Cell Free Conversion of Pyruvate to 2,3-Butanediol Using Co-Substrate Feed as pH Control Strategy
Bilal Jilani1, Wheaton L. Schroeder2, Costas D. Maranas2,3, and Daniel G. Olson1,3
1Dartmouth College; 2 The Pennsylvania State University; and 3Center for Bioenergy Innovation
Cell-free systems are promising tools for production of chemicals and to improve understanding of biochemical pathways. Researchers are interested in developing well-characterized modules whose behavior can be mathematically predicted, allowing them to be combined into larger systems either as production modules or indicator reactions. The conversion of pyruvate to 2,3-butanediol is a good model system due to interest in 2,3-butanediol as a commodity chemical, thermodynamic favorability of all reaction steps, and prior demonstration of high titer production both in vitro and in vivo.
In the present work, the team demonstrate development of a high-performance pyruvate to 2,3- butanediol conversion system. Researchers start by characterization of individual enzymes in the pathway and demonstrate that none of them are subject to significant inhibition by substrates or products. Researchers then demonstrate conversion of 1063.1 (±19.0) mM (~93.7 g/L) acetoin to 1017.3 (±2.0) mM (~91.7 g/L) 2,3-butanediol, which represents 95.7% of the theoretical maximum yield, high titer and yield using a 2-enzyme system consisting of butanediol dehydrogenase and formate dehydrogenase. Team members subsequently extended the system to allow conversion of pyruvate to 2,3-butanediol using a 4-enzyme system. Researchers were able to convert 2045.5 (±30.7) mM (~225.0 g/L) pyruvate to 929.1 (±20.4) mM (~83.7 g/L) 2,3-butanediol, which represents 90.8% of the theoretical maximum yield. Achieving high titer production required careful attention to proton recycling. Further increases to product titer were limited by experimental limitations (substrate solubility, foaming due to gas formation, etc.) rather than intrinsic limitations of the enzymatic pathway.
The team subsequently developed mechanistic kinetic models for each enzyme individually and showed that these models (1) can be combined to predict the behavior of the 4-enzyme system, or (2) can be used to predict targeted modifications to minimize enzyme concentration (while maintaining the overall conversion rate) or to minimize concentration of a particular metabolic intermediate.