Study On The Mechanism Of Synechococcus Elongatus PCC 7942 For CO₂ Fixation And Synthesis Of Novel Liquid Fuel Isobutanol

Under the severe situation of global warming and energy shortage,our country has put forward the grand goal of "strive to achieve carbon neutrality by 2060",emission reduction and sink increase are two fundamental ways to achieve carbon neutrality.The proposal of the third-generation biorefinery technology based on the utilization of CO₂ by microorganisms to synthesize fuels and chemicals provides a feasible solution for achieving "carbon neutrality" through sink enhancement.Cyanobacteria can utilize CO₂ in the atmosphere for photoautotrophic metabolism,and have the characteristics of simple structure and easy genetic manipulation,so they have become excellent chassis organisms for the third-generation of biorefinery technology.Cyanobacteria have been genetically modified to synthesize butanol liquid fuels with high octane number and low hygroscopicity using CO₂,which has become a promising technology for bioenergy refining.However,the main problems of this technology are low liquid fuel yield,low carbon sequestration efficiency and lack of stable and convenient means to increase butanol yield.The main contents and results of this study are as follows:（1）Salt stress is the easiest way to implement environmental stress,appropriate addition can stimulate the stress resistance of cyanobacteria,and achieve the accumulation of various target products such as lipids and alcohols.Therefore,in this study,sea salt stress was used to improve isobutanol production of engineered cyanobacteria,and the cellular morphology,metabolic pathway,oxidative stress and other endogenous physiological response mechanisms of cyanobacteria were investigated.The results showed that 2%sea salt increased the isobutanol production of engineered cyanobacteria by 4 times to 0.637 g/L,the Calvin cycle and TCA cycle were inhibited,and the anabolism of amino acids,polypeptides and nucleic acids were weakened,resulting in the inhibition of the growth of cyanobacteria.However,sea salt significantly accelerated the breakdown of lipids,accompanied by the production of more NADH,which is an important coenzyme in the process of isobutanol synthesis and an important reason for promoting the production of isobutanol.Secondly,the cells shrank and the permeability of the membrane increased,which was more conducive to the release of isobutanol.Under salt stress,cyanobacteria produced large amounts of sucrose,raffinose,carotenoid and reduced glutathione to resist high salt and oxidative damage.In addition,the experiment of using simulated seawater and municipal sewage to cultivate engineered cyanobacteria to synthesize isobutanol with CO₂ was explored in this study,achieving a similar isobutanol yield as the culture medium,and realizing the resource reuse of salt in seawater and nitrogen and phosphorus in wastewater.（2）Carbonic anhydrase（CA）can promote efficient reversible conversion of carbon dioxide and bicarbonate,participate in the acquisition of extracellular inorganic carbon in cyanobacteria and carboxylation of carbon fixation in carboxysome,and is an important factor affecting the CO₂ concentration mechanism（CCM）and carbon fixation efficiency of cyanobacteria.In this study,the extracellular carbonic anhydrase gene ecaA was successfully knocked out from S.elongatus PCC 7942,and the wild and mutant strains were cultured under three inorganic carbon sources to explore the influence mechanism of the change of carbon uptake mode on cyanobacteria physiological metabolism.The results showed that the catalytic effect of EcaA was not obvious under the conditions of 0.04%CO₂ and 40 mM NaHCO₃,and there was no difference in the growth conditions;under the condition of 3%CO₂,both the wild-type and mutant strains grew rapidly,and it was found that the cyanobacteria had higher CCM energy efficiency and carbon fixation rate when the intracellular pH value was in the range of 8.0-8.4;the expression of HCO₃-transporter gene bicA was up-regulated after the removal of Ci restriction;supplemented with 40 mM NaHCO₃,the expression of transporter genes sbtA,cmpA and carbonic anhydrase genes ccmM and icfA were up-regulated in mutant strains,while the expression of CA gene ecaA was inhibited in wild strains.