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Metabolic Engineering And Physiological Study Of Cofactor Metabolism Of An Industrial Erythromycin Producer Guided By Comparative Omics Analysis

Posted on:2021-02-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:X B LiFull Text:PDF
GTID:1521306032958959Subject:Biochemical Engineering
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Erythromycin is one kind of macrolide antibiotic produced by Saccharopolyspora erythreaea and plays irreplaceable roles in pharmaceutical industry.Strategies of metabolic engineering have been widely employed to improve the bio-production of secondary metabolites in streptomycetes.However,it is still a long way to fully engineer S.erythraea and exploit its full potential to increase theproduction of erythromycin.The lack of manufacturing methods and tool kits for S.erythraea and the superficial understanding regarding the regulatory mechanisms of erythromycin biosynthesis are the two major reasons of limiting the improvement the production of erythromycin.In order to further investigate the biosynthesis and regulation of erythromycin production at the molecular level,and to propose feasible strategies to improve the biosynthesis of erythromycin in an industrial strain,research was carried out into S.erythraea as follows:(a),the optimization and expansion of genetic manipulation platform for metabolic engineering;(b),the integration of comparative genomics and transcriptomics for discovering new strategies of metabolic engineering;(c),the investigation of effects of cofactor metabolism on the biosynthesis of erythromycin and the related mechanisms.Firstly,the inter-species transconjugation process to transfer genes into an industrial erythromycin-producing strain,HL3168 E3 was optimized with respect to the pre-germination of S.erythraea spores,ratio of S.erythraea to E.coli,components of transconjugation medium,and duration of transconjugation.As a consequence,the ratio of S.erythraea to E.coli was a most important factor to generate more exconjugants.Next,a synthetic promoter library was successfully constructed.Semi-consensus sequences at-35 and-10 region of promoters were set up based on the alignment between the identified promoter sequences of 16s rRNA gene in Streptomyces coelicolor and the putative 16s rRNA genes in S.erythraea.The up-and downstream and the spacers region of-35 and-10 regions were synthesized randomly.Consequently,a synthetic promoter library were constructed with varying strength ranging from 0.1%to 90%of ermE*p.Secondly,comparative genomics and transcriptomics were integrated to discover new strategies for engineering S.erythraea.The whole genome of E3 was re-sequenced using Illumina Hiseq2000.Compared to the reference genome of NRRL23338,the genome of E3 had mutations with respect to the increase of copy numbers of several genes,large deletions inside of coding sequences,and 256 single nucleotide polymorphisins(SNPs).These mutations made E3 as a high erythromycin-producing strain.At the transcriptional level,compared to NRRL23338,the transcription of erythromycin biosynthesis gene clusters in E3 was induced significantly,while the transcription of gene clusters of other secondary metabolites was repressed in E3.Furthermore,the mechanisms by which E3 enhances its erythromycin production were proposed at three aspects,i.e.,supply of precursors,regulatory networks and intracellular biochemistry environment.As for the supply of precursors,the genes sucA and sucB coding for α-ketoglutarate dehydrogenase were overexpressed in E3,which enhanced the erythromycin production by 40%.When it came to the regulatory network,SACE0101/0102 were identified as genes encoding a novel two component system for responding to the concentration of copper ions.In addition,in this study,it was found that the mutation or the deletion of SACE0101 could enhance the copper tolerance of S.erythraea,and the addition of copper ions could enhance the biosynthesis of erythromycin.Thirdly,effects of the change of intracellular biochemical environment,especially the perturbation of intracellular cofactors,on the biosynthesis of erythromycin were investigated.Through decreasing intracellular energy level by overexpressing F1-ATPase at attB-attP site and increasing the level by adding sub-lethal concentration of antibiotic,the positive correlation between the intracellular energy level and the biosynthesis of erythromycin was uncovered in the present research.It revealed that the intracellular redox status was changed as the energy level was perturbed.In order to maintain the energy and the redox balance,metabolic shifts occurred at the node of acetyl-CoA,from which more carbon flux was switched into the biosynthesis of the reddish pigment,7-O-rahmnosyl flaviolin,with a lower NADH yield.Based on the correlation between the energy level and the biosynthesis of erythromycin,the entire F1F0-ATPase was overexpressed in E3,which resulted in improving the erythromycin production by 28%.In addition,the change of intracellular redox status induced by the energy perturbation also probably affected the biosynthesis of erythromycin.Therefore,the effects of redox status on the biosynthesis of erythromycin were further determined.The heterologous expression of NADH oxidase(NOX)from synthetic promoters decreased the intracellular[NADH]/[NAD+]level in a gradient.The expression of NOX in S.erythraea resulted in an increased production of erythromycin by 19~29%and this increment rose to 60%as more oxygen was supplied,whereas the biosynthesis of the reddish pigment was repressed accordingly.Next,the effect target of NADH on the biosynthesis of erythromycin was preliminarily determined around the formation of bis-(3’-5’)-cyclic dimeric guanosine monophosphate(c-di-GMP)using transcriptomic data and enzymatic assay.NADH inhibited the activity of diguanylate cyclase,which thereby repressed the formation of c-diGMP.BldD has been proved a positive transcriptional regulator of erythromycin biosynthesis gene cluster.c-di-GMP could activate the binding of BldD to the erythromycin gene cluster.Therefore,the expression of NOX alleviated the inhibition of c-di-GMP formation by NADH,which resulted in the stimulation of erythromycin biosynthesis gene cluster.In addition,the comparison among the strains with different cofactor metabolism emphasized the redox regulation as the prior role of oxygen consumption in S.erythraea.In conclusion,the genetic manipulation platforms in S.erythraea were optimized and expanded.Afterwards,new strategies of metabolic engineering for erythromycin enhancement in S.erythraea were proposed in the present research using comparative omics analysis,particularly regarding the connection between the intracellular energy/redox status and the biosynthesis of erythromycin.This study has laid a foundation for further metabolic engineering including cofactor engineering to improve the production of erythromycin by S.erythraea.
Keywords/Search Tags:Saccharopolyspora erythraea, metabolic engineering, comparative omics, energy metabolism, redox status
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