Optimization Of 5-aminolevulinic Acid Biosynthesis Pathway And Development Of Its Biosensor In Escherichia Coli

5-Aminolevulinic acid(ALA)is an important precursor for the synthesis of tetrapyrrole compounds and has a wide range of applications in agriculture and medicine.The biosynthesis of ALA relies on two pathways,C4 and C5.The C5 pathway was first constructed in Escherichia coli by the researchers in our laboratory,so that glutamate can generate ALA through three enzymatic reactions.Although many metabolic engineering regulation strategies have been applied to ALA biosynthesis,there is still a lot of room for manipulating the engineering of key enzymes in the biosynthetic pathway and the optimization of carbon flux,which has led to a further increase in ALA production to reach a bottleneck that is difficult to break through.Breaking through the bottleneck requires us to further precisely balance and optimize pathway carbon flux.However,it is more complicated to precisely adjust the carbon flux,and it is more difficult to screen high-yielding ALA strains.Therefore,it is urgent to develop ALA-responsive biosensors for application as high-throughput screening tools.On the one hand,this study used a variety of strategies to metabolically regulate the glutamate node of ALA C5 pathway to increase ALA production.To enhance the endogenous glutamate synthesis pathway,we replaced the promoters of icd(encoding isocitrate dehydrogenase),gltBD(encoding glutamate synthase),gltA(encoding citrate synthase)and gdhA(encoding glutamate dehydrogenase)with the strongest promoter BBA_J23100 from iGEM(www.igem.org).Transferred into the ALA fermentation plasmid,it was found that only replacing the promoter of icd gene could only slightly increase the ALA yield,which proved the disadvantage and inefficiency of rigid regulation on the genome.In order to simplify the difficulty of operation,we overexpressed icd,gdhA and acnB(encoding aconitase),and it was found that overexpression of these genes can increase the production of ALA.Overexpression of gdhA has the best effect,increasing the production of ALA by about 70%,proving the usefulness and potential of rationally optimizing the carbon flux of the C5 pathway for accumulation of ALA.In addition,we used CRISPRi technology to inhibit four key genes of glutamate catabolism and sucA(encoding ?-ketoglutarate decarboxylase),but did not achieve the effect of increasing ALA production,and the simultaneous inhibition of the two genes resulted in bacterial growth.Obvious defects proved that it is difficult to achieve reasonable optimization and increase the output of ALA with a single suppression intensity.A series of results indicate that the ALA synthesis pathway has room for optimization,but there is a lack of tools for dynamic regulation and screening of high-yielding strains,so the development of ALA biosensors is urgently neededThe other is the design and development of ALA biosensors,including the development of ALA biosensors based on transcription factors(TF)and the development of biosensors based on the chimeric two-component system(TCS).For the development of transcription factor-based ALA biosensor,we first selected L-arabinose regulator AraC as the initial transcription factor and used computer-assisted rational design and modification to obtain 19 AraC unit point mutants;then all mutants were characterized and screened for ALA responsiveness,and 5 ALA responsive AraC single point mutants were obtained.;then combined mutations and characterization of the responsive mutants to obtain 3 ALA-responsive AraC double-site mutants;finally,the ALA dose-response characterization of the ALA biosensor based on 8 AraC mutants and determination the performance parameters,of which the ALA biosensor with good performance can be directly applied or optimized for use in the metabolic engineering of the ALA synthesis pathway.For the development of ALA biosensor based on the chimeric two-component system,the chemoreceptor PctC(Pseudomonas aeruginosa)was selected for fusing with the TCS EnvZ/OmpR in E.coli.After determining the responsiveness of the biosensor based on the PctC/EnvZ chimera to y-aminobutyric acid(GABA),it was found that the chimera was sensitive to the acidity of the ALA solution.Therefore,we constructed a chimera between PctC and the other four TCS,and characterized that the PctC/PhoQ chimera was more effective.Based on the GABA-responsive chimeric TCS,the PctC sensing domain was rationally designed and modified,and the obtained mutants were verified as ALA biosensors,and the ALA-responsive mutants were preliminarily determined.It was also found that the ALA-responsive mutants produced by the PctC/EnvZ and PctC/PhoQ chimeras are not consistent,which proves that the ligand responsiveness of the two chimera sensing domains is different.Due to the high background fluorescence of the two chimera systems and the problem of leakage,the ALA biosensor based on this system needs to be further optimizedIn summary,this study optimized the ALA C5 pathway in E.coli,which increased ALA production by up to 70%,which proved the usefulness of reasonable optimization and balance of carbon flux and the necessity of developing ALA biosensors.Then the ALA biosensor was designed and developed,TF-based and chimeric TCS-based ALA biosensor were obtained.The ALA biosensor based on the TF AraC mutants has been well characterized and can be used in the metabolic engineering of ALA synthesis pathway;while the biosensor based on the chimeric TCS needs to be further optimized for application.The design and development of the ALA biosensor in this study is the key starting point for the subsequent optimization and application of the ALA biosensor,laying the foundation for its application in the regulation and optimization of the ALA synthesis pathway and the screening of high-yielding strains.