Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 591349, 16 pages http://dx.doi.org/10.1155/2015/591349 Review Article Antibacterial Discovery and Development: From Gene to Product and Back VictorFedorenko,1 OlgaGenilloud,2LiliyaHorbal,1GiorgiaLetiziaMarcone,3,4 FlaviaMarinelli,3,4YossiPaitan,5andElioraZ.Ron6,7 1DepartmentofGeneticsandBiotechnology,IvanFrankoNationalUniversityofLviv,Lviv79005,Ukraine 2Fundacio´nMEDINA,HealthSciencesTechnologyPark,18016Granada,Spain 3DepartmentofBiotechnologyandLifeSciences,UniversityofInsubria,21100Varese,Italy 4TheProteinFactory,InteruniversityCentrePolitecnicodiMilano,ICRMCNRMilano,andUniversityofInsubria,21100Varese,Italy 5ClinicalMicrobiologyLaboratory,MeirMedicalCenter,44281KfarSaba,Israel 6DepartmentofMolecularMicrobiologyandBiotechnology,TelAvivUniversity,6997801TelAviv,Israel 7GalileeResearchInstitute(MIGAL),11016KiryatShmona,Israel CorrespondenceshouldbeaddressedtoFlaviaMarinelli;[email protected] Received30October2014;Revised30December2014;Accepted13January2015 AcademicEditor:MetinGuru Copyright©2015VictorFedorenkoetal. This is an open access article distributed under the Creative Commons Attribution License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly cited. Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in themedia,accompaniedbycommentsontheriskthatwemaysoonrunoutofantibioticsasawaytocontrolinfectiousdisease. InfectionscausedbyEnterococcusfaecium,Staphylococcusaureus,Klebsiellaspecies,Clostridiumdifficile,Acinetobacterbaumannii, Pseudomonasaeruginosa,Escherichiacoli,andotherEnterobacteriaceaespeciesrepresentamajorpublichealthburden.Despite thepharmaceuticalsector’slackofinterestinthetopicinthelastdecade,microbialnaturalproductscontinuetorepresentoneof themostinterestingsourcesfordiscoveringanddevelopingnovelantibacterials.Researchinmicrobialnaturalproductscreening anddevelopmentiscurrentlybenefitingfromprogressthathasbeenmadeinotherrelatedfields(microbialecology,analytical chemistry,genomics,molecularbiology,andsyntheticbiology).Inthispaper,wereviewhownovelandclassicalapproachescan beintegratedinthecurrentprocessesformicrobialproductscreening,fermentation,andstrainimprovement. 1.Introduction andGram-positivebacteriarepresentthedominantprocess bywhichbacteriabecomemultiresistant.Theselectivepres- Antibacterialtherapyhassavedmillionsoflivesandconsid- sureofantimicrobialuseinhospitals,incommunities,andin erably reduced the rate of premature death from bacterial agriculturecomprisestheenginedrivingthisprocess.Nowa- infections. These achievements led to the assumption that days we are aware that bacterial resistance to all currently pathogenicbacteriaandthehighmortalityduetoinfectious used antibiotics has emerged for both Gram-positive and diseases would be a thing of the past. Unfortunately, soon Gram-negative bacteria. This threatening situation urgently after the introductionof antibiotics,reports concerningthe callsforaconcertedinternationaleffortamonggovernments, emergence of resistance started to accumulate. Antibiotic thepharmaceuticalindustry,biotechnologycompanies,and resistance mechanisms, which appear de novo or are trans- the academic world to react and support the development mittedamongbacteria,havebeenwellstudiedanddescribed of new antibacterial agents. One example of such initiative in many reviews. These include detoxification of antibiotic effort is the Infectious Diseases Society of America (IDSA) molecules and mutations in the designated target or, as calltodevelop10newsystemicantibacterialdrugsby2020[2] described recently, are mediated by population-level resis- by targeting drug development against both Gram-positive tancemechanisms[1].Itisnowapparentthatinterspeciesand and Gram-negative bacteria. Unless serious action is taken, intraspecieshorizontalgenetransferofbothGram-negative theacuteanddangeroussituationthatexiststodaymaysend 2 BioMedResearchInternational usbacktothepreantibioticera,whentherewasnocurefor Although the prevalence of Gram-negative bacteria is bacterial infections. If this happens, the prophecy of Louis currentlysomewhatlowerthanthatofGram-positivebacte- Pasteurwillbefulfilledand“microbeswillhavethelastword.” ria,itiswellrecognizedthatGram-negativeMDRinfections areemergingasathreattohospitalizedpatientswithasignif- icantimpactonlengthofhospitalization,mortality,andcost 2.MedicalNeedsforNovelAntibacterials [14,15].Theseincludemultiresistantnonfermenters,suchas Pseudomonas aeruginosa and Acinetobacter baumannii, or Multidrug-resistant bacterial infections represent a major multiresistant, extended-spectrum 𝛽-lactamase-producing public health burden, not only in terms of morbidity and Enterobacteriaceae and more recently carbapenem-resistant mortality, but also in increased expenses for managing Enterobacteriaceae(CRE)ofdifferenttypes.Emergingresis- patientsandimplementingextensiveinfectioncontrolmea- tanceisduetothespreadoftheKlebsiellapneumoniaecarba- sures. Mortality due to multidrug-resistant bacterial infec- penemase (KPC) and to the novel New Delhi metallo-𝛽- tionsishigh.In2002itwasreportedthat1.7millionhealth- lactamase(NDM-1). Therisingcrisisofmultidrug-resistant care-associatedinfectionsoccureachyearinAmericanhos- Gram-negative bacteria has prompted the use of salvage pitalsandwereassociatedwithabout99,000deaths[3].This therapywithcolistin,anolderpolymyxinknowntobeneuro- representsahugeincreasefromapreviousestimation,which toxic and nephrotoxic [16, 17]. However, there are already reportedthatin1992about13,300peoplediedfromhospital- reportsdescribingisolatesofseveralGram-negativebacteria acquiredinfection[4].ItisestimatedthatintheEU,Iceland, that are resistant to all available antibiotics, including poly- andNorwayabout37,000patientsdieasadirectresultofa myxins[18,19]. hospital-acquired infection each year; an additional 111,000 dieasanindirectresultofhospital-acquiredinfection[5];and 3.NaturalProductDiscovery:TheScreening about25,000patientsdiefromamultidrug-resistantbacterial IngredientstoExploitMicrobialDiversity infection. Presently,themostfrequentmultidrugresistance(MDR) Despite the pharmaceutical sector’s lack of interest in bacteria are Enterococcus faecium, Staphylococcus aureus, addressing the topic in the last decade, microbial products Klebsiellapneumoniae,Acinetobacterbaumannii,Pseudomo- continue to represent one of the most interesting sources nas aeruginosa, and Enterobacter spp. which therefore were for the discovery of novel antibacterials today and research termed “ESKAPE” after initially being reported [6], with in the field is currently benefiting from progress that has several reports adding Clostridium difficile or other Enter- beenmadeinotherrelatedfields(microbialecology,metage- obacteriaceae[7].Gram-positivepathogens,suchasStaphylo- nomics, metabolomics, or synthetic biology), fields which coccus,Streptococcus,Enterococcus,andClostridium,account haveprovidedadeeperunderstandingofthemicrobiomeand for a large proportion of serious infections worldwide. An thusthedevelopmentofnewtoolstofosterthediscoveryof increasingpercentageofsuchGram-positiveisolatesexhibit novelcompounds.Amonglivingorganisms,microorganisms reducedsusceptibilitytofirst-linetherapies[8–10],resulting (actinobacteria, cyanobacteria, myxobacteria, and fungi) in poor clinical outcomes in both community and hospital representoneofthemostprolificsourcesfortheproduction settings [10–13]; this has a significant impact on overall of antibiotics. For decades, exploitation of their specialized healthcareutilizationandcosts[10,11].Staphylococcusaureus (commonly termed secondary) metabolism has guaranteed and Enterococcus spp. were found to be among the most thediscoveryofnovelantibioticsandothercompoundswith commonly isolated pathogens in the hospital environment, unprecedentedchemicalcharacteristicsandbiologicalprop- andbeingfrequentlyresistanttomultipledrugscomplicates ertiesthatdonotexistinthescreeninglibrariesofsynthetic therapy.Therepresentativehospital“superbugs,”methicillin- compounds[20,21].Inthissection,weexaminethecurrent resistant S. aureus (MRSA) and vancomycin-resistant ente- trendsinmicrobialproductscreeningfordiscoveringnovel rococci(VRE),frequentlyattractmass-mediaattentionand, antibiotics. A flow diagram showing the overall screening in many countries, pressure is increasing to reduce MRSA operationisreportedinFigure1. andVREinfectionrates.Resistancetoanti-MRSAandanti- VRE drugs is uncommon; however, infections by MRSA 3.1. Microbial Product Libraries. Microbial natural product strains resistant to glycopeptides, daptomycin, or linezolid librariesrelyonthequalityanddiversityofnovelmicrobial (common anti-MRSA drugs) and by VRE strains resistant strains and the approaches used to exploit their metabolic to daptomycin or linezolid (common anti-VRE drugs) are diversity.Accesstomicrobialdiversitytraditionallyfocused increasinglybeingreported,includingreportsoftransferable on intensive sampling and isolation using general methods resistance mechanism to these drugs among staphylococci from a wide range of geographical locations and habitats, and enterococci. In addition, reports regarding the emer- with recurrent isolation and screening of the predominant genceandspreadofvirulentclonesofMRSAandClostridium speciesandalowprobabilityofisolatingnovelcompounds. difficileinthecommunityandinhospitals,respectively,have Althoughestimatesforthepotentialproductionofunknown been published often. Moreover, multidrug-resistant Strep- novel molecules by Streptomyces spp. [22] were high, the tococcus pneumonia clones are currently considered major reality is that species spread widely in different environ- communitypathogensinmanypartsoftheworld,although mentsproducethesamewell-knownandstructurallyrelated theyarebeingchallengedbynewconjugatevaccines. antibacterial molecules. Current approaches oriented to BioMedResearchInternational 3 ∙Terrestrial and marine sources: soils, rhizospheres, plants, rocks, Sources: environmental lichens, sediments and invertebrates samples n ∙Isolation (actinomycetes, proteobacteria, fungi, o ati cyanobacteria, myxobacteria, etc.) ner Culture collection ∙Identification (morphology,16S rRNA) e y g ∙Strain selection (de-replication:, DNA and LCMS fingerprinting) ar br Li ∙Nutritional conditions: OSMAC, elicitors, stress factors Fermentations temp, time, formats (microplates, tube, flasks) ∙Extraction (solvents, SPE, fractionation) NP libraries Extracts and fractions: ∙Screening (bioassays): enzymatic, target based, whole cell, S Hits ∙Early MS dereplication (in-house known NP databases) HT ∙Secondary assays/counterscreens ∙Compound isolation (bioassay-guided fractionation) ∙Structural elucidation (HRMS and NMR) Active molecules Compound development: ∙Biological profiling, MOA scale-up production ∙Pharmacology PKPD L H2 ∙Medicinal chemistry ∙Strain improvement ∙Preclinical safety ∙Media optimization Lead (H2L, hit to lead) Figure1:Earlystagesofantibioticdiscoveryfrommicrobialproductlibraries. discovering novel molecules mostly aim to target specific 3.2. Tools for Strain Selection. Strain selection criteria are and minor microbial communities in unique or underex- essential for building a strain collection and ensure the plored environments, including specific terrestrial niches, uniqueness of the isolates and that the widest microbial plant host-microbe associations, and marine environments. diversity is represented. Phenetic and molecular tools that Environmentalconditionscomprisestrongselectingfactors canbeappliedhierarchicallyonthelargenumbersofisolates andthedistributionofsomemicrobialspecies,eveninhighly normallyrecoveredfromenvironmentalsampleshavebeen occurringtaxa,presentsbiogeographicpatternsdetermined intensively developed. These can include a simple mor- by microenvironmental conditions that can be translated phological characterization of the growth and sporulating intonovelcompounds.Manyresearchgroupshaverecently characteristicsofactinomycetesandfilamentousfungiatthe emphasizedtheexplorationofuntappedmicrobialcommu- macroscopic and microscopic levels, allowing preliminary nitiesthatareassociatedwithrhizospheres,plantendophytes, assignmenttoataxonomicgroupthatcanbecomplemented lichens, endolithic microbial communities, insect parasites, with the ribosomal gene sequencing of isolates in a large and endosymbionts and marine sediments and inverte- proportionofthecases.PartialribosomalrDNAsequencing brates.Theseapproacheshavefavoredtheisolationofnovel isfrequentlyusedtoconfirmthetaxonomicaffiliationandto microbialcommunitiespotentiallyproducingnovelchemical assessinmoleculardatathemicrobialdiversityandindivid- scaffolds [23–27]. The search for novel sources has been ualphylogeneticrelationshipswithinstrainsinacollection. combinedwiththeuseofnovelisolationmethodstargeting The existing intraspecies heterogeneity in microbial taxa thecultivationofspeciesunderrepresentedorpreviouslynot cannot be resolved in phylogenetic inner branches, which cultivatedunderlaboratoryconditions[28–32].Mostofthese require the introduction of additional fingerprinting tools methods are focused on selective isolation of the members forselection.Othermethodscurrentlyusedcanincludethe of minor occurring taxa by using poor nutritional media application of high-throughput chemotaxonomic profiling devoid of carbon sources, subinhibitory concentrations of methodssuchasthosebasedonthewhole-cellfattyacidcom- antibioticsthatmightfavorthedevelopmentofslow-growing position[33]andtheuseofMALDI-TOFMSproteinprofiles, representatives of these microbial communities after weeks a promising alternative to conventional identification tech- ofincubation,alternativegellingsubstratestoagarshownto niques [34], and molecular fingerprinting techniques based prevent the growth of some microbial groups in laboratory ontherandomamplificationofgenome-conservedrepetitive conditions, in situ incubation chambers, or isolating endo- regions (AFLPs, RAPDS, and REP fingerprinting) [35–37]. phytesthatgerminatedirectlyonthesubstrateusinghumid The generation of rapid fingerprints based on the restric- chambersorbysurfacesterilization. tionpatternofamplifiedconservedsequencesinpolyketide 4 BioMedResearchInternational synthase or nonribosomal peptide synthetase biosynthetic indifferentfungalspecies[55,56].Similarlytofungi,HDAC systemsprovidesadditionalinformationaboutthediversity inhibitors such as sodium butyrate or splitomicin have andthebiosyntheticpotentialofthenewisolates[38]. been reported to activate cryptic pathways in Streptomyces coelicolor, and HDAC orthologues have been identified to 3.3.CultivationandExtraction. Traditionallythegeneration be broadly distributed in actinomycetes [57], offering new ofmicrobialproductlibrarieswasbasedontheempiriccul- avenues to induce cryptic or poorly expressed specialized tivationofmicrobialstrainsinseveralnutritionalconditions metabolitesinthesetaxaandexpandthechemicaldiversity using different liquid and solid formats in varying volumes ofmicrobialproductlibraries. andbyextractingthefermentationbrothstogeneratecrude Whereas the production conditions are key to promot- extracts or semipurified fractions containing mixtures of ing the biosynthetic potential, microbial product libraries specializedmetabolites.Theuseofalimitednumberofthree comprise a collection of extracts and are also defined by tofourconditionsatonce,employingdifferentmediacompo- the type of extraction used in their preparation. Extraction sitions,cultivationformats,orincubationperiodsortemper- proceduresshouldbedesignedtoensurethewidestdiversity atures,wasgenerallyacceptedasbeingsufficienttoproduce of compound polarities in the extracts. These can range new, specialized metabolites, withoutrealknowledge ofthe from simple whole-broth extraction with solvents of differ- nutritional requirements and physiology of most of the ent polarity (from aqueous methanol or acetone miscible groupsofstrainsbeingscreenedandthekeyelementsinvolved with the broth to more nonpolar solvents such as ethyl inregulatingtheirspecializedmetaboliteproduction.Nowa- acetate or methyl-ethyl-ketone, providing cleaner extracts days,thecontinuouslyincreasingnumberofwhole-genome of mid-polarity compounds) to solid-phase extraction with sequencesofknownproducersshowsthatalargefractionof ion exchange resins that directly enrich metabolites from thegenomeremainssilentandthatswitchingoncrypticpath- the broth (cross-linked polystyrene Diaion HP20 or XAD ways might trigger the production of novel molecules [39– resins) [58, 59]. Orthogonal fractionations that are used to 42]. The OSMAC (one strain, many compounds) approach generate prefractionated libraries reduce the complexity of has been proposed as an alternative way of exploring each theextracts,enablingscreeningathigherconcentrationsand straininmultipleconditionstobetterexploittheirspecialized simplifyingthefollowingdereplicationphase[60]. metabolismandtotriggerpartofthismicrobialbiosynthetic potential[43].Theuseofmultiplenutritionalconditionshas 3.4.AntibacterialScreeningAssays. Antibioticscreeningstrat- recently been explored by many groups to generate large egiesofnaturalproductshaveseenanimportantevolutionin screeningextractslibrariesindifferentformats(tubes,flasks), thepastfewdecades,fromthelow-throughput,earlypheno- but miniaturized, parallel fermentation in deep-well plates typicassays—usedtoidentifycompoundsonlytargetingpath- representedamajorbreakthroughinthescaleandnumbers ogens without any previous potential mode of action of conditions that can be tested [44]. All major taxonomic hypothesis—to high-throughput, whole-cell, target-based groups of actinomycetes and filamentous fungi can be cul- assays and structured-based design derived from in silico tivated in a large variety of complex and synthetic liquid screening [61, 62]. High-throughput screening of microbial media of diverse composition in carbon sources, inorganic product libraries continues to be commonly based on phe- orcomplexnitrogensources,traceelements,andphosphate- notypic assays that have the advantage of utilizing intact controlled levels [20]. By testing in parallel a high number bacteriaandensurethattheactivecompoundcanpenetrate of nutritional conditions, minor groups of isolates can be the bacterial membranes and reach their target. Nowadays, exploredandscreenedfortheproductionofantibioticactiv- theseassaysofferthepossibilityofintegratingreportergenes ities.Identifyingproductionmediathatcanfurtherpromote to run whole-cell, target-based screens, in liquid- or agar- their microbial biosynthetic potential increases the chances based format, including single- or two-plate assays, which ofproducingnovelmoleculesandidentifyingactiveextracts aim to identify differential activity. The different types of thatcanbethenpursuedonalargerscaleinchemicalisola- assays targeting classical bacterial functions and essential tionprojects[45,46]. pathways,includingDNAreplication,cellwallbiosynthesis, Theproductionofspecializedbacterialmetabolitescanbe and protein biosynthesis, have been extensively described stimulated by using known chemical inducers (e.g., sidero- in recent papers [63, 64]. Among these approaches, one of phores, rare earths, or metabolism intermediates) [47–50], thebreakthroughsistheuseofStaphylococcusaureusgenes small, diffusible, bacterial, hormone-like molecules such as essential for growth to develop a series of screens based the𝛾-butyrolactones,andotherbutenolides[51].Otherelici- on reducing the expression of targets to identify bacterial torsofspecializedmetabolismincludeN-acetyl-glucosamine inhibitors. The induction of antisense RNAs to selectively that when added to production media modulates the N- decrease the production of intracellular gene products has acetyl-glucosamine-responsiveproteinDasR[52]orgenerat- beendevelopedasaprimaryscreeningprocedurefordiscov- ingribosomalmutationsthatresultinalteredppGppbiosyn- ering new antibiotics [65] and was effectively employed to thesisandcataboliterepressionthatfavorbiosynthesis[53]. find novel classes of inhibitors with novel modes of action, Epigeneticmodulationoffungalexpressionbyhistoneacety- suchasthefattyacidsynthesisinhibitorsplatensimycinand lation and methylation has a strong influence on antibiotic platencin,andalonglistofnewproteinsynthesisandprotein production [54], and small-molecule epigenetic inhibitors secretion inhibitors [64]. An effective screening approach of histone deacetylase (HDAC) or DNA methyltransferase hasconsistedintheuseofmechanism-basedprofilingusing (DMAT)areusedtoactivatesilent,naturalproductpathways the S. aureus fitness test-based genome-wide screening for BioMedResearchInternational 5 upfront empiric evaluation of the antimicrobial activities growingconditions(upstreamprocess)remainthemaintools derived from the screening of microbial product libraries since any purification scheme (downstream) at this stage is on a wide panel of bacterial pathogens [66]. The S. aureus hardtoimproveandchangeduetotherigorousmanufactur- fitness test consists of a collection of inducible S. aureus ingregulations. antisenseRNAstrainsengineeredforreducedexpressionof For the majority of antibiotics, the only feasible supply asingletargetthatcorrespondstoessentialgenesforwhich process continues to be fermentation, total synthesis being inducible antisense RNA expression determines a growth toocomplicatedortooexpensive.Table1showsthatthevast phenotype.Thisassaygeneratesaprofileofstrainsensitivities majority of the antibiotic drugs introduced into the market specificforthemechanismofaction(MOA)ofthecompound since 2000 are microbial products and are still produced beingtestedandithasbeenusedtoprofileandrevealnovel byfermentation.Mostnaturalproductsaresocomplexand activitiesincrudemicrobialproductextracts[63]. contain so many centers of asymmetry that they probably willneverbeproducedcommerciallybychemicalsynthesis. 3.5.ChemicalDereplicationProcess. Giventhatknownmol- Asanexample,totalchemicalsynthesisoftheglycopeptide ecules continue to be rediscovered in microbial product teicoplaninwasperformedbysubstantiallyinventinganew extracts,alltheHTSscreeningstrategieshavebeenaccompa- chemistry[70],butitistooexpensiveandmicrobialfermen- niedbytheimplementationofefficient,earlyLC-MSderepli- tationremainstheonlywaytoproducethisvaluabledrug[71, cation platforms to identify known compounds in natural 72].However,comparedtosyntheticprocesses,manufacture products databases containing known antibiotic compound byfermentationismoredifficulttocontrol;thus,itcanleadto spectra [67]. For identification of the bioactive compounds theformationofmorevariableantibioticproductswithmore inmicrobialproductsextracts,bioassay-guidedfractionation complicated and less predictable composition and impurity and further purification of the active molecule from new, profiles.Thisisduetothefactthat(a)thepurityoftheactive large-scalerefermentationoftheoriginalmicrobialproducer substancesisdependentonthefungalorbacterialstrainsthat are required. To miniaturize the production conditions in producetheantibiotic;(b)theconditionsunderwhichstrains HTS,thedesiredmetabolitesneedtobereproducedinlarger areprocessedmayvary;(c)therawmaterialsthatareutilized, fermentationformats(tubes,flasks,andbioreactors;seethe includingthequalityofwaterinwhichthestrainsgrow,may followingsectiononfermentation)lateron.Afterconfirming also vary; and (d) the extraction and purification processes theoriginalhitactivityinthenewextract,severalroundsof mayhavelimitedselectivity[73]. chromatographicseparationsfollowingthebiologicalactivity Hence,thecrudeproductobtainedbyfermentationmight in the enriched fractions ensure that the active component not be a single antibiotic substance or entity, but rather a has been enriched. Analysis of the active fractions by LC- complexmixtureofanalogues,asisthecasewithteicoplanin MS in each round of fractionation permits dereplication of (acomplexoffiverelatedcompoundsdesignatedteicoplanins anyknowncomponentsthatcanberecognizedinreference A2-1–A2-5 characterized by five different linear or branched naturalproductdatabasesandexplainstheobservedactivity. ten-oreleven-carbonfattyacids)[72],colistin(amulticom- Normally,threetofourroundsoffractionationareneededto ponentpolypeptideantibiotic,comprisedmainlyofcolistins obtainthedesiredmoleculeasapurecompoundwith>95% A and B) [74], and gentamicin (oligosaccharide antibiotic 󸀠 purity[68,69].NMRandLC-MSanalyticalmethodsarethen composedofamixtureofthreecomponentsdesignatedasC , 󸀠 applied not only to assess the purity of the compounds but C a,andC2)[75].Therefore,itmightbedifficulttocompare alsotogeneratethedossierofspectraneededtoelucidatethe apparently identical active ingredients unless they originate structureofthenovelcompounds[60]. fromthesamemanufacturer. Theneedtoimprovethefermentationprocess(andreduce 4.FermentationIsOftentheOnlyWayto thecostofamultistepprocess)isparticularlydemandingfor ProduceNovelNaturalMicrobialProducts producingthosenaturalscaffoldsthatundergosemisynthetic modification, as in the case of the second-generation gly- Antibioticsarethemostimportantcategoryofbioactivecom- copeptides(dalbavancin:tradenameDalvance,DurataTher- poundsextractedfrommicroorganismsusingfermentation. apeutics; oritavancin: trade name Orbactiv, The Medicins Duringthediscoveryprocess,whichisbasedonbiologically Company; telavancin: trade name Vibativ, Theravance) re- guidedscreening(seethesectionabove),sufficientamounts centlyapprovedbytheFoodandDrugAdministration(FDA) ofactivefractionsneedtobeproducedbyselectedmicrobial [76]. strains for the initial biological profiling and to elucidate the chemical structure. During the development and clin- 4.1. Antibiotic Fermentation Process. Notwithstanding the ical phases, the large-scale production of antibiotics from key role of the fermentation process, not very much has microbialfermentationsiscoupledwithanintensiveeffortto changedsincethefirstsubmergedfermentationprocesswas improvethestrain(seethesectionbelow)inordertoreduce developedtomeetthedemandforpenicillinsaftertheSecond production volumes and costs and guarantee quality and WorldWarandtheprocessesforproducingantibioticstoday reproducibilityofthedrugbulks.Later,whenmarketingthe areverysimilartothoseemployed60yearsago.Thefermen- antibiotic, which is driven by profitability and competitive- tationprocessusuallystartswithaworkingcellbank(WCB) ness,loweroperationalcostswithconcurrentlyhigheryields inoculated in a flask containing a vegetative medium (in are required for microbial production [21]. To achieve that whichproductiondoesnotoccur)toallowthestraintogrow. goal,manipulatingandimprovingmicrobialstrainsandtheir Afteraperiodthatcanvarydependingonthestrain,oneor 6 BioMedResearchInternational Table 1: Examples of natural products (NP), semisynthetic modified natural products (SNP), natural product-derived but produced by chemicalsynthesis(NP-derived),ortotallysyntheticantibiotics(S)launchedsince2000:productionmethod,chemicalclass,activityagainst Gram-positiveand/orGram-negativebacteria,leadsource,andproducingorganism. Antibacterial Production Class NP-leadsource Leadsource Drugname Yearapproved spectrum Chemical Oxazolidinone S G+ Linezolid 2000 synthesis Actinomycete Fed-batch Lipopeptide (Streptomyces SNP(A21978C) G+ Daptomycin 2003 fermentation roseosporus) Chemical Carbapenem NP-derived G+/G− Doripenem 2005 synthesis Fed-batch Fungus SNP Pleuromutilin G+ Retapamulin 2007 fermentation (Pleurotusspp.) (pleuromutilin) Fed-batch Actinomycete Glycopeptide SNP(vancomycin) G+ Telavancin 2009 fermentation (Amycolatopsisspp.) Fungus Fed-batch 𝛽-lactam (Cephalosporium SNP G+/G− Ceftaroline 2010 fermentation (cephalosporin) fosamil acremonium) Actinomycete Fed-batch Tiacumicin (Dactylosporangium NP G+ Fixadomicin 2011 fermentation aurantiacum) Fed-batch Actinomycete Glycopeptide SNP(A40926) G+ Dalbavancin 2014 fermentation (Nonomuraeasp.) Frozen stock Shake flask (WCB) Pre-seed fermentor Seed fermentor Production fermentor Figure2:Flowdiagramfortheclassicalfermentationprocess:thenumberofseedstepsmayvaryaccordingtothefinalscaleoftheproduction fermentor. a series of increasing volume reactors containing vegetative up the fermentation process usually constitutes the final medium are serially inoculated to obtain enough material step in any research and development program for large- to start the last-vessel fermentation within the production scale industrial manufacture of fermentation products [81]. medium(Figure2).Submergedfermentationsforproducing Productionreactorsizesrangefrom40to100cubicmeters. antibacterialsareusuallyperformedinstirredtankreactors It is importantto understand that the process ofscaling up and are operated in batch or fed-batch mode. In batch a fermentation system is frequently governed by a number reactors all components, except gaseous substrates such as ofimportantengineeringconsiderationsandisnotsimplya oxygen,pH-controllingsubstances,andantifoamingagents, matterofincreasingcultureandvesselvolume. areplacedinthereactoratthebeginningofthefermentation. Batchprocessesaresimpleandrobust,buttheonlyway 4.2.RegulationofAntibioticSynthesisandMediumComposi- to reach a high cell density is the fed-batch mode, which tion. Antibioticsareusuallynotproducedduringthephase is more complex but allows the metabolism of the strain of rapid growth but rather are synthesized during a subse- to be controlled [77]. In a fed-batch process, one or more quent stationary phase. Antibiotic production starts when nutrients are added in order to control the reaction rate growthislimitedafteronekeynutrientsourceisexhausted: accordingtoitsconcentration,avoidingcataboliterepression carbon, nitrogen, or phosphate. For example, penicillin (seebelow)[77].Mostantibioticsareproducedwiththefed- biosynthesisbyPenicilliumchrysogenumstartswhenthereis batchsystem(e.g.,teicoplanin[72],daptomycin[78],tylosin nolongeranyglucoseintheculturemediumandthefungus [79], and 𝛽-lactams [80]) (see Table1). Continuous culture startsconsuminglactose,alessreadilyutilizedsugar[82]. is not common in the pharmaceutical industry because the Themainregulationeffectinspecializedmetabolismis, probabilityofmutationandcontaminationishigher.Scaling in fact, carbon catabolite repression, defined as the control BioMedResearchInternational 7 (inactivation) of specific operons in favor of a primary and molecules,andN-acetyl-glucosamine,epigeneticmodulators efficientutilizationofasimplecarbonsource(commonly,but thatarebeingusedtoactivatecrypticorsilentgeneclusters notalways,glucose).Theoperons/genes/enzymesinvolvedin during the screening processes (see previous paragraph crucial steps of biosynthesizing specialized metabolites are on cultivation and extraction), can be also added to the under catabolite repression. Catabolite repression is strictly productionmediatoimproveantibioticproduction[47–57]. linked to growth rate and growth phases since only after Limits in their use during scaling up of the fermentation easilyutilizablesubstrateshavebeenconsumedcantheeffi- processandproductdevelopmentconsistintheircostandin cientproductionofspecializedmetabolitesbegin.Therefore, theriskofchemicalcross-contaminationduringthepurifica- tion phase (downstream). Recent molecular studies have regulating metabolite biosynthesis ensures that precursors provided new insight into the role of catabolite carbon and metabolic energy are invested in the manufacture of control.Theydemonstratedarelationshipbetweenantibiotic specialized metabolites only under environmental circum- production and morphological development involving stances and at developmental stages where those molecules N-acetyl-glucosamine, which, when added to production contributetothefitnessoftheorganism[83,84]. media, modulates the N-acetyl-glucosamine-responsive Glucose represses the production of many antibiotics protein DasR and pleiotropic regulation of both antibiotic (e.g.,daptomycin[78],clavulanicacid[85],andaminoglyco- synthesisandsporeformation[52].Molecularinvestigations sideantibioticssuchasstreptomycin,kanamycin,neomycin, alsoelucidatedtheroleofribosomal/RNApolymerasemuta- and gentamicin), but the molecular mechanism underlying tionsresultinginalteredppGppbiosynthesisandinstringent glucoserepressionhasresistedmolecularanalysisforalong response interplaying with catabolite repression [49, 101]. time, although more recently this topic was thoroughly A thorough understanding of how global regulators (see elucidated and widely covered in the literature [49, 84–87]. section below on strain improvement) respond to a variety Readily utilizable nitrogen sources repress enzymes of spe- of nutritional or environmental stress signals, for example, cializedmetabolismduringthebiosynthesisofcephalosporin phosphate,carbon,nitrogenstarvation,heatshock,pHstress, [54, 88], cephamycin [89], tylosin [90], and erythromycin andcellwalldamage,iscurrentlyprovidingamorerational [91]. Similarly, free inorganic phosphate depletion from approach for defining medium and process conditions for the growth medium is required to trigger production of antibioticproduction[49,91,93]. tetracyclines[92,93],𝛽-lactams,andglycopeptides[93,94]. WhereasthemolecularmechanismforPhoP-mediatedphos- 5.StrainImprovementinthePostgenomicEra phate control is partially understood at the molecular level [93,94],thesignalsensorsandsignaltransductioncascades With the development and advent of genome sequencing involvedinregulatingmetabolismbyotherstressfactorsneed technologies[102,103],itbecameobviousthatmostbacterial tobefurtherelucidated[49]. genomescontainahiddenwealthofclustersresponsiblefor Toimprovetheproductionofantibiotics,slow-metabo- the biosynthesis of potential bioactive compounds [39–41] lizing carbon, nitrogen, and phosphorous sources are used: that await discovery. The main reason for the existence of complexsubstratessuchaspolysaccharides(e.g.,starch),olig- such a plethora of undiscovered biosynthetic pathways is osaccharides (e.g., lactose), and oils (e.g., soybean oil) are that many gene clusters are dormant or not expressed in oftenpreferredtoglucose,andyeastextract,cornsteepliquor, sufficientquantitiestobedetectedundertypicalfermentation and soybean flour are commonly essential components for conditions [104–106]. As discussed above, this is related supplying nitrogen, phosphorous, vitamins, and trace ele- to the existence of tight regulatory networks that precisely mentstoantibiotic-producingstrains.Inmediacontaininga orchestratespecializedmetaboliteproductioninbacteriaand mixtureofrapidlyusedcarbon,nitrogen,andphosphorous respondtodifferentenvironmentalandintracellularsignals sources and slowly used sources, the former are used first [49, 86, 107]. Undoubtedly, a low yield of natural products toproducecellsandthelatteremployedoncetherapidlyassim- representsaserioushurdleonthewaytocommercialproduc- ilatedcompoundsaredepletedtosustaintheproductionof tion. Therefore, exploring and understanding the interplay specializedmetabolitesduringthestationaryphaseofgrowth. betweenantibioticproduction,regulatorynetworks,environ- Recent examples of how optimization of medium compo- mentalandintracellularsignalswillprovideuswithkeysto sition contributes to improving the final product concen- understandingspecializedmetaboliteoverproduction. tration, yield, and volumetric productivity have been Nowadays, numerous strategies for improving strains reportedondaptomycin,nisin,cephalosporinC, clavulanic havebeenandcontinuetobedeveloped.Classicalapproaches acid,andA40926,theprecursorofsemisyntheticdalbavancin for strain improvement were based on recursive rounds of [72, 78, 95–100]. In the case of daptomycin produced by mutagenesis and further selection [108, 109]. Despite the StreptomycesroseosporusNRRL11379,Ngandcoworkershave drawbacks(unwantedmutationsandbeingtimeconsuming successfully established a cost-effective medium and feed- andlaborious),thisstrategywassuccessfulandwidelyused back-controlling approach by utilizing dextrin as the major for rapidly increasing the production yield of antibiotic- carbonsourceinfed-batchfermentation[78].Forglycopep- producing microbes. Most of the industrial overproducers tide antibiotics such as A40926 and teicoplanin, optimized currentlyinuseweredevelopedinthisway[110,111].How- media and processes have recently been proposed [65, 98– ever,withthedevelopmentofmolecularbiology,biotechnol- 100]. The increasing list of specialized metabolism elicitors ogy, bioinformatics, sequencing technologies, and synthetic and chemical inducers, such as siderophores, rare earths, biology, new strategies have come to the scene and provide metabolismintermediates,diffusiblebacterialhormone-like theopportunityforrationalstrainimprovement(Figure3). 8 BioMedResearchInternational Bioinformatic-guided engineering based on omics Optimization of data and genome-scale culture conditions metabolic network models Heterologous Random Strain expression of clusters improvement in surrogate hosts Random mutagenesis Rational Amplification of clusters and further screening xn ( ) Manipulations with Manipulations with primary metabolism structural, transporter and Manipulations with resistance genes in global and cluster the cluster situated regulatory Flux of genes Sigma factor, precursors ribosome O engineering C Over-expression of H3C SCoA O O positive regulators using Inactivation of native or heterelogous negative CoA promoters regulators HO S Figure3:Approachesusedforimprovingsecondarymetaboliteproductioninactinobacteria.Solidarrowsindicatestrategiesdescribedin thisreview;dash-dottedarrowsdenoteotherstrategiesthatareused. Overall,alloftheserelativelynewapproachesarebased genes,promoters,andheterologoushostsforrationalstrain on spatial, temporal, and quantitative regulation of gene improvement will be reported. Many superb and in-depth expression at the transcriptional or translational level, or reviewshavebeenpublishedrecentlythatdescribedifferent both,therebyenablingproductionofhigheramountsofspe- approachesformetabolicengineeringofactinobacteria[104, cialized metabolites by overcoming bottlenecks, optimizing 112,113,126,127].Wereferthereaderstothemforafurther expression of genes, and redirecting the flux of precursors. comprehensiveintroductiontothesetopics. Therefore, titer can be elevated by overexpressing positive regulatorsordeletingrepressors[94,104,112,113];amplifying 5.1. Regulatory Genes as Basic Keys to Metabolite Overpro- geneclusters[114];redirectingthefluxofprimarymetabolites duction. Genesinvolvedintheproductionofantibioticsare and precursors [104–115]; overexpressing structural genes located together on a chromosome or plasmid and form thatconstitutebottlenecksonthewaytometaboliteproduc- biosynthetic clusters. Such clusters usually contain struc- tion[116,117];manipulatingresistancegenesandtransporters tural,resistance,transporter,andregulatorygenes.Therefore, responsible for the flux of antibiotic [118–120]; ribosomal regulatory genes that are associated with cluster and con- engineering[101,105];andsoforth.Substitutingnativepro- trol biosynthesis of certain compound are named pathway- motersinaclusterwithwell-defined,strongpromoters,either specific or cluster-situated regulators (CSR). They form constitutiveorinducible,givesanopportunitytobypassexist- the lowest level in the hierarchically organized regulatory ingregulatorymachineryofthehoststrainandimprovepro- network of antibiotic production in bacteria [49]. Since duction[121,122].Insomecasesappreciableyieldsofmetabo- production of specialized metabolites is tightly connected litescanbeobtainedbyexpressinggeneclustersinsurrogate tomorphologicaldifferentiationanddependsonaplethora hosts which are easy to manipulate (Streptomyces lividans, of environmental conditions, expression of CSRs hinges on Streptomyces albus) or which are industrial strains (Strep- a variety of other pleiotropic, higher-level regulators that tomyces avermitilis) or which are genetically engineered, senseandtransmitsignalstothem.Inturn,CSRs,whichare versatile hosts with reduced genomes (S. avermitilis SUKA, usuallyfinalcheckpoints,transferthesesignalstostructural Streptomyces coelicolor M1154) [123–125]. In the following genesandswitchbiosynthesisofnaturalproductsonandoff sectionofthereview,onlysomeexamplesofusingregulatory [49, 86]. However, like for every rule, there are exceptions BioMedResearchInternational 9 inthestructureofbiosyntheticgeneclusters.Elucidationof family, led to a 5.6-fold increased production of frederi- thegeneticorganizationofnumerousbiosyntheticpathways camycin A in S. griseus [135]; (e) amplifying the tcp28 or revealedthattherearesomewhichlackCSRs[128,129].These tcp29genes,whichencodeStrRandLuxRfamilyregulators, findingsindicatethatthecluster-situatedlayerofregulationis respectively, in the Actinoplanes teichomyceticus wild-type notmandatoryandisabsentinsomeclusters.Insuchclusters, strainboostedteicoplaninproduction1.5-3-fold[136,137]. theexpressionofstructuralgenesiscontrolledbyubiquitous regulatorygenesthatoccupyhigherlevelsintheregulatory 5.3.ManipulationswithNegativeCluster-SituatedRegulatory web[128,129]. Genes. An effective and promising alternative method to According to how specialized metabolite production is overexpressingcluster-situatedactivatorstoboostantibiotic influenced,allregulatorscanbeconventionallyclassifiedinto productionistoinactivatepathway-specificrepressors.This twogroups:positiveregulators,whichactivate,andnegative is exemplified by the disruption of the lipReg3 gene coding regulators, which repress the biosynthesis of natural prod- for the MarR-type regulator that controls lipomycin export ucts. With the aim of enhancing the titer, both pleiotropic inS.aureofaciensTu¨117,whichledtoa4-foldimprovementin and CSRs, native and heterologous ones, are used. CSRs lipomycinproductionincomparisontothewild-typestrain usually give an opportunity to manipulate one biosynthetic [138]. pathway, whereas global regulators might affect production Otherexamplesthathaveproventheeffectivenessofthis ofseveralspecializedmetabolitesand/ormorphologicaldif- strategy are as follows: (a) inactivation of the jadR2 gene, ferentiation.Therefore,theeffectofapleiotropicregulatory coding a “pseudo” 𝛾-butyrolactones receptor, in S. venezue- geneveryoftendependsonitspositioninthehierarchically lae generated the mutant that produces jadomycin without organized regulatory network and in some cases might be stress treatments (toxic concentration of ethanol, etc.) [139, unpredictable. 140]; (b) inactivation of another deduced 𝛾-butyrolactone receptor coding gene tylP led to a 1.5-fold improvement 5.2. Manipulations with Positive Cluster-Situated Regulatory in tylosin production in S. fradiae [141]; (c) deletion of Genes. Overexpressionofpositive,pathway-specificregula- the ptmR1, encoding GntR type repressor, in S. platensis tors mainly enhances the transcription of structural genes MA7327 resulted in, on average, 100-fold overproduction responsible for the production of certain metabolites and of platensimycin and platencin compared to the wild-type therefore is a commonly used, single-step strategy for strain [142]; and (d) inactivation of the TetR type regulator improvingantibioticyield.Herein,wewilldescribeexamples alpW in S. ambofaciens triggered constitutive production demonstratingtheeffectivenessofthisapproachforrational of kinamycin, a compound with antibacterial activity [143]. strainimprovement. Thus,inactivationofrepressorcodinggenesisusefulnotonly Streptomyces globisporus 1912 is used to produce the for elevating antibiotic production, but, in some cases, for angucyclineantibioticlandomycinE(LaE).Thelandomycin wakeningsilentgeneclusters. biosynthetic gene cluster contains only one regulator gene, lndI, whose product is highly similar to the OmpR-PhoB 5.4. Manipulations with Pleiotropic Regulatory Genes. Suc- subfamilyofproteins.Byinactivatingit,antibioticproduction cessfulapplicationofomnipresentpositivepleiotropicregu- waspreventedintheI2-1mutant,whichconfirmstheroleof latorstoimprovethetiterofcompoundswhosebiosynthetic LndIasanactivatorofLaEbiosynthesis.Complementation geneclusterscontainCSRs,orwhicharefreeofthem,hasalso oftheI2-1mutantwiththreeadditionalcopiesoflndI gene beenshown.Inmostcases,apositiveeffectoftheiroverex- resultedin15-foldincreaseinLaEproductionincomparison pressionisduetotheactivationofcluster-situatedregulatory tothewild-typestrain1912[130],demonstratingtheeffective- gene expression or direct activation of the expression of nessofsuchanapproachforimprovingthestrain. structuralgenesinthecluster.Forinstance,overexpressionof SimocyclinoneD8isanaminocoumarincompoundthat thepleiotropicregulatorafsRsvinS.venezuelae,S.peucetius, is produced by Streptomyces antibioticus Tu¨6040. simReg1, andS.lividansTK24ledtoa4.85-,8-,and1.5-foldincrease which belongs to the OmpR-PhoB subfamily of regula- in pikromycin, doxorubicin, and actinorhodin production, tors, is one of three regulatory genes in the simocyclinone respectively, relative to the wild type [144]. In the case of biosyntheticgenecluster.Itsinactivationabolishedantibiotic S. venezuelae, the increase in pikromycin production was production,whileoverexpressionofsimReg1inanintegrative causedbyenhancedexpressionofthepathway-specificregu- pSET152-derived plasmid increased the simocyclinone titer latorgenepikDandtheketosynthasegene[144].Byintroduc- 2.5-fold[131]. ingadditionalcopiesoftheafsRorafsSgenesintoS.coelicolor, Other examples are as follows: (a) the C-1027 titer in actinorhodinproductioncouldalsobeincreased[145]. S. globisporus was improved 5-fold after overexpressing the Streptomyces ghanaensis is a producer of phosphogly- sgcR1gene,codingforaStrR-likeprotein[132];(b)amplifying colipid antibiotic moenomycin A [146]. The moenomycin theclaRgeneencodingtheLysRfamilyproteininmulticopy biosynthetic gene cluster does not contain CSRs; therefore, plasmids resulted in a threefold increase in clavulanic acid different pleiotropic regulators were used to improve the biosynthesis and in a sixfold increase in alanylclavam pro- moenomycintiter.OverexpressionoftheadpAghgene,aplei- duction [133]; (c) inserting a single copy of pimM, a LuxR otropicregulatorofantibioticproductionandmorphological type regulator, into the S. natalensis wild-type strain ele- development,ledtoa2.5-foldimprovementinmoenomycin vatedpimaricinproduction2.4-fold[134];(d)overexpressing productioninS.ghanaensiscomparedtothewild-typestrain fdmR1,theencodingpathway-specificactivatoroftheSARP [129]. Introduction of the second copy of bldAgh, a leucyl 10 BioMedResearchInternational tRNA coding gene, into the wild-type strain S. ghanaensis 5.6.HeterologousExpressionofClustersasaWaytoOverpro- led to a 1.6-fold increase in moenomycin production [129]. duction. Withtheadventofgenomesequencingandmetage- Overexpression of the relA, a ppGpp synthetase gene from nomics, a plethora of clusters coding for putative biologi- S. coelicolor, led to a 2-fold improvement in moenomycin cally active compounds which previously eluded discovery productioninS.ghanaensisrelativetothewildtype[147]. because of silencing or low product yield have become and continuetobecomeavailable.Inaddition,therearegrowing Similarly, inactivation of negative pleiotropic regulators numbersofactinobacteriathataredifficulttocultureandto inS.ghanaensisincreasedmoenomycinproduction.Thegene manipulategeneticallybutwhichproduceormightproduce absB codes for the RNAseIII endoribonuclease involved in interestingchemicalcompounds.Thereasonsoutlinedabove globalregulationofmorphologicaldifferentiationandantibi- drove the development of a new approach in metabolic oticproductioninS.coelicolor [148].Bydeletingit,moeno- mycinproductionwasimproved2.7-foldcomparedwiththe engineering for developing surrogate high-producing hosts parentalstrain[129].Inactivationofanotherglobalregulator fortheheterologousexpressionofgeneclusters.Therearea gene, wblA(gh), encoding a homologue of the WhiB-family number of potential surrogate hosts. Some of them derive of proteins, produced a 2.3-fold increase in moenomycin from well-studied Streptomyces strains such as S. lividans, biosynthesisinS.ghanaensis[149]. S.coelicolor,orS.albus;othersareobtainedfromindustrial strains or are genetically engineered, versatile hosts with Disruption of the (p)ppGpp synthetase gene, relA, in S. reduced genomes. However, the main aim of this approach clavuligerusboostedclavulanicacidproduction3-to4-fold is still relevant and aims to build an ideal and universal and that of cephamycin C increased about 2.5-fold [150], surrogate host that will be easy to genetically manipulate, confirmingthattheremightbeapleiotropiceffectofglobal is fast growing and devoid of competitive sinks of carbon regulatoramplificationorinactivation. andnitrogenandantibioticactivity,andwillbesuitablefor overproductionofdifferentspecializedmetabolites. 5.5.PromotersasBio-BricksforTiterImprovement. Another common metabolic engineering approach to induce or To improve moenomycin production, several Strepto- enhancetheexpressionofsilentorpoorlyexpressedpathways myces strains were used as heterologous hosts, namely, S. isbasedonreplacingnativepromotersinaclusterwithwell- coelicolor M145, M512 (ΔactII-ORF4, ΔredD), S. lividans defined,strongpromoters,decouplingthemetabolicpathway TK24, 1326, S. albus J1074, S. venezuelae ATCC10712, and from the existing cellular regulatory networks. Examples S. thermospinosisporus NRRL B24318. The highest moeno- describedbelowclearlyprovetheeffectivenessofthecombi- mycin titer was found in S. albus strains, the lowest in S. nationoftwometabolicengineeringstrategies:amplification coelicolor [147]. These data show a high variation between ofpositiveregulatorsandtheirexpressionunderthecontrol differenthosts.Worthyofnoteisthattheyieldofantibiotic ofheterologouspromotersofvariousstrengths. inS.albuswasonaverage4timeshigherthaninthenative Forthispurposedifferentnaturalorsyntheticconstitutive producerS.ghanaensis[147]. or inducible promoters may be used [137, 151]. One of the There are several genetically engineered heterologous most widely employed promoters in streptomycetes is the hosts that were obtained by controlled minimization of erythromycinresistancegeneermEpfromSaccharopolyspora genomes. For example, S. coelicolor M1154 was constructed erythraeaoritsupregulatedvariant𝑒𝑟𝑚𝐸𝑝∗[152].Forexam- by deleting four gene clusters (actinorhodin, prodiginine, ple,simultaneousoverexpressionofthednrN,dnrI,andafsR calcium-dependent antibiotic, and cryptic polyketide) and regulatorygenesunderthecontrolof𝑒𝑟𝑚𝐸𝑝∗inS.peucetius subsequently introducing point mutations in the rpoB and led to a 4.3-fold increase in doxorubicin production [153]. rpsL genes that enhance specialized metabolite production Anotherprominentexampleoftheuseofthispromoteristhe [125].Expressionofthegeneclustersforchloramphenicolor improvementintylosinproductioninS.fradiae.Biosynthesis congocidine in this strain led to a 40- and 30-fold increase of tylosin is orchestrated by the complicated interplay of inproduction,respectively,incomparisontotheS.coelicolor fiveregulators[154].Tobypassexistingregulatorynetwork- M145 strain. Another surrogate host was developed on the positiveregulators,tylSortylRwasplacedunderthecontrol basis of the industrial strain S. avermitilis [124]. A region of the 𝑒𝑟𝑚𝐸𝑝∗ and overexpressed in the S. fradiae wild- of more than 1.4Mb that contains nonessential genes and typestrain.Thisboostedtylosinproduction3.8-and5.0-fold, gene clusters was deleted stepwise from the chromosome respectively[154].Productionofteicoplanininthenonstrep- ofS.avermitilis.ExpressionofcephamycinC,streptomycin, tomycetesactinomyceteA.teichomyceticuswasimproved2.8- andpladienolidebiosyntheticgeneclusterswastestedinthe fold and 10-fold by overexpressing the StrR-type regulator obtainedstrains.ProductionlevelofstreptomycininSUKA5 tcp28underthecontrolofthepromoteroftheSARPregulator strain was approximately 3 times higher than in the native gene actII-ORF4 and apramycin gene resistance promoter producer. Biosynthesis of cephamycin C was also greatly (aac(3)IVp), respectively, which appeared to be stronger in improved. However, the biosynthesis was switched on only this strain than the widely used ermEp [136, 137]. This in the presence of the activator CcaR. Substitution of the reflects the necessity to test the activity of heterologous native promoter of the ccaR gene with the alternative rpsJ promoters in a particular strain since their activity might promoter led to an additional increase in cephamycin C differinvariousspecies.Therefore,therepertoireofavailable production[124],underscoringtheurgencyandneedtouse promotersshouldbeextended. theapproachesoutlinedabovetofurtherimproveantibiotic