Constructing Building Blocks for a Cell-Free Synthetic Pathway Biotransformation: a High Yield and Low-Cost Bio-Manufacturing Platform

The production of hydrogen from low-cost renewable biomass is appealing because biomass is an enriched, collectable renewable energy source.  The use of carbohydrates as high-density hydrogen carriers and energy sources for hydrogen production is possible due to cell-free synthetic biology technology.  Synthetic pathway biotransformation (SyPaB) is the implementation of complicated biochemical reactions by the in vitro assembly of a large number of enzymes and coenzymes.  Hydrogen can be produced from carbohydrate with an overall reaction of C₆H₁₀O₅ (aq) + 7H₂O (l) --> 12H₂ (g) + 6CO₂ (g).  As a result, nearly 12 moles of dihydrogen are produced per glucose equivalent of polysaccharides and water by this non-natural synthetic pathway, which contains 13-14 enzymes.   For facilitating cascade reactions among three enzymes: Thermus thermophilus triose phosphate Isomerase (TtcTIM), Thermotoga maritima fructose bisphosphate aldolase (TmALD), and Thermotoga maritima fructose 1,6-bisphosphatase (TmFBP), we need find optimal conditions and proper enzyme ratios for co-immobilized enzymes (combi-CLEAs). In this talk, we will present our recent progresses in enzyme purification, immobilization, and substrate channeling among cascade enzymes.