Construction Of Engineered Mixotrophic Cyanobacteria And Saccharomyces Cerevisiae For Carbon Dioxide Fixation With High Efficiency

Global warming is one of the major environmental issues that jeopardizes human society.In order to alleviate global warming caused by excessive emissions of greenhouse gases(GHGs),reducing atmospheric GHGs with the goal of "carbon peak" and "carbon neutrality"is of great necessity.The utilization of biofuels can efficiently reduce CO2 emissions,while promoting CO2 fixation to balance the emission and removal is a major way to realize carbon neutrality,thus alleviating global warming effectively.Photoautotrophs fix CO2 via Calvin cycle,and co-utilization of CO2 and organic carbons would accelerate microbial growth,however,the efficiency of CO2 fixation in the engineered mixotrophic photoautotrophs remains unclear.In addition,heterotrophs as the platforms of most biofuel production could fix CO2 when Calvin cycle from photoautotrophs is introduced,thus realizing CO2 fixation and bioproduction simultaneously,while CO2 fixation in the engineered heterotrophs needs to be improved.In order to solve these problems,the obligate photoautotrophic cyanobacterium Synechococcus elongatus PCC7942 was selected as the host strain.Since abundant glucose and xylose could be obtained from the hydrolysis of lignocellulose,two engineered S.elongatus capable of utilizing glucose or xylose were constructed for lignocellulose resource.The effect of glucose or xylose utilization on CO2 fixation in the engineered strains under continuous light and light/dark cycles to simulate day and night was studied,and transcriptomics and metabolomics were performed to interrogate the metabolic mechanism.Moreover,metabolic response to circadian rhythm was explored to have a comprehensive view of the mechanism.In addition,the heterotrophic microorganism Saccharomyces cerevisiae which has the endogenous bioethanol synthesis pathway was then studied.The effect of CO2 as a metabolic product of yeast during sugar fermentation was investigated.RuBisCO(ribulose-1,5-bisphosphate carboxylase/oxygenase)in Calvin cycle from S.elongatus was then optimized to increase the carboxylation activity,and expressed together with PRK(phosphoribulokinase)in S.cerevisiae capable of utilizing both glucose and xylose to realize efficient CO2 fixation in the engineered S.cerevisiae,and ethanol yield was significantly improved during xylose fermentation.Moreover,Calvin cycle was optimized to further improve CO2 fixation in the engineered S.cerevisiae.This study provides theoretical basis and technical support for efficient CO2 fixation by mixotrophs as well as realization of carbon neutrality during bioproduction.The main conclusions are as follow.(1)The engineered S.elongatus capable of utilizing glucose or xylose were constructed by expressing galactose transporter,which could transport glucose,or xylose transporter,xylose isomerase and xylulokinase involved in xylose degradation pathway.Growth profiles showed that the engineered mixotrophic S.elongatus grew faster under either continuous light or light/dark cycles,while the reliance on light energy decreased,however,CO2 fixation enhanced in the engineered strains with 57%under continuous light and 388%under light/dark cycles due to glucose utilization,and 51%under continuous light due to xylose utilization.(2)Transcriptomics and metabolomics revealed that efficient utilization of sugar increased the carbon flux as well as generated energy in the engineered mixotrophic S.elongatus,thus decreasing the reliance on light energy.The sufficient metabolites and extra energy provide substrates and energy for Calvin cycle,thus promoting CO2 fixation in the mixotrophic strains under continuous light and light/dark cycles.In addition,circadian clock had little effect on the metabolism in the engineered strain due to glucose utilization during light/dark cycles,thereby causing negligible light effect on CO2 fixation in the engineered mixotrophic S.elongatus.(3)CO2 as the metabolic product affected sugar metabolism in heterotrophic S.cerevisiae.Specifically,productions of glycerol reduced in S.cerevisiae,which may be due to the repressed expression of GPP1 involved in glycerol synthetic pathway in response to CO2.The redox imbalance caused by decreased glycerol could be alleviated by ethanol synthesis from acetate,thus decreasing acetate production but increasing ethanol yield in S.cerevisiae.In addition,CO2 affected the transcriptional regulatory network of S.cerevisiae.However,since S.cerevisiae could not fix CO2 efficiently,the total carbon flux in metabolites was not increased.(4)To realize efficient CO2 fixation and bioethanol production simultaneously in S.cerevisiae,form I RuBisCO in Calvin cycle from S.elongatus with optimized carboxylation activity by co-expression of HSP60 chaperone GroE from Escherichia coli and RuBisCO chaperone RbcX was expressed in S.cerevisiae capable of fermenting glucose and xylose.PRK from S.elongatus was co-expressed to construct the engineered S.cerevisiae with efficient CO2 fixation.The engineered strain in high-level CO2 had a higher yield of ethanol during xylose fermentation with an increase of 41.63%and 5.91%under anaerobic and aerobic conditions,respectively,and CO2 fixation was further improved by overexpression of carbonic anhydrase and phosphoglycerate kinase to optimize Calvin cycle and higher ethanol yield was obtained.