12.2 Biosynthetic Applications of DNA Scaffold 245and with two copies of the TS ([1 : 1 : 2] 8 bp), reduced the production time for the
l‐threonine by more than 50%, with the maximum yield produced within 24 h of
fermentation. For the strain without the DNA scaffold, it took 2 days to produce
the same maximum yield of l‐threonine. In addition, the concentration of the
intermediate homoserine, which might inhibit the growth of the host cell, was
reduced 15‐fold.
12.2.2 trans‐Resveratrol
We examined the ability to assemble trans‐resveratrol (trans‐3,5,4′‐trihydrox
ystilbene) biosynthetic enzymes on DNA in the cytoplasm of E. coli using zinc
finger DNA‐binding domains, recognizing a 9‐bp‐long nucleotide sequence, as
DNA‐binding proteins [11]. The metabolic pathway for resveratrol has already
been reconstituted in yeast, mammalian cells, and bacteria [7, 12, 16]. The pro
duction of the trans‐resveratrol from 4‐coumaric acid is a two‐step reaction in
which 4‐coumaric acid is converted to 4‐coumaroyl‐CoA by 4‐coumarate–CoA
ligase (4CL). trans‐Resveratrol is formed by the condensation of one molecule of
4‐coumaroyl‐CoA and three molecules of malonyl‐CoA by stilbene synthase
(STS) (Figure 12.3). We used a low copy number expression plasmid with genes
encoding for 4CL and STS, which were fused to the C‐terminus of Zif268 and
PBSII zinc finger domains, respectively. The DNA scaffold was present on sepa
rate high copy number plasmids. Different DNA programs with various spacer
lengths (2, 4, and 8 bp) and numbers of program repeats (4 and 16) (Table 12.2)
were tested, and almost 10 mg l−1 of trans‐resveratrol was produced when the
number of scaffold repeats was 4 and the spacer length between the DNA‐target
sites was 2 bp, which is 10 times more than with the fusion protein of 4CL and
STS (Figure 12.3b) [11].
AE1
BE2
DE3n
[1 :1:1] 1
8,18, 28 bpAE1
BE2
DE3
DE3n
[1 :1:2] 1
8 bpAE1
BE2
DE3
DE3nDE3[1 :1:3] 1
8 bpAE1
BE2
DE3
DE3nDE3
DE3[1 :1:4] 1
8 bpAspartate HDH HK
semialdehyde Homoseri nePhospho-
homoseri ne ThreonineTS
E1 E2 E3< = <(a)
(b)
Figure 12.2 The biosynthesis of l‐threonine in E. coli is enhanced by the DNA scaffold.
(a) The three‐step conversion of aspartate semialdehyde to l‐threonine. (b) Arrangements of
DNA‐target sites on the DNA program with indicated production rates for l‐threonine are
depicted. The DNA scaffold includes the chimeric proteins, homoserine dehydrogenase (HDH;
E1), homoserine kinase (HK; E2), and threonine synthase (TS; E3), fused to DNA‐binding
domains (ADB). Consecutive arrangements of DNA‐target sites for threonine synthase (E3), the
third enzyme in the biosynthesis of l‐threonine, improved the production rate for l‐threonine.
The DNA‐target sites specific for the individual chimeric proteins are separated with 8‐, 18‐, or
28‐bp spacers between each DNA‐target site. The fastest rate of l‐threonine production in
E. coli was obtained with the DNA program [1 : 1 : 2], with DNA‐binding sites separated by 8 bp
(see also [10]).