The Function Research Of The Pathway-specific Activator OtcR Of Oxvtetracvcline Biosynthesis And Its Applications For Improved Oxytetracvcline Production In Streptomvces Rimosus

Oxytetracycline(5'-hydroxytetracycline,OTC)and related tetracyclines are potent inhibitors of bacterial protein synthesis with broad-spectrum activity against both grampositive and negative pathogens.Although the clinical use of the tetracyclines has declined in recent years due to their side effects and reduced efficacy,OTC had found wide application in animal feeds and aquaculture,with annual production exceeding 100,000 tons.And the second-generation semisynthetic analogs of tetracyclines as well as third-generation derivatives with improved pharmacokinetic and chemical properties have shown potential for wide application in human against tetracycline resistant infections and for other purposes.These new generation of tetracycline drugs are chemically synthesized from OTC-like starting molecules;for example,to synthesize doxycycline,OTC is first converted to methacycline,which is further modified to obtain doxycycline.Therefore,in the foreseeable future,OTC still has a tremendous market demand.OTC is an aromatic polyketide antibiotic commercially produced by Streptomyces rimosus.In 1950,Pfizer published the discovery of a similar antibiotic produced by S.rimosus that they named Terramycin(later renamed OTC).Despite its importance,little is known about the regulation of OTC biosynthesis,which hampered any effort to improve OTC production via engineering regulatory genes.A gene encoding a Streptomyces antibiotic regulatory protein(SARP)was discovered immediately adjacent to the otrB gene of Oxytetracycline biosynthesis gene cluster(oxy)in S.rimosus and designated otcR(Oxytetracycline Regulator).Deletion and complementation of otcR abolished or restored OTC production,respectively,indicating that otcR encodes an essential activator of OTC biosynthesis.Reverse transcription PCR analysis showed that transcriptive levels of the five oxy structural genes(oxyA,oxyI,oxyJ,oxyR and oxyS)were increased by overexpression of otcR.Then,the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster,two or three direct hexameric repeats were identified in the promoter regions of oxyA,oxyI,oxyJ,oxyR and oxyS by MEME software.It is important to note that the identified repeats(6bp or 9bp deoxyribonucleotides)are separated by 11 bp,corresponding to one complete turn.It is a typical feature of the binding sites of SARP regulators,as SARPs normally form homodimers in vivo and are expected to bind two direct repeats at the same side of DNA.Transcriptional analysis in a heterologous green fluorescein protein(GFP)reporter system demonstrated that OtcR directly activated the transcription of five oxy structural genes(oxyA,oxyI,oxyJ,oxyR and oxyS)in Escherichia coli,fluorescences were enhanced ranging from 1.3 to 4 folds compared to that without otcR.Further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats.Engineering the regulation of antibiotic biosynthesis is an effective strategy to improve antibiotic production.Simple overexpression of pathway-specific activator gene using a constitutive promoter was often used to improve antibiotic production.This simple genetic engineering has been successful in some cases.Therefore,otcR was chosen as an engineering target,OTC production was significantly increased by overexpression of otcR under the control of strong promoter.Overexpression of OtcR enhanced OTC production dramatically by 4-6 times compared to the parental strain S.rimosus M4018.For a further improvement of the OTC production,the intracellular pool of malonyl-CoA by overexpressing acetyl-CoA carboxylase in M4018 were increased.Herein,for the OTC production,that by overexpressing both the pathway-specific activator OtcR and acetyl-CoA carboxylase in S.rimosus had been shown a maximum of 9.09g/L was achieved in recombination strains,while only 1.37g/L in the wild type strain M4018.The work has an important significance for engineering industrial strains to improve OTC production.The fermentation of OTC production by S.rimosus takes a long time(about 8-10 days),and the genetic manipulation of S.rimosus is also difficult due to its intrinsic high resistance to many commonly used antibiotics,including kanamycin and apramycin.To address this issue,we considered heterologous expression of oxy cluster in a fastgrowing Streptomyces strain.Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites.Here,it is employed to activate and manipulate the oxytetracycline gene cluster(oxy)and to alter OTC fermentation process.To achieve these goals,a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts.It shows rapid and dispersed growth and intrinsic high resistance to OTC.By manipulating the expression of two cluster-situated regulators(CSR)OtcR and OtrR and precursor supply,the OTC production level was significantly increased in this heterologous hostfrom 75 to 431 mg/L only in 48 h,a level comparable to the native producer S.rimosus M4018.This work shows that S.venezuelae WVR2006 is a promising chassis for the production of secondary metabolites,and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.In the era of synthetic biology,fine-tuning gene expression to achieve optimal level of target production has been accepted as key to successful practice.Therefore,as a prelude to an engineering attempt,in this work,a series of experiments were performed to engineer antibiotic-producing strain to give the best yield improvement and optimize the fermentation process.