12.5 Applications of DNA‐Guided rogramming 255Many biosynthetic pathways are also branched, which means that the enzymes
at the initial steps are shared and the enzymes after the branch differs, which
determines the end products and their ratios. With DNA scaffolds where the
order of enzymes can be changed, the end product could be determined by scaf
folding the selected pathway. This leads to the production of cleaner end prod
ucts and less unwanted products (Figure 12.7a). In addition, with a substrate to
product channeling, which is achieved by the DNA scaffold, the accumulation of
intermediate products that are toxic for the cell or that can significantly slow
down the production rate is consumed faster.
Moreover, the DNA‐guided assembly could also be used outside the cell to
support biosynthetic reactions in vitro, comprising (i) functional units, for exam
ple, biosynthetic enzymes linked to DNA‐binding domains or linked to single‐
stranded oligonucleotides by chemical modification [13–15]; (ii) a DNA scaffold
comprising one or more target site sequences; and (iii) a substrate for the first
enzyme and cofactors for the enzymes provided to the mixture. Erkelenz et al.
[31] generated a hybrid DNA–protein device based on the two cytochrome P450
BM3 subdomains conjugated to oligonucleotides. The two conjugates arranged
on a switchable DNA scaffold form active monooxygenase, which could be
turned off by DNA strand displacement.
DNA scaffolds could also be used to control the flow of different classes of
biological information mediators that extend beyond the metabolic pathways
and small molecule products. For example, DNA scaffolds could be used to
rewire intracellular signaling pathways or to coordinate other assembly‐line pro
cesses, such as protein folding, degradation, and posttranslational modifications
(Figure 12.7a,b). Thus, we anticipate that DNA scaffolds should enable the con
struction of reliable protein networks to program a range of cellular events. Even
though the beauty of nature’s most elegant compartmentalization strategies,
such as a protected tunnel [32] or intracellular organelles [33, 34], has yet to be
recapitulated by engineers, the use of DNA scaffolds is an important early step
toward this goal.
Strain development is still hampered by the intrinsic inefficiency of enzymatic
reactions caused by simple diffusion and the random collision of enzymes and
metabolites. Scaffolding strategies that promote the proximity of metabolic
enzymes and direct metabolic intermediates through the catalytic assembly
steps are promising solutions for the named problem [7–12]. Regardless of scaf
fold type, the enzyme assembly increases the local concentration of intermedi
ates around the enzyme on the scaffold, preventing the loss of intermediates by
competing reactions and overcoming the problem of toxic intermediates due to
the rapid conversion of inhibitors.
Definitions
The DNA scaffold is a DNA molecule that serves as a platform for the spatial
organization of DNA‐binding protein domains. The sequential order of the
DNA‐binding protein domains with their fusion partners is defined through
the DNA‐target sites positioned along the DNA molecule. The ordering of the