A dynamic pathway analysis approach reveals a limiting futile cycle in N-acetylglucosamine overproducing Bacillus subtilis.
Recent advances in genome engineering have further widened the gap between our ability to implement essentially any genetic change and understanding the impact of these changes on cellular function. We lack efficient methods to diagnose limiting steps in engineered pathways. Here, we develop a generally applicable approach to reveal limiting steps within a synthetic pathway. It is based on monitoring metabolite dynamics and simplified kinetic modelling to differentiate between putative causes of limiting product synthesis during the start-up phase of the pathway with near-maximal rates. We examine the synthetic N-acetylglucosamine (GlcNAc) pathway in Bacillus subtilis and find none of the acetyl-, amine- or glucose-moiety precursors to limit synthesis. Our dynamic metabolomics approach predicts an energy-dissipating futile cycle between GlcNAc6P and GlcNAc as the primary problem in the pathway. Deletion of the responsible glucokinase more than doubles GlcNAc productivity by restoring healthy growth of the overproducing strain.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.|Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland.
Yanfeng
Y
Liu
Max Planck Institute for terrestrial Microbiology, Marburg 35043, Germany.
Hannes
H
Link
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.
Long
L
Liu
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.
Guocheng
G
Du
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.|Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi 214122, China.
Jian
J
Chen
Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland.
Uwe
U
Sauer
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002952:Citric Acid Cycle / Q000235:genetics; D003198:Computer Simulation; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005941:Glucokinase / Q000172:deficiency / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000235:genetics; D007700:Kinetics; D060847:Metabolic Engineering / Q000379:methods; D055432:Metabolomics / Q000379:methods; D008956:Models, Chemical; D015219:Substrate Cycling
D001426:Bacterial Proteins; D003470:Culture Media; C010004:N-acetylglucosamine 6-phosphate; D005941:Glucokinase; D005947:Glucose; D000117:Acetylglucosamine