figure  12.4). The DNA scaffold [1 : 4 : 2] combines both the bidirectional and
consecutive arrangement of DNA‐target sites and functional units. For meva­
lonate production, the [1 : 4 : 2] 2 12 bp DNA scaffold gave the best yield, followed
closely by the [1 : 2 : 2] 2 ,4,16 12‐bp scaffold [11].
In some biosynthetic pathways, the bottleneck is the conversion rate of a sub­
strate into a product, which can be a substrate for the next enzyme in the meta­
bolic pathway. By changing the arrangement of biosynthetic enzymes on a DNA
scaffold, the imbalances in the enzyme kinetics can be overcome. It might be
expected that the multimerization of functional enzymes could interfere with
the formation of functional scaffolds; however, the biosynthesis of l‐threonine
depends on enzymes that are active as homotetramers and homodimers, and
still, the DNA scaffold improves the production rate of l‐threonine [10]. The
fact that multimeric proteins might facilitate DNA scaffold cross‐linking, there­
fore building regions with locally elevated concentrations of metabolites (metab­
olite microdomains), is dedicated for bioconversion. Moon and coworkers [30]
showed a positive correlation between a glucaric acid titer and the number of
scaffold interaction domains targeting upstream myo‐inositol‐1‐phosphate syn­
thase. In the mevalonate pathway, protein scaffolding generating microdomains
enabled faster growth rates, likely minimizing the cellular accumulation of the
toxic intermediate HMG‐CoA in E. coli [8].
Taken together, the DNA scaffold is a useful tool to improve biosynthesis. The
predictable nature of DNA enables the fine‐tuning of metabolic biosynthesis and
production yields.

12.5 Applications of DNA-Guided Programming


By studying different DNA scaffold architectures, enzyme stoichiometry, and
flux balanced or imbalanced biosynthetic pathways, it should be possible to
determine when the enzyme co‐localization is most beneficial. This, in turn, will
be very useful for guiding the future design of these systems and in envisioning
new applications for enzyme co‐localization. It is also worth mentioning that the
DNA scaffold approach is highly complementary to many of the existing meth­
ods for enzyme, pathway, and strain engineering that are already in the cellular
engineer’s toolkit. Therefore, a successful strategy for achieving the production
yields near theoretical maximum is necessary for the commercial viability of
production processes and will likely involve a combination of these approaches.
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