242 12 Metabolic Channeling Using DNA as a Scaffold
anchoring of the DNA‐binding protein to the DNA‐target site is well character
ized. Due to the close proximity of the chimeric proteins bound to the pro
grammed nucleic acid sequence, other enzymes that might redirect synthesis are
spatially excluded from the multi‐protein complex. Designed DNA‐binding
domains can be used as fusion partners of biosynthetic pathway enzymes. These
domains can share the same type of protein fold; they have similar affinity to the
scaffold, and, therefore, the binding of all of the components of the biosynthetic
pathway proceeds under the same reaction conditions.
RNA scaffolding is in many aspects similar to that of the DNA; however, only
a limited number of well‐characterized RNA binding domains is available [7, 9]
(Chapter 13) (Figure 12.1d, Table 12.1). The advantage of the RNA over the
DNA scaffold is that it could be used in eukaryotes to organize the metabolic
pathway into the cytosol, whereas the DNA scaffold is probably limited to the
prokaryotes.
Based on the pros and cons, the DNA scaffold localizes primarily in the nuclei
in eukaryotic cells; therefore for eukaryotes, the protein and RNA scaffolds are
the only choices. Moreover, the protein scaffold could be directed to micro‐
locations within the cells. The main advantages of the DNA scaffold are the
simple DNA program design and well‐characterized anchoring of the DNA‐
binding proteins to the DNA‐target site, as well as orthogonality; therefore, they
are recommended for use in bacteria over both the RNA and protein scaffolds.12.2 Biosynthetic Applications of DNA Scaffold
DNA scaffold‐assisted biosynthesis is a viable strategy for enhancing the meta
bolic product yield or production rate. This enhancement appears to arise from
the proximity of metabolic enzymes bound to the DNA scaffold that increases
the effective concentrations of the intermediary metabolites. In every tested
case, the DNA scaffold‐assisted biosynthesis implemented on existing meta
bolic pathways improved either the product yield or rate of product synthesis
(Table 12.2).12.2.1 l‐Threonine
Lee et al. [10] devised a DNA scaffold to facilitate the production of l‐threonine
in Escherichia coli (Figure 12.2). The biosynthetic pathway composed of the
homotetramer homoserine dehydrogenase (HDH), homotetramer threonine
synthase (TS), and homodimer homoserine kinase (HK) was assembled on
the DNA program using 4‐fingered zinc finger domains binding to 12 bp DNA‐
target sequences, named artificial DNA‐binding domains (ADBs). Metabolic
enzymes were linked to the N‐terminal site of the ADBs, and they report testing
several designs of the DNA program. Initially, the influence of 8, 18, and 28‐bp
spacers between individual DNA‐target sites on the l‐threonine product rate
was analyzed. In addition, the impact of the target sites from one to four, for a
third chimeric enzyme in the l‐threonine metabolic pathway (TS‐ABD3), was
evaluated. The DNA scaffold with an 8 bp spacer between the DNA‐target sites,