Synthetic Biology Parts, Devices and Applications

240 12 Metabolic Channeling Using DNA as a Scaffold

and organelle targeting more closely imitate the formation of natural enzyme

complexes and were used in the first attempts to improve the biosynthetic path­

way efficiency by designed substrate channeling [8–12].

Although the number and distribution of the enzymes in a multi‐protein com­

plex [8–12] (Chapter 13) could be programmed by the sequence of a polypeptide

backbone, the three‐dimensional arrangement of the polypeptides may be

unpredictable due to the flexibility in the peptide linkers between the interaction

domains (Figure 12.1a,b, Table 12.1). Designing the polypeptide backbone with

the scaffold‐guided protein domains is limited by the number of available protein

dimerization domains. Finally, each protein interaction domain has different

conditions under which it folds and forms the functional interactions.

E3

DNA sequence governs ordering

of biosynthetic enzyme pathway

through interaction of DNA-binding

domains with DNA scaffold

Bundle of biosynthetic enzymes

linked to a polypeptide scaffold

through dimerization domains

Without scaffold a

position of biosynthetic

enzymes is random

(a) (b)

(d)

(c)

RNA aptamers used to localize

biosynthetic enzymes to an

RNA scaffold

E1 E2

E1

E3

A B C

a b c

E1 E2 E3

Product 1

A C B

a c b

E1 E3 E2

Product 2

E2

E1

E2

E3

Product Product Product

Figure 12.1 Spatial and temporal organization of biosynthetic enzymes based on different

types of scaffolds. (a) Biosynthetic enzymes are typically randomly distributed inside the cell.

The conversion of the substrate may therefore be limited by the diffusion rate and the

concentration of the substrate and localization of the enzymes. (b) Immobilizing biosynthetic

enzymes using synthetic protein scaffolds can bring the enzymes into close proximity and

therefore enhance the metabolic flux. In the absence of a large superscaffold, the precise

arrangement of enzymes is unpredictable and is limited by the tertiary structure of the

protein scaffold. (c) Biosynthetic enzymes with predictable RNA binding sites have been

assembled using synthetic RNA aptamers. The enzymes are in close proximity and in the

predefined order, which enables faster conversion of the substrate into the end product.

(d) An assembly line based on the DNA scaffold promotes positioning of biosynthetic

enzymes in close proximity and the predefined order. The substrate conversion is faster with

less unwanted side products. The enzymes are linked to DNA‐binding domains, which

recognize specific nucleotide sequences. The order of the enzymes can easily be changed by

changing the order of the specific nucleotide sequence on the DNA program, which can lead

to different end products.