DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.
Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Robert J
RJ
Conrado
Gabriel C
GC
Wu
Jason T
JT
Boock
Hansen
H
Xu
Susan Y
SY
Chen
Tina
T
Lebar
Jernej
J
Turnšek
Nejc
N
Tomšič
Monika
M
Avbelj
Rok
R
Gaber
Tomaž
T
Koprivnjak
Jerneja
J
Mori
Vesna
V
Glavnik
Irena
I
Vovk
Mojca
M
Benčina
Vesna
V
Hodnik
Gregor
G
Anderluh
John E
JE
Dueber
Roman
R
Jerala
Matthew P
MP
DeLisa
D001665:Binding Sites; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D060847:Metabolic Engineering; D008798:Mevalonic Acid / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D019946:Propylene Glycol / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D004798:Enzymes; D013267:Stilbenes; D019946:Propylene Glycol; D004247:DNA; D000077185:Resveratrol; D008798:Mevalonic Acid