CO₂ Sequestration Engineering Of Microorganisms For Production Of L-malate

Elevated CO₂ is a partial cause of the complex phenomenon of climate change.Conventional methods for CO₂ sequestration,including CO₂ capture,CO₂ separation,and CO₂ storage,have obvious shortfalls,such as energy-intensive processes and high operational costs.Microbial CO₂ sequestration provides a green and sustainable approach for to solve this problem,which can simultaneously alleviate environmental problems,simultaneously produces value-added chemicals,helping ameliorating resource shortage.In this study,microbial CO₂ sequestration to produce L-malate was choosen as the research model.Autotrophic and heterotrophic synergistic CO₂ fixation pathway,synthetic light-driven CO₂fixation system and artificial light-driven CO₂ mitigation system were designed according to stoichiometric calculation and literature mining.Using metabolic engineering and synthetic biology techniques,these devises was realized to improve the efficiency of CO₂ fixation and L-malate titer in Synechococcus elongatus UTEX2973,and enhance L-malate titer and yield in E.coli MG1655.The main results are as follows:1.Based on stoichiometric calculation,the energy imbalance between light reaction and dark reaction of CO₂ to L-malate in cyanobacteria was diagnosed,and synergistic CO₂fixation pathway was designed;Using cell-free system,efficient enzymes for synergistic CO₂fixation pathway were screened,including phosphoenolpyruvate carboxykinase?As PCK?and L-malate dehydrogenase?As MDH?of Actinobacillus succinogenes;the introduction of As PCK and As MDH into Synechococcus elongatus UTEX2973 realized the construction of the synergistic CO₂ fixation pathway,resulting in the CO₂ fixation efficiency increased by73%,and the titer of L-malic acid increased from 0 to 200?M;After further optimizing the expression ratio of key enzymes in synergistic CO₂ fixation pathway and blocking primary L-malate degrading pathway,the titer of L-malate in engineered strain SH108 was increased to 600?M;Finally,L-malate titer by strain SH108 was further improved to 950?M by optimizing fermentation conditions.2.Based on stoichiometric calculation,synergistic CO₂ fixation pathway for L-malate production in E.coli was designed.The pathway is mainly composed of an ATP-dependent1,5-bisphosphate ribulose carboxylase/oxygenase?Ru Bis CO?branch and ATP regenerating PCK reductive pathway.Using E.coli MG1655 as the host,the 5-phosphoribulose kinase,Ru Bis CO and carbonic anhydrase from Synechocystis sp.PCC6803 were introduced to construct functional Ru Bis CO branch.By further blocking L-malate degradation pathway and competition pathways,and introducing the enzymes of As PCK and As MDH to reconstruct the PCK reduction pathway,the CO₂ fixation efficiency of engineered E.coli GH089 was increased by 49 fold,with the titer of L-malate increased from 7.9 m M to 106 m M;After optimizing the expression level the key enzymes,the CO₂ fixation efficiency was further increased from 36.5 mg/g DCW to 49 mg/g DCW.Finally,the titer of L-malate was achieved at387 m M when strain GH389 was fermented in a 3.6-L bioreactor.3.Based on literature mining,kinetics and thermodynamic analysis,a novel CO₂ fixation pathway was designed and named as HWLS pathway.This pathway was made up of formate dehydrogenase?FDH?,formyltetrahydrofolate cyclohydrolase?Fhs?,methyltetrahydrofolate dehydrogenase?Fch A?,methylenetetrahydrofolate reductase?Fol D?,formaldehyde lyase?FLS?and dihydroxyacetone kinase?DHAK?,which was demonstrated to be feasible in cell-free system.FLS was diagnosed as the rate-limiting step,and a novel high-throughput screening method was developed protein engineering of FLS.After achieving the mutant FLS*with a 4-fold increase in activity,the efficiency of HWLS pathway was increased by 2.3times.The optimized HWLS pathway was further introduced into an L-malate-producing E.coli that equipped with self-assembled Cd S nano-light-harvesting system.Under blue anaerobic conditions,the yield of L-malate from glucose was increased from 1.26 mol/mol to1.64 mol/mol by the engineered strain LF-2.Finally,the yield of L-malate from glucose can be achieved at 1.85 mol/mol within 72 hours anaerobic conversion after optimizing fermentation conditions.4.Based on in silico simulation of flux balance analysis,about 34%of the carbons is lost in the form of CO₂ when E.coli growing on glucose under aerobic conditions,and the TCA cycle is the main source of CO₂ release.According to literature mining,a novel light-driven mitigation system was designed based on a cyanobacteria transcription factor and marine plankton rhodopsin protein,to dynamically regulate the CO₂ release of the TCA cycle.The transcriptional regulatory units P_N and Ndh R from Synechocystis sp PCC 6803 were cloned and rationally modified,resulting in a functional molecular switch P_N2-Ndh R that can respond to intracellular?-ketoglutarate in E.coli;the switch P_N2-Ndh R was demonstrated to be capable of regulating the TCA cycle when it was used to control the gene glt A,resulting in a decrese of overall CO₂ release.By further constructing rhodopsin-based ATP regenerating system,the engineered strain can achieve higher carbon yield of biomass from glucose under aerobic green light condition.Finally,integrating this aerobic CO₂ reduction system with the anaerobic CO₂ fixation system in Chapter 4,the overall yield of L-malate from glucose can be at 1.48 mol/mol by engineered strain LFM-2,exceeding the constraint model theoretical yield.