Synthetic protein scaffolds provide modular control over metabolic flux.
Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.
Dueber
John E
JE
California Institute of Quantitative Biomedical Research (QB3), University of California, Berkeley, California, USA. jdueber@berkeley.edu
Wu
Gabriel C
GC
Malmirchegini
G Reza
GR
Moon
Tae Seok
TS
Petzold
Christopher J
CJ
Ullal
Adeeti V
AV
Prather
Kristala L J
KL
Keasling
Jay D
JD
D000818:Animals; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005937:Glucaric Acid / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D008798:Mevalonic Acid / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D015202:Protein Engineering; D051381:Rats; D017186:Titrimetry
D029968:Escherichia coli Proteins; D005937:Glucaric Acid; D008798:Mevalonic Acid