Study On The Synthesis Of Metabolites In The Tricarboxylic Acid Cycle Based On The Microdroplet Environment

The issue of the origin of life was a worldwide problem.Studying the origin of life is beneficial to understand the details in the origin of life periods,such as the time,geographical environment,climatic conditions,conversion forms of matter and heat,nature of metabolism,heritable variation,self-replication and other physiological phenomena.This would guide the scientific and technological development,and promote the advancement of human science and technology.The tricarboxylic acid(TCA)cycle is a central anabolic biochemical pathway,whose origins are thought to date back to geochemistry.It was also thought as the one long before the emergence of enzymes,RNA,or cells,which is significant to the core metabolism.Therefore,the study of the synthesis mechanism of abiotic substances transfer in the TCA cycle of pre-life periods not only promotes the development of research on early metabolism and other pre-biological organic molecules,but also plays an important role in guiding the synthesis of organic materials at present.Based on the patent named"a kind of chemical reaction intermediate detection-mass spectrometry device"in our laboratory,the microdroplets reaction system was set up to verify the feasibility of the method and achieve the non-biosynthesis and substances transformation in the TCA cycle.After that,the parameters of microdroplets system and instrument were optimized to ensure the best experimental conditions.Based on the microdroplets reaction system,the non-biosynthetic reaction in the TCA cycle was achieved without enzymes and catalysts:oxaloacetic acid is reduced to malic acid,fumaric acid is reduced to succinic acid,malic acid is dehydrated to fumaric acid,citric acid is dehydrated to citraconic acid,isocitrate is dehydrated to citraconic acid,isocitrate is decarboxylated to?-ketoglutaric acid,?-ketoglutaric acid is decarboxylated to succinic acid.It was also found that the reaction which was difficult to occur in the biological system(i.e.,carboxylation of?-ketoglutarate to produce isocitrate,citrate to oxaloacetic acid)could be carried out in the microdroplets environment.Under the mixed conditions of different reaction types,good experimental results can still be obtained,which proved that the microdroplets could bear the possibility of multiple reaction types at the same time at the origin of life.The reaction mechanism of non-biosynthesis was studied by changing the gas pressure,the concentration of the reaction solution,and whether the external charge was applied.It was found that by changing the gas pressure and thus the microdroplets sizes,the reaction conversion efficiency was improved concerning to the decrease of microdroplets sizes at a certain microdroplets diameter.This is because the specific surface area of the microdroplets was increasing by reducing the microdroplets sizes.The conversion rate reached 13%at 100 psi.In the concentration-dependance experiments,it was found that the conversion efficiency is low concerning to a higher substrate concentration.Accordingly,the reaction conversion efficiency was significantly enhanced by reducing the concentration of reactants.This is because the hydrophobicity of microdroplets surface,the reagents were easy to concentrate on/near the air-water interface.In addition,it was easier to aggregate at the interface for reaction when the substrate concentration is low.When the concentration is 10^-5mol·L^-1,the conversion rate can reach 10%.By comparing the experiment data with and without applying external charges,it was found that the external charges were not the main driver of the non-biosynthesis reaction in microdroplets.However,by decreasing the microdroplets sizes and thus increasing the specific surface area,applying external charges could increase the conversion efficiency.This experimental evidence provided and explained how does the non-biosynthetic reactions occur at or near the air-water interface of microdroplets,which is beneficial to further shed light on the abiotic pathways of complex biomolecules in the study of the origin of life.