Application Of Metabolomic Analyses In Photosynthetic Cyanobacteria Under Butanol Tolerance

Butanol is highly toxic to cyanobacterial cells, which could restrict the future application of this renewable system in producing carbon neutral biofuel butanol. To seek knowledge regarding butanol tolerance in cyanobacteria, a library of response regulator(RR) gene mutants of Synechocystis sp. PCC 6803 was screened. The results showed that the deletion mutant of an orphan RR-encoding gene sll0039 was more sensitive to butanol than the wild type, while complementation of the ?sll0039 mutant with the sll0039 gene recovered its tolerance to the same level as the wild type, suggesting that the sll0039 gene was involved in the regulation of tolerance against butanol.Further analysis employing an integrated liquid chromatography-mass spectrometry(LC-MS)-based and gas chromatography-mass spectrometry(GC-MS)-based metabolomics was conducted to determine the possible regulatory network of butanol tolerance mediated by Sll0039.To accurately analyze metabolites in cyanobacteria, in this study, an LC-MS based metabolomics method was optimized for Synechocystis sp. PCC 6803. With the optimized extraction, liquid chromatographic and mass spectral parameters, the method was capable of detecting 24 important metabolites related to central carbohydrate and energy metabolism in Synechocystis sp. PCC 6803.LC-MS analysis allowed the identification of several metabolites, such as adenosine 5?-diphosphate(ADP)-glucose, dihydroxyacetone phosphate(DHAP), Dribose 5-phosphate(R5P), D-glucose 6-phosphate(G6P), which were differentially regulated between the wild type and the ?sll0039 mutant grown under butanol stress, while GC-MS analysis identified 1, 2, and 2 metabolic modules associated with the sll0039 gene deletion at 24, 48, and 72 h, respectively, suggesting that they were under control directly or indirectly by Sll0039 RR.In addition, a metabolomic comparison of the metabolic responses to butanol stress was conducted in the ?sll0039 mutant and the ?slr1037 mutant previously found to be involved in butanol tolerance[1], and the results showed that the regulatory networks mediated by Sll0039 and Slr1037 could be functionally independent in Synechocystis.This study optimized a LC-MS based metabolomics method for cyanobacteria Synechocystis sp. PCC 6803, which laid an important foundation for the metabolomics analysis of cyanobateria. In addition, this study made a metabolomic comparison of the metabolic responses to butanol stress in the ?sll0039 mutant and the ?slr1037 mutant, which provided a metabolomic description of the butanol tolerance network regulated by Sll0039.