Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembling a number of enzymes or their complexes and coenzymes. Assembly of numerous enzymes without cellular membrane, gene regulation, or undesired pathway can circumvent some of the obstacles to modifying living microorganisms. Several synthetic pathways for the production of liquid biofuels--alcohols and hydrocarbon precursors (polyols) as well as gaseous biofuel--hydrogen have been presented. The present constraints to SyPaB include the lack of stable enzymes as Lego-like building blocks, the different optimal reaction conditions for individual enzyme, and the use of costly labile coenzymes. It is expected that high-yield SyPaB will be an important platform for producing low-cost biofuels and biochemicals.

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