Construction And Application Of A Dynamic Regulation System And A High-throughput Screening Tool Based On The Heme Biosensor

Microbial metabolism engineering aims to use microorganisms as hosts to maximize the production of valuable compounds.It provides an alternative synthetic strategy for biofuels,medicines,and natural products.To achieve the desired yield and titer of the target chemical requires complex optimization and modification of the biosynthetic pathway.The development and utilization of biosensors in metabolic engineering has drawn more attentions in recent years.Suitable biosensors can detect,screen or dynamically regulate metabolic pathways to achieve the optimal state of the pathways.Heme is a typical tetrapyrrole compound,which can be used as a flavoring agent in the synthesis of meat products.At the same time,heme is also an FDA-approved drug in the treatment of porphyria.Its biosynthetic pathway involves a variety of valuable chemicals,such as 5-aminolevulinic acid(ALA),siroheme,chlorophyll,etc.In the research on heme biosynthesis pathways,the effects of traditional metabolic engineering strategies are relatively limited.Heme participates in a variety of physiological and biochemical reactions including respiration,cell differentiation,signal transduction,circadian rhythm and gas induction,is necessary for cells to maintain normal physiological functions.However,excessive free heme is toxic to cells.The necessity and toxicity of heme increase the difficulty of optimizing the heme biosynthetic pathway.In addition,a study has shown that the expression of several heme synthesis gene may have a negative impact on heme biosynthesis,which may be related to the complex regulation mechanism of heme.Therefore,the development of biosensor that responds to heme is of great significance for solving complex problems involving heme synthesis pathway.In this study,we developed a heme biosensor HrtR derived from Lactococcus lactis.We used HrtR to construct a heme-responsive dynamic regulation system and growth-coupled high-throughput screening tool in Escherichia coli,which could realize the high-efficiency production of chemicals with the steady state of heme and the screening of the optimal heme biosynthesis gene expression,respectively.1.Construction and application of heme response dynamic regulation systemWe first characterized the heme biosensor HrtR in E.coli,using green fluorescent protein(GFP)as the reporter to show the heme response of HrtR.Since E.coli DH5? does not own heme input system,we have overexpressed a variety of heme importer proteins into DH5?.Finally,the transporter ChuA derived from E.coli O157:H7 EDL933 was used to measure the dose response of the heme biosensor in DH5?.In addition,strains with different heme production capabilities were used to further confirm the linear relationship between the gfp expression and the intracellular heme concentration.In order to fine-tune the affinity of HrtR,we selected three residues related to heme binding in HrtR for saturation mutations,namely Val131,His 149 and Thr68.According to the fluorescence intensity,five mutants(H149S,V131L,T68L,V131I,H149D)were selected from 57 mutants to determine the dose-response curve.The dose-response curves of H149S,T68L and V131L were determined after measurement,and their heme affinity and EC50 were calculated,but V131I and H149D could not be measured by adding heme externally.We used molecular dynamics simulation and purified protein for heme titration,and successfully determined the heme affinity of five mutants.We designed a dynamic regulatory system that responds to heme using the characterized HrtR and gene silencing tool CRISPRi.This system uses HrtR as a sensor to sense the intracellular free heme and transmit the signal to CRISPRi.CRISPRi acts as an effector to precisely inhibit target genes.Inhibition of the target gene will reduce the heme concentration.When heme is enrichment,CRISPRi will inhibit the expression of target genes.When heme is deficient,HrtR will inhibit the expression of CRISPRi to liberate the target gene.Therefore,the system can regulate the target gene dynamically.Firstly,we used the dynamic regulation system act on ALA dehydratase(ALAD,encoded by hemB)and the red fluorescent protein mKate2 to characterize the dynamic regulation system.By optimizing the inhibition intensity of CRISPRi,no dynamic fluctuation of red fluorescence was observed.We optimized the promoter and RBS intensity of mKate2 and the dynamic fluctuation of red fluorescence were successfully observed.Using real-time quantitative PCR to measure the expression intensity of das9 and hemB,it also successfully proved that the dynamic regulation system can achieve the desired expectations at the transcription level.Finally,the dynamic regulation system was applied to produce ALA.The dynamic regulation system increased the ALA accumulation from 2.4 g/L to 3.75 g/L.At the same time,by measuring the intracellular heme concentration during fermentation process,it can be found that the intracellular heme concentration fluctuates continuously during different periods of fermentation.This result confirms that dynamic regulation system can exert the expected effect in the production of the target chemical.Substituting wild-type HrtR with different mutants can obtain different ALA yields.The mutant H149D increased the ALA yield to 5.35 g/L,further indicating that the precise tuning of the biosensor ligand affinity can improve the productions of target chemicals.The dynamic regulation system has also played better function in the production of porphobilinogen(PBG)and porphyrin.The use of precisely regulated biosensors has increased the production of PBG and total porphyrin by 55.7%and 41%,respectively.2.Construction and application of growth-coupled high-throughput screening toolsIn this section,HrtR was used to construct a high-throughput screening tool by constructing a genetic circuit to couple growth and in vivo heme levels.Since heme and its biosynthetic intermediates would affect the determination of fluorescence,we did not select fluorescent protein as the reporter for flow fluorescence cell sorting(FACS)high-throughput screening.We used HrtR to regulate the tetracycline resistance gene tcR.High level of intracellular free heme will increase the strain's resistance to tetracyclines.The growth difference was used to screen out high levels of accumulated heme in successive iterations.We added a certain concentration of ALA or heme into the medium containing tetracycline and the strain containing the selected plasmid was cultured in this medium for 12 h and measured the OD600.The results showed that the addition of ALA or heme can improve the growth of the strain under tetracycline.By adding 0-250?g/mL tetracycline,the optimal concentration for screening was determined to be 140?g/mL.Based on the constructed high-throughput screening tool,the optimal gene expression intensity for the synthesis of heme precursor protoporphyrin IX(PPIX)was screened.The synthesis of PPIX from ALA requires the co-catalysis of hemB/C/D/E/F/Y.We used four different intensities of RBS to control the expression of each gene.Then,we obtained a combination library of six genes with different expression intensities.The obtained library was transferred to the strain containing the selected plasmid and subcultured in LB medium with 140 ?g/mL tetracycline.After four passages,high-producing strains of PPIX were enriched,and the yield of PPIX reached to 160.78 mg/L.The sequence of the high-yielding strains showed the expression levels of hemD and hemY were lower than hemB,hemC,hemE and hemF.This proves that a reasonable metabolic flux balance can achieve better results than high-level expression pathway genes.Overexpression of ferrous ion chelator(FECH,coded by hemH)derived from Bacillus subtilis enabled the strains to produce heme.We further optimized the start codon of hemH and overexpressed the heme exporter protein to increase the heme production.After the optimization of fermentation conditions,the final extracellular heme production reached 8.34 mg/L.This proves that the growth-coupled high-throughput screening tool can be successfully used for the metabolic optimization of the heme biosynthesis pathway,and proves that the metabolic flux balance is of great significance for the synthesis of heme.In summary,this paper constructed a dynamic regulation system and a high-throughput screening system based on the heme biosensor,which solved the different problems in the metabolic engineering of the heme synthesis pathway,and further enhances the ability of E.coli to produce heme and chemicals related to the heme synthesis pathway.