Simon Boecker1, Björn-Johannes Harder1, Regina Kutscha2, Stefan Pflügl2, Steffen Klamt3. 1. Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstraße 1, 39106, Magdeburg, Germany. 2. Institute for Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, Technische Universität Wien, Gumpendorfer Straße 1a, 1060, Vienna, Austria. 3. Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstraße 1, 39106, Magdeburg, Germany. klamt@mpi-magdeburg.mpg.de.
Abstract
BACKGROUND: The alcohol 2,3-butanediol (2,3-BDO) is an important chemical and an Escherichia coli producer strain was recently engineered for bio-based production of 2,3-BDO. However, further improvements are required for realistic applications. RESULTS: Here we report that enforced ATP wasting, implemented by overexpressing the genes of the ATP-hydrolyzing F1-part of the ATPase, leads to significant increases of yield and especially of productivity of 2,3-BDO synthesis in an E. coli producer strain under various cultivation conditions. We studied aerobic and microaerobic conditions as well as growth-coupled and growth-decoupled production scenarios. In all these cases, the specific substrate uptake and 2,3-BDO synthesis rate (up to sixfold and tenfold higher, respectively) were markedly improved in the ATPase strain compared to a control strain. However, aerobic conditions generally enable higher productivities only with reduced 2,3-BDO yields while high product yields under microaerobic conditions are accompanied with low productivities. Based on these findings we finally designed and validated a three-stage process for optimal conversion of glucose to 2,3-BDO, which enables a high productivity in combination with relatively high yield. The ATPase strain showed again superior performance and finished the process twice as fast as the control strain and with higher 2,3-BDO yield. CONCLUSIONS: Our results demonstrate the high potential of enforced ATP wasting as a generic metabolic engineering strategy and we expect more applications to come in the future.
BACKGROUND: The alcohol 2,3-butanediol (2,3-BDO) is an important chemical and an Escherichia coli producer strain was recently engineered for bio-based production of 2,3-BDO. However, further improvements are required for realistic applications. RESULTS: Here we report that enforced ATP wasting, implemented by overexpressing the genes of the ATP-hydrolyzing F1-part of the ATPase, leads to significant increases of yield and especially of productivity of 2,3-BDO synthesis in an E. coli producer strain under various cultivation conditions. We studied aerobic and microaerobic conditions as well as growth-coupled and growth-decoupled production scenarios. In all these cases, the specific substrate uptake and 2,3-BDO synthesis rate (up to sixfold and tenfold higher, respectively) were markedly improved in the ATPase strain compared to a control strain. However, aerobic conditions generally enable higher productivities only with reduced 2,3-BDO yields while high product yields under microaerobic conditions are accompanied with low productivities. Based on these findings we finally designed and validated a three-stage process for optimal conversion of glucose to 2,3-BDO, which enables a high productivity in combination with relatively high yield. The ATPase strain showed again superior performance and finished the process twice as fast as the control strain and with higher 2,3-BDO yield. CONCLUSIONS: Our results demonstrate the high potential of enforced ATP wasting as a generic metabolic engineering strategy and we expect more applications to come in the future.
Entities:
Keywords:
Biofuels; Butanediol; Enforced ATP wasting; Escherichia coli; Productivity; Two‐stage process