252 12 Metabolic Channeling Using DNA as a Scaffold
the DNA program, in which individual DNA‐target sites were separated with
spacer lengths of 2 or 8 bp, while a spacer length of 4 bp (where the enzymes are
oriented to the opposite directions from the DNA duplex) showed a smaller yet
measureable improvement over the free soluble enzymes. The impact of 4‐ and
12‐bp‐long spacers between the DNA‐target sites for the 1,2‐propanediol and
the mevalonate DNA scaffolds was also analyzed [11]. All scaffolds with 4‐bp
spacers between zinc finger binding sites were less effective than their 12‐bp
counterparts.
Lee et al. [10] constructed scaffold plasmids to position ADB–enzyme fusions
every 20 bp (8 bp spacer), 30 bp (18 bp spacer), and 40 bp (28 bp spacer), so that all
scaffold‐bound enzymes were on the same side of the DNA program in three‐
dimensional space. The scaffold with the 8‐bp spacer sequence was associated
with the most efficient l‐threonine production, confirming the finding that close
proximity of the metabolic enzymes enhances the product synthesis, most likely
through substrate channeling.
As demonstrated with the trans‐resveratrol and the other biosynthetic
pathways, the spatial orientation and clustering of the enzymes on the DNA
scaffold are important. Due to the predictable nature of the DNA, it is possible
to predict the enzyme orientation in situ that simplifies designing the DNA
scaffold, which is important for larger enzymes that, due to the steric effect,
might prevent binding of other enzymes on a DNA scaffold.12.4.2 Number of DNA Scaffold Repeats
In addition to the length of a spacer, the number of repeats of the DNA scaffold
is important for fine‐tuning the biosynthetic metabolic pathway.
Conrado et al. examined in detail the effect of increasing the number of scaf
fold repeats. They constructed scaffolds with enzyme–scaffold ratios in range of
40 : 1 to 1 : 3 (e.g., [1 : 1 : 1] 1 to [1 : 1 : 1] 16 ). A DNA program with DNA‐target
sequences for each of three‐enzyme pathways for producing 1,2‐propanediol
was placed on the same plasmid as zinc finger chimeras. The best 1,2‐propane
diol yield was obtained when the number of scaffolds was 4, regardless of the
arrangement of the DNA‐target sites (see Section 12.4.3), with 12‐bp spacers
between the binding sites. For DNA scaffolds with 4‐bp spacers between the
DNA‐target sites, the number of repeats played no role [11].
For mevalonate production, which is also a three‐enzyme metabolic path
way, the genes encoding the chimeric biosynthetic enzymes were not on the
same plasmid with the DNA program, enabling alternations not only through
out the number of scaffold repeats but also with the copy number of plasmids
with the DNA scaffold. The largest yield enhancement came from the 16
repeats of the [1 : 4 : 2] scaffold. This was followed closely by several of the scaf
folds [1 : 2 : 2] with 2, 4, or 16 repeats (Figure 12.4). In agreement with the previ
ous results for 1,2‐propanediol and mevalonate, a yield enhancement for the
trans‐resveratrol was observed when the number of scaffold repeats was
decreased from 16 to 4.
These improvements highlight the ability to impact biosynthesis via simple
changes in scaffold design. The number of scaffold repeats can easily be