Construction of a cell-free synthetic pathway for the production of lactic acid from spent coffee grounds

Coffee is the second largest traded commodity after petroleum but almost half of it ends up as waste in form of spent coffee grounds. Over 50% of the spent coffee grounds are composed of carbohydrates, with mannose representing the most abundant sugar. However, a holistic approach for its efficient utilisation has not been addressed yet. Cell-free synthetic biology is developing into a platform technology enabling a rapid construction of enzymatic pathways for the production of platform chemicals and pharmaceuticals from various substrates. Here, we designed a cell-free synthetic pathway for the utilisation of mannose derived from spent coffee grounds to produce the platform chemical lactic acid. The concept for the synthetic pathway is based on a putative mannose metabolic pathway from the thermoacidophilic archaeon Thermoplasma acidophilum. We were able to reconstruct this metabolic route by heterologous overexpression of mannose dehydrogenase and mannonate dehydratase genes, and successfully convert mannose into 2-keto-3-deoxygluconate (2-KDG) in a one-pot enzymatic reaction. Further addition of two more enzymes resulted in a conversion of 2-KDG into lactic acid in a controlled and stereo-selective manner. Solid binding peptides (SBP) show great binding capacity towards a wide range of solid materials for the directed immobilisation of proteins and enzymes on solid supports. By co-expression of a silica-specific SBP, the pathway enzymes can be immobilised onto cheap silica-based supports like zeolite. This creates a range of reusable biocatalatyic modules, which can be rapidly assembled for future constructions of cell-free production pathways.