TY - JOUR
T1 - Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering
AU - Fehér, Tamás
AU - Planson, Anne-gaëlle
AU - Carbonell, Pablo
AU - Fernández-castané, Alfred
AU - Grigoras, Ioana
AU - Dariy, Ekaterina
AU - Perret, Alain
AU - Faulon, Jean-loup
PY - 2014/11/1
Y1 - 2014/11/1
N2 - Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer‐aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top‐ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top‐ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17‐fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.
AB - Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer‐aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top‐ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top‐ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17‐fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.
UR - https://onlinelibrary.wiley.com/doi/abs/10.1002/biot.201400055
U2 - 10.1002/biot.201400055
DO - 10.1002/biot.201400055
M3 - Article
SN - 1860-6768
VL - 9
SP - 1446
EP - 1457
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 11
ER -