Metabolomic Basis Of Laboratory-evolved Butanol Tolerance In Photosynthetic Synechocystis Sp.PCC 6803

Compared with bio-ethanol, butanol has better physical and chemical properties and has been proposed as a next-generation biofuel. Recent efforts demonstrated the potential application of cyanobacteria as a “microbial cell factory” to produce butanol directly from CO2. However, cyanobacteria have very low tolerance to the toxic butanol, which limits the economic viability of this renewable system.Recently, laboratory-based adaptive evolution has been proposed as a valuable mean to enrich favorable genetic changes and achieve better biofuels tolerance in various microbes, which we applied to increase the butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803.Through a long-term experimental evolution process, we achieved a 225% increase of the butanol tolerance in Synechocystis sp. PCC 6803 after a continuous 126 passages for 600 days in BG11 media amended with gradually increased butanol concentration from 0.20% to 0.65%(v/v).To decipher the molecular mechanism responsible for the tolerance increase, we employed an integrated GC-MS and LC-MS approach to determine metabolomic profiles of the butanol-tolerant Synechocystis strains isolated from several stages of the evolution, and then applied PCA and WGCNA network analyses to identify the key metabolites and metabolic modules related to the increased tolerance. The results showed that unstable metabolites of 3-phosphoglyceric acid(3PG), D-fructose 6-phosphate(F6P), D-glucose 6-phosphate(G6P), DHAP, phosphoenolpyruvic acid(PEP), D-ribose 5-phosphate(R5P), and stable metabolites of glycerol, L-serine and stearic acid were differentially regulated during the evolution process, and 9 models, which could be related to tolerance increase to butanol in Synechocystis.The study provided the first time-series description of the metabolomic changes related to the gradual increase of butanol tolerance, and revealed metabolomic basis important for further rational engineering in Synechocystis, and provided a list of potential targets to engineer Synechocystis against butanol stress.