Synthetic Biology Parts, Devices and Applications

12.3 Design of DNA‐Binding roteins and Target Sites 247

hydroxymethylglutaryl‐CoA reductase (HMGR). The biosynthesis of meva­
lonate in E. coli, as such or assisted by a protein scaffold, has already been pub­
lished [8, 18]. The chimeric proteins between the enzymes of the mevalonate
pathway and zinc finger domains were constructed [11] (Figure 12.4b). For the
DNA scaffold design, the DNA‐target sequences corresponding to each of the
DNA‐binding domains were placed on a separate plasmid, and the influence of
the DNA‐target sites arrangements on mevalonate production was tested.
Similar to the resveratrol and 1,2‐propanediol scaffolds, the mevalonate yield
was increased up to threefold in the presence of [1 : 2 : 2]n scaffolds (n = 2, 4, and
16) with 12‐bp spacers, compared with the random scaffold control; however,
the best mevalonate yield was achieved with the DNA scaffold containing the
[1 : 4 : 2] 16 program (Figure 12.4c,d).

12.3 Design of DNA‐Binding Proteins and Target Sites

A self‐replicating DNA plasmid in one or more copies is an ideal scaffold for
any information processing; for example, the DNA sequence represents a pro­
gram consisting of a series of blocks (DNA‐target sites), which determine the

n

A

E1

B

E2

B

E2

D

E3

(a)

[1:1:1]n [1:2:1]n [1:2:2]n

A

E1

B

E2

D

E3

n

A

E1

B

E2

D

E3

B

E2

D

E3

n

DHAP MgsA Methylglyoxal Acetol

E1

DkgA

E2

GldA

E3

Acetyl-CoA Acetoacetyl-CoA Mevalonate

AtoB

E1

HMGS

E2

HMGR

(b) E3

(c)

n = 1, 2, 4, 8, 16
4 and 12 bp spacer

Consecutive

arrangement

Bidirectional

arrangement

Bidirectional and

Consecutive arrangement

[1:2:1]4,16,32 12bp [1:2:2]2,4,16 12bp [1:4:2] 16 12bp

[1:1:1] 16 12bp <<[1:1:1] 4 4bp [1:1:1] 4 12bp

[1:2:1]1,2,8 12bp<[1:2:1] 4 12bp

(d)

1,2-Propanediol

Mevalonate

<<

A

E1

B

E2

B

E2

B

E2

D

E3

n

D

E3

B

E2

[1:4:2]n

< [1:2:2] 4 12bp =[1:4:2] 2 12bp

[1:2:1] 4 4bp =[1:2:2] 4 4bp =

[1:1:1]4,8 12bp=

1,2-Propanediol

HMG-CoA

Figure 12.4 Biosynthesis of (a) 1,2‐propanediol and (b) mevalonate in E. coli. (c) Schemes of
consecutive, bidirectional, and mixed consecutive and bidirectional arrangements of DNA
scaffolds with different stoichiometry and positions of DNA‐target sites that were tested for the
improved biosynthesis of 1,2‐propanediol or mevalonate. DNA scaffolds can be used to overcome
the limitations in biosynthetic pathways that occur because of individual enzymes with lower
activity, compared with other enzymes in the same biosynthetic pathway. By changing the order
or number of DNA‐target sites, we can increase reaction yields, fine‐tune biosynthesis production,
and minimize side products. If the first enzyme in the biosynthetic pathway is most active, others
can be distributed on both sides around the first, resulting in 1 : 2 molar ratios in favor of enzymes
with low activity. Such groups of enzyme binding sites can then be multiplied on the DNA
scaffold to achieve better molar ratios between the DNA scaffold and enzymes. (d) Impact of
different scaffold architectures on 1,2‐propanediol and mevalonate production [11].