(211f) Cell-Free Enzymatic Hydrogen: Conversion of Biomass Sugars to Biocommodity

Biohydrogen has tremendous potential to minimize the greenhouse gas emissions and other environmental issues associated with current hydrogen production methods, which use natural gas.  The economic potential of this alternative is tremendous as well.  By harnessing the power of thermostable enzymes assembled into the proper pathway, sugars derived from cellulosic biomass can be used to create biohydrogen at a cost competitive with current fossil production methods.  Synthetic Pathway Biotransformation (SyPaB) is one such scheme.  In this method, twelve heterologously expressed, thermostable enzymes are assembled in one pot, to form a synthetic enzymatic pathway that fully oxidizes glucose to hydrogen and carbon dioxide.  Industrial examples exist of enzymes with weight-based total turnover numbers (kg product produced per kg enzyme) of more than 1,000,000.  Once this stability is achieved for the SyPaB enzymes, cost of the biocatalyst becomes a very small fraction of the biocommodity cost.  Between this and other advantages over whole-cell catalysis, such as no cell wall transport required and better ability to monitor reaction conditions, SyPaB conversion has the potential to produce hydrogen from biomass sugars at a faster rate and higher yield than any other method.  Previous studies by our group have demonstrated the feasibility of SyPaB conversion of starch and soluble cellodextrins to hydrogen.  Here, we report a broadened substrate range and increases in reaction rate and yield.  Our yield is now the highest of any biohydrogen production method, and our reaction rate is now greater than dark fermentation.  Substrates now include glucose, fructose, xylose, and insoluble cellulose, the last of which is made possible by combining SyPaB and with cellulose hydrolysis.  Rate and yield improvements were achieved using a metabolic model informed by enzyme kinetics and metabolomics assays of the reaction mixture throughout the time course of the reaction.  These important results highlight the excellent potential of cell-free conversions for future biocommodity production, and demonstrate the power of metabolomics and enzyme kinetic modeling to greatly improve the performance of these systems.