Synthetic Biology Parts, Devices and Applications

246 12 Metabolic Channeling Using DNA as a Scaffold

12.2.3 1,2‐Propanediol

A biosynthetic pathway for 1,2‐propenediol composed of methylglyoxal synthase

(MgsA), 2,5‐diketo‐d‐gluconic acid reductase (DkgA), and glycerol dehydroge­

nase (GldA) in E. coli is well established [17] (Figure 12.4a). The biosynthetic

enzymes were fused to the N‐terminus of the zinc finger domains ZFa, ZFb, and

ZFc, recognizing a 9‐bp target, and the corresponding chimeras were placed on

the same plasmid as the target DNA sequence [11]. Several enzyme–scaffold

ratios were tested (Figure 12.4c,d), and the E. coli with the [1 : 1 : 1] 4 12‐bp spacer

1,2‐propanediol system produced almost five times more product than the

unscaffolded control (Table 12.2).

12.2.4 Mevalonate

The biosynthesis of mevalonate is a three‐step reaction composed of acetoa­

cetyl‐CoA thiolase (AtoB), hydroxymethylglutaryl‐CoA synthase (HMGS), and

Zif PBS

4CL STS

2 bp

[1:1] 4 2 bp

(Produced up to 10 mg/l resveratrol)

4 x

4CL STS

(a)

4CL STS

4-Cumaric acid Resveratrol

(b)

E1

OO

OH

HO OH

HO CoAS

OH OH

E2

p-Cumaroyl-CoA

4CL-STS

Fusion protein

(Produced ~1 mg/l resveratrol)

Figure 12.3 A DNA scaffold enhances the biosynthesis of trans‐resveratrol in E. coli. (a) In the

biosynthetic pathway of resveratrol, the 4‐cumaric acid is converted to resveratrol in a two‐

step reaction with the biosynthetic enzymes 4‐coumarate–CoA ligase (4CL) and stilbene

synthase (STS). (b) Close proximity of the 4CL and STS enzymes can be achieved by fusing the

enzymes with linker polypeptides or by introducing DNA scaffolds where the enzymes

(4CL or STS) are fused to the DNA‐binding domains (Zif268 or PBSII). The chimeric protein of

the enzyme and DNA‐binding domain binds to a specific nucleotide sequence present on the

DNA program. The DNA‐target sites specific for the individual chimeric proteins are separated

with a 2‐bp spacer between each of four tandem repeats [11].