Synthetic Biology Parts, Devices and Applications

12.4 DNA rogram 253

changed, not only by changing the DNA program repeats on plasmid DNA but
also by changing the number of plasmids in a cell. This can be achieved by
introducing different origins of replication, from low to high copy number
properties. The biosynthesis of a metabolite represents an additional burden for
the cell. If the burden is too high for the cell, the production of a metabolite will
not lead to the maximal yield. By changing the number of scaffold repeats, we
can determine the state where the production yield of the wanted biosynthetic
product is maximal.

12.4.3 DNA‐Target Site Arrangement

In addition to the length of a spacer between DNA‐target sites and the number
of DNA scaffold repeats, the stoichiometry of DNA‐target sites for individual
enzymes forming biosynthetic pathways could also be varied. This is beneficial
for biosynthetic pathways with enzymes with different kinetics.
It should be noted that different enzyme arrangements on plasmid DNA are
possible. Different architectures are described as, for example, [E1a:E2b:E3c]n for
a three‐enzyme scaffold, whereas a, b, and c describe the enzyme stoichiometry
within a single scaffold unit and n is the number of times the scaffold unit is
repeated on the plasmid (Figure 12.7a).
For the l‐threonine scaffold, Lee et al. [10] used the following architectures
[1 : 1 : 1], [1 : 1 : 2], [1 : 1 : 3], [1 : 1 : 4] with an increasing number of homodimer TSs

E 1 E 2

E 4

E 3

E 5

p 1 p 2

p 3 p 4

p 5

s 1 Biosynthetic flux channeling

A B

E1 E2

C

E3

D

E3

E

E3

P P P P

Information processing

(e.g., cascade of protein kinases)

(a)

(b)

A

E1

B

E2

D

E3

E

E4

A

E1

B

E2

F

E5

Figure 12.7 Applications of DNA‐guided programming. (a) For many biosynthetic pathways,
the first enzymes in the cascade are the same, and the end product is determined by the
enzymes that are lower in the cascade. By immobilization of specific enzymes on a DNA
scaffold, when others are left out, we can determine which end product will be preferentially
synthesized. This is a powerful tool for influencing the biosynthetic flux to produce less
unwanted products and a cleaner end product. (b) Similar to protein scaffolds, DNA scaffolds
can be used for defining the order in which protein kinases phosphorylate each other or a
chain of other posttranslational protein modifications. Signaling pathways can, therefore, be
modulated using different scaffolds. The DNA scaffold could also be used for information
processing such as rewiring intracellular signaling pathways and designing new protein
networks for constructing new biological devices with selected features.