12.4 DNA rogram 251spacer of four to five nucleotides positions the neighboring two functional
domains to the opposite sides of the helix (Figure 12.6c).
Initially, the impact of the clustering of metabolic pathway enzymes [11] was
examined, and the DNA‐target sites within the [1 : 1] 4 scaffold were separated on
the plasmid by either 2 or 850 bp (Figure 12.6a). The [1] 4 ‐850 bp‐[1] 4 scaffold
provided the same number of binding sites on the plasmid for both enzymes but
prevented the close proximity of the bound enzymes to one another. The fivefold
enhancement in resveratrol production observed for the [1 : 1] 4 scaffold was
abolished when the binding sites for each enzyme were positioned far apart on
the plasmid, indicating that close proximity of the pathway enzymes is important
(Figure 12.6b).
In the example of resveratrol biosynthesis, we [11] examined whether the
three‐dimensional positioning of individual enzymes effects production yields
(Figure 12.6c). DNA scaffolds with DNA‐target sites separating 2, 4, or 8 bp were
constructed. Considering the standard DNA topology, the 2‐ and 8‐bp spacer
position functional units were on the same site of the DNA scaffold, and the 4‐bp
spacer position functional units to the opposite site of the DNA program. In the
case of the [1 : 1] 16 resveratrol system, the best product yields were obtained with
ABE2AE1BE2BE2AE1DNAA BE1
E1
E2DNASide view Front viewA BE1 E2850 bp 4 x(a) (c)[1] 4 -850 bp-[1] 4[1:1] 4 2 bp2 bp
8 bp4 bp01234trans-Resveratrol (mg/l)[1]-850 bp-[1] 44[1:1]^4
2 bpA BE1 E22 bp^4 x(b)Figure 12.6 Spatial position of biosynthetic enzymes is defined by DNA program.
(a) Scheme of two types of DNA scaffolds that differ in spacer lengths separating the DNA‐
target sites. A first scaffold plasmid (left) carries four copies of Zif268 and four copies of
PBSII binding sites, separated by an insertion of 850 bp along part of the plasmid backbone.
A second scaffold plasmid carries four copies of the Zif268 and PBSII binding sites separated
by 2‐bp‐long spacers. (b) Enzyme clustering improves the production of trans‐resveratrol,
which was measured in Escherichia coli‐expressing fusion enzymes (Zif268‐4CL and PBSII‐
STS) with different DNA program plasmids [1] 4 ‐850 bp‐[1] 4 and [1 : 1] 4 2‐bp spacers
(for details see Figure 12.4a) [11]. (c) The spatial orientation of the enzymes is governed with
a spacer between the DNA‐target sites. The 2‐ and 8‐bp spacers orientate chimeric enzymes
on the same side of the DNA program (up). The 4‐bp spacer between the target sites
orientates the enzymes on opposite sides of the DNA program (below). The best production
of trans‐resveratrol in E. coli was achieved when the binding sites for Zif268 and PBSII were
separated with 8 bp [11].