Review Article Antibacterial Discovery and Development: From Gene to Product and Back

17 Pages·2015·2.09 MB·English

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