Mechanism And Application Of Leucine-responsive Regulatory Protein (Lrp) Controlling Antibiotic Biosynthesis

Leucine-responsive regulatory proteins?Lrps?are a group of transcriptional regulators that regulate diverse cellular processes in bacteria and archaea,such as peptide transport,energy,central metabolism,DNA repair and recombination,bacterial persistence and virulence.However,the regulatory role of Lrps in antibiotics biosynthesis remains poorly understood in anbiotic-producing actinomycete.Saccharopolyspora erythraea is an important filamentous actinomycete utilized for production of erythromycin.Erythromycin and its derivatives are broad-spectrum polyketide antibiotics that are widely used clinically against pathogenic Gram-positive bacteria.According to bioinformatic analysis,SACE_Lrp shares the highest similarity to the Lrp of Corynebacterium glutamicum?56%identity?,known to directly control expression of its divergently transcribed brnFE encoding the branched-chain amino acid ABC transporter.In S.erythraea,SACE_Lrp is divergently oriented from SACE₅387-5386 that is also annotated as a putative branched-chain amino acid ABC transporter.In this study,we expressed His6-tagged SACE_Lrp in E.coli BL21?DE3?,and examined its affinity to PLrp-5387?SACE_Lrp-5387-int?with electrophoretic mobility shift assays?EMSAs?.SACE Lrp directly controlled the expression of the divergently transcribed SA CE 5387-5386 operon by interacting with SACE_Lrp-5387-int.Gene disruption of SACE_Lrp in S.erythraea A226??SACE_Lrp?resulted in a 25%increase in erythromycin production,while overexpression of SACE_Lrp decreased erythromycin production by 23%.These findings demonstrated that SACE_Lrp negatively regulates erythromycin production.ASACE Lrp showed similar growth rates in R5 culture by the mycelium dry weight and sporulation rates on R3M agar medium to the parent strain S.erythraea A226,indicating that SACE Lrp was not involved in growth and morphological differentiation.Additionally,the transcription of eryAI?SACE 0721,encoding polyketide synthase I?,ermE?SACE₀733,encoding rRNA methyltransferase?,ilvE[SACE₁646,putative encoding branched-chain amino acid?BCAA?aminotransferase]and SACE₅387-5386?putative encoding BCAA ABC transporter?in ?SACE Lrp were increased by 4-,2-,3-and 6-folds in comparison to A226 respectively.To investigate the effect of deleting SACE_Lrp on the transport of BCAAs,we measured the extracellular and intracellular concentrations of free BCAAs in A226 and ?SACE_Lrp.Both extracellular and intracellular concentrations of leucine,isoleucine and valine were reduced in ?SACE_Lrp compared to A226,indicating the deletion of the SACE_Lrp not only promoted the transport of BCAAs into the cell,but also increased the catabolism of intracellular BCAAs,thus providing more precursors for erythromycin biosynthesis.We therefore introduced pIB-5387-5386 into S.erythraea A226,and as expected,the A226/pIB-5387-5386 strain enhanced production of erythromycin by 31%relative to A226/pIB139.We examined the effect of adding BCAAs to the growth media on erythromycin production,and found that addition of valine,isoleucine or leucine to the growth media increased erythromycin production by 47%,35%and 16%in S.erythraea A226,respectively.To determine the conserved binding sequence of SACE Lrp protein,we performed DNase I footprinting using DNA-protein complexes digested by DNase I.The results showed that a 14bp sequence?5'-CTCCGGGCAACATT-3'?signature was indispensable for SACE_Lrp binding activity.Given that SACE_Lrp could directly regulate the expression of the SACE₅387-5386,our work further indicated that SACE_Lrp also acted as an auto-regulator responding to three amino acid-effectors based on a biosensor system.Lrp family regulators are responsive to a variety of amino acids.To determine the potential amino acid effectors of SACE_Lrp,a series of EMSA analyses were carried out with SACE_Lrp binding to the PLrp-5387 probe.Only lysine,arginine and histidine from the 20 protein amino acids influenced the affinity,with lysine and arginine reducing the binding affinity,whereas histidine increasing it.To our knowledge,SACE_Lrp is the first reported Lrp showing opposing responses to different effectors.Deletion of SACE_Lrp?WB?SACE_Lrp?in the industrial strain S.erythraea WB enhanced the production of erythromycin by 19%.Deletion of SACE Lrp combined with simultaneous overexpression of SACE₅387-5386 in S.erythraea WB increased erythromycin production by 41%and 48%when grown in a medium without or with extra 10mM valine respectively.In a 5-L fermenter,the erythromycin accumulation in the engineered strain S.erythraea WB?SACE_Lrp/5387-5386 with extra 10 mM valine in the industrial culture media reached 5001mg/L,a 41%increase over S.erythraea WB.These insights into the molecular regulation of antibiotics biosynthesis by Lrps in S.erythraea are instrumental in improving industrial increases of secondary metabolite production.Sequence alignment showed that SC03361 shares the highest similarity?44%identity?to the SACE_Lrp in Streptomyces coelicolor.This information implies a physiological role of SC03361 as an important regulator for secondary metabolism and?or?morphological differentiation.In this study,Deletion of SC03361 led to dramatic reductions in actinorhodin?Act?production and delayed aerial mycelium formation and sporulation on solid media.SC03361 stimulates Act production by altering the transcription of the cluster-situated regulator gene actII-ORF4 and directly bind to the promoter of actII-ORF4.SC03361 directly repressed the expression of its own gene and activated the transcription of adjacent divergently transcribed gene SC03362.Phe and Cys were identified as the effector molecules of SC03361,Phe reducing the binding affinity,whereas Cys increasing it.Moreover,cross-talk between SC03361 and SACE_Lrp was discovered for binding each other's target site in this work.Our results indicate that SC03361 functions as a pleiotropic regulator positively controlling secondary metabolism and morphological development in S.coelicolor.These findings indicate that Lrp-mediated regulation of antibiotic production is common in antibiotic-producing actinomycetes.