University of Groningen
Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus
subtilis cell factories
van Tilburg, Amanda Y.; Cao, Haojie; van der Meulen, Sjoerd B.; Solopova, Ana; Kuipers,
Oscar P.
Published in:
Current Opinion in Biotechnology
DOI:
10.1016/j.copbio.2019.01.007
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van Tilburg, A. Y., Cao, H., van der Meulen, S. B., Solopova, A., & Kuipers, O. P. (2019). Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories. Current Opinion in Biotechnology, 59, 1-7. https://doi.org/10.1016/j.copbio.2019.01.007
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Metabolic
engineering
and
synthetic
biology
employing
Lactococcus
lactis
and
Bacillus
subtilis
cell
factories
Amanda
Y
van
Tilburg
2,
Haojie
Cao
2,
Sjoerd
B
van
der
Meulen,
Ana
Solopova
1and
Oscar
P
Kuipers
Metabolicengineeringandsyntheticbiologyapproacheshave
prosperedthefieldofbiotechnology,inwhichthemainfocus
hasbeenonEscherichiacoliandSaccharomycescerevisiaeas
microbialworkhorses.Inmorerecentyears,improvingthe
Gram-positivebacteriaLactococcuslactisandBacillussubtilis
asproductionhostshasgainedincreasingattention.This
reviewwilldemonstratethedifferentlevelsatwhichthese
bacteriacanbeengineeredandtheirvariousapplication
possibilities.Forinstance,engineeredL.lactisstrainsshow
greatpromiseforbiomedicalapplications.Moreover,we
provideanoverviewofrecentsyntheticbiologytoolsthat
facilitatetheuseofthesetwomicroorganismsevenmore.
Address
DepartmentofMolecularGenetics,GroningenBiomolecularSciences andBiotechnologyInstitute,UniversityofGroningen,Nijenborgh7, Groningen9747AG,TheNetherlands
Correspondingauthor:Kuipers,OscarP(o.p.kuipers@rug.nl)
1Presentaddress:APCMicrobiomeInstitute,UniversityCollegeCork,
CollegeRd.,Cork,Ireland.
2
Theseauthorscontributedequallytothiswork. CurrentOpinioninBiotechnology2019,59:1–7
ThisreviewcomesfromathemedissueonTissue,cellandpathway engineering
EditedbyMarjanDeMeyandEvelinePeeters
https://doi.org/10.1016/j.copbio.2019.01.007 0958-1669/ã2018ElsevierLtd.Allrightsreserved.
Introduction
Inthelastfewdecades, metabolicengineeringand syn-theticbiologyapproachesto improve industrial applica-tions of microbes have delivered many breakthrough results [1]. However, most of this work has been per-formed with the main model organisms Escherichia coli and Saccharomyces cerevisiae.Here, we will focus on two otherworkhorsesinbiotechnologythatisthefood-grade bacteriumLactococcuslactis,andindustrialchassisBacillus subtilis. L. lactis is indispensable in dairy and health applications, beingaproductionorganismfor antimicro-bials, polyphenols, oral vaccines, and flavor-compound and texturizing compound. B. subtilis is an efficient
metabolite and enzyme producer for various industrial applications.Moreover,thesporulationpropertiesofthe latter also offeropportunitiesfor vaccine production by expressingantigensatthesurfaceofspores.These micro-organisms have some unique properties, which make them particularly suited for specific applications. Both L.lactisandB.subtilislackimmunogenic lipopolysacchar-ides that render them better hosts for expression of health-relatedproducts,likeantimicrobialdeliveryororal vaccines,than E.coliand otherGram-negativebacteria. Moreover,L.lactismodelstrainMG1363producesonlya low number of exoproteins and noexoproteases, while genome-reducedB.subtilisstrains[2]alsolack extracel-lularproteaseactivity,whichmakethesehostsexcellent enzymeproducers,sincetheproductswillstablyremain in theculturesupernatants.
MetabolicengineeringofL.lactis
Formorethan20years,therelativelysimplemetabolism ofL.lactishasservedasatargetformetabolic engineer-ing. The ability to metabolize a broad range of carbon sources, high glycolyticflux and tolerance to high con-centrations of organic acids and alcohols makes it an excellent candidate for bioproduction of fine chemicals and food ingredients. Rewiring the pyruvate node by blocking competing enzymesand modifying the native glycolytic flux via ATP (adenosine triphosphate) and cofactor recycling, lead to strains efficiently producing diacetyl,acetaldehyde,acetoin,andsoon.Theabilityof L.lactis toswitchbetweenfermentation andrespiration whenheminispresentwaselegantlyexploitedasawayto regenerate NAD (nicotinamide adenine dinucleotide) during acetoinand 2,3-butanediol production [3].The most recent studies notonly obtained the highest 2,3-butanediollevelreportedforL.lactistodate[4],butalso demonstrated its potential for converting dairy waste streams into the value-added products (3R)-acetoin, 2,3-butanediol and biofuel ethanol with high yields encouraging furtherbioprocessoptimization[4,5].
Many engineering efforts were put in food industry-related strain improvement, such as stress and phage resistance[6–8].Reroutingandoverexpressionofvarious nativeandheterologouspathwaysinL.lactis(Figure1a,b) haveyieldedefficientvitamin,polyol,EPS(extracellular polymeric substances), hyaluronic acid producers (reviewedinRef.[6]).Developmentofprotocolsforcell propagationinemulsiondropletscoupledtomicrofluidics
andotherscreeningmethodsallowedselectionofstrains withhigherbiomassorbettervitaminproducerswithout anytargeted geneticmanipulation[9,10].
SyntheticbiologytoolsforL.lactis
Genetic engineering forstrain optimization in L. lactis has been focused for many years on traits useful to improve industrial milk fermentations. Because of its beneficialpropertiesandtheexpandinggenetictoolbox (Table1),L.lactishasalsogainedinterestasan expres-sionhostfortheproductionofheterologousproteinsand therapeutic/antimicrobial peptides [11]. Various tools have been instrumental to enable the use of L. lactis asaproductionhost.Dependingonthepurpose,either
(artificial) constitutive or inducible promoters are employedto (over)expressthe gene or gene clusterof interest.Thedevelopmentofinduciblegeneexpression systems has been extremely powerful for functional characterizationorproductionpurposes.Varioussystems have been developed, of which the NIsin-Controlled Expression system (NICE) is mostly used [12]. Also othersystemssuch asZirex(Zinc-regulated expression system)[13]andACE(agmatine-controlledexpression) [14]weredeveloped thatcanbeused aloneorin com-binationto enable sequential expression.Riboswitches canbeusedasanOFF-switchtoenablegenesilencing and are currently underdevelopment for L. lactis [EP Hernandezet al., unpublisheddata].
2 Tissue,cellandpathwayengineering
Figure1
(a)
(b)
(c)
Single element engineering
Combinatorial engineering
Global metabolic optimization
Expression element engineering Transporter engineering
Cofactor engineering
Secretion machinery engineering
NADH/NADPH, ATP, other metabolites or enzymes Nutrients Byproducts Products Global transcription factor
Current Opinion in Biotechnology
MetabolicengineeringstrategiesfortheproductionofindustrialrelevantproductsbyL.lactisandB.subtilis.
(a)Rationalengineeringofexpressioncassettes(fromlefttoright,differentcoloredboxesrepresentvariouspromoters,RBSs,CDSsand terminators,respectively),transporters,cofactors,orsecretionmachinerycomponents.(b)Modularorcombinationaloptimizationofdifferent rationalapproachesinamulti-geneproductsyntheticpathway.(c)Globaltranscriptionmachineryengineering(gTME)allowssystem-wide pathwaymodificationforimprovingthemetaboliccapacityorchemicaltolerance.Allthethreelevelsofmetabolicoptimizationapproacheshave beendoneinB.subtilis,whileonlystrategy(a)and(b)havebeenappliedfortheoverexpressionofproductsinL.lactis.
TointroduceforeignDNAintoL.lactis,electroporationis currently the golden standard. However, DNA transfer betweentwoL.lactisspeciesbyconjugationwasdeveloped [15]andanon-conjugativeverticalgenetransferhasbeen observedaswell(LMorawska,OPKuipers,personal com-munication).Recently,theinductionofcompetencegenes createdfunctionalcompetenceinL.lactis[16].Different genemodificationsystemshaveenabledefficient chromo-somalmutationsbyusingrecombineering[17].Gene inser-tionsor deletionswere facilitated by double cross-over using the pCS1966plasmidthatisunableto replicateandthat containsaselectableantibioticmarkeranda counter-select-ableorotatetransporter.Thiscanleadtomarkerless dele-tionsorinsertions[18].Anextensiveoverviewofcloning vectorsandtoolsforgene expression/modificationis pre-sented elsewhere [19]. Currently, alternative natural methods are being developed that open up application optionsin,forexample,thedairyindustry.Phage contami-nationcanbethecauseof significantloss of bacterialactivity in food fermentations andduring the productionof bio-chemicals.AntisenseRNAsagainstcrucialbacteriophage geneswerepreviouslyusedinL.lactis[20]toinhibitthe efficiencyofplaquingandburstsize.Inthesestudies,there wasalimiteddesignoftheseantisenseRNAs,otherthan cloningdifferentpartsofthegenessuchthattheopposite strandistranscribedasanantisenseRNA.Recently, hun-dreds of novel RNAs with putative regulatory functions have been discovered [21] and advances in the field of regulatory RNAs havegainedtremendous insightin the molecularmechanismsofhowmRNAscanbesilencedby degradationorbyblockingoftheShineDalgarnosequence to prevent translation, creating possibilities for a more designerapproachusingregulatoryRNAsasatooltoaffect gene expression.Cell penetratingpeptides(CPPs)could provideawaytodeliverphosphorodiamidatemorpholino oligonucleotides(PMOs)toenablegenesilencing.These CPP–PMO’sarenowallowedtherapeutically[22],butcan beappliedtosteerfermentationduringtheprocessitselfin
order to eliminate cell functions such as transporters or pathways. An illustrative example of synthetic biology makinguseofpeptidemodularityinsteadofDNA modu-laritywasprovidedbylanthionineringshuffling of lanti-biotics,makinguseofover25differentring,hinge,andtail modulesandamicro-alginatebead-basedhighthroughput screening method to obtain new-to-nature lantibiotics. Some of the analyzed peptides possessed antimicrobial activity andwere shownto havean unprecedented host range[SSchmittetal.,unpublisheddata].
UseofL.lactisforbiomedicalapplications
An established history of safe use in the food industry makes L. lactis an appealing organism for a number of biomedical applications. Absence of lipopolysaccharides and only a low number of exoproteins makeit a better deliveryvehicleorexpressionhostthanE.coliandother Gram-negative bacteria. Genetically modified (GM) L. lactisisusedto(1)preventandtreatinflammatorybowel disease(IBD),diabetes,cancerbymodulating inflamma-tionviacytokine,anti-protease,antioxidantenzyme, anti-bacterial, and anti-antigenic peptide secretion; (2) fight infectious diseasesand allergic reactionsvia modulation ofimmuneresponsesandasasafervaccine.GML.lactis mayevenbeusedtodeliverDNAmoleculestomammalian cellsasvaccinesorasaformofgenetherapy[23].
Steidleretal.haveestablishedproofofprincipleusingL. lactisengineered toproducetheanti-inflammatory cyto-kine interleukin-10 (IL-10) to treat IBD [24]. Using animal models, theyshowed thatdietaryadministration of engineered bacteria is therapeutically effective. An elegantsystemforthecontainmentoflive GMbacteria wasdesignedreplacingthethyAgenewithanexpression cassetteresultinginastrainthatproduceshIL-10whenis strictly dependent onthymidineor thymine for growth andsurvival.Thisstrainwasthefirstgeneticallymodified organism (GMO) to reach clinical trials [25]. To date,
Table1
ExistingandnovelsyntheticbiologytoolsforL.lactis
Purpose Syntheticbiologytool Reference
Controlledgeneexpression Nisin-controlledgeneexpressionsystem(NICE)
[19] Zinc-regulatedexpressionsystem(Zirex)
Agmatine-controlledexpressionsystem(ACE) Stress-induciblecontrolledexpressionsystem(SICE) pH-responsiveexpression(P170)
Constitutivegeneexpression Constitutivenativepromoterlibrary [55] Genomeengineering pSEUDO.Site-specificintegrationbasedonhomologousrecombination [18] Recombineering.Marker-freemethodforchromosomalmutations/deletionsusingssDNAoligo’s [17] Cre-loxPrecombinationsystem.Site-specificrecombinationsystemthatallowsmultiplegene
deletionsinL.lactis
[56]
CRISPR-Cas9/CRISPRi-basedgenomeediting [57]
Improvedexpressionhost L.lactisNZ9000-4(9k-4)withminimizedgenomeandenhancedheterologousproteinproduction [58] Improvedproteinsecretion Signalpeptides(SP).Alibraryofusp45-derivedSPforefficientproteinsecretion [59]
severalengineeredlactococcihavereachedclinical stud-ies using similar safe containmentstrategy. Recently,a phaseIb/IIastudywasannouncedtotesttheabilityofL. lactissecretingIL-10andproinsulin(AG019)[26]totreat earlyonsettype1diabetes.Aphase2clinicaltrialofan oral rinse composed of a recombinant L. lactis strain engineered to secrete the mucosal protectant human trefoilfactorhTFF1wasinitiated[27].Anoral adminis-trationofL.lactisengineeredtosecreteanti-TNF-alpha nanobodiesprovedtobeeffectiveandsafeagainstIBDin aphaseItrial[28].
AlthoughtheNICEsystemprovedtobeusefulinmany cases, synthetic biology approaches required develop-mentofpromotersthatdonotinvolveanexternalinducer andareconstitutiveorrespond tofactorswhichbacteria encounterinthemammalianbody (Table1).
A resource-conserving and environmentally sustainable productionofplant naturalproductswith health-benefi-cialpropertiesusingmicrobialcellfactoriesisan attrac-tivealternativetoplantextractionorchemicalsynthesis. L. lactis was shown to be an excellent host for the expression of plant and fungal membrane proteins and solubleenzymesinvolvedinpolyphenol,terpenoid,and estersynthesis [6,29].Functional pathways for the pro-duction of nutraceuticals resveratrol and anthocyanins were assembled. Employment of metabolic biosensors formalonyl-CoAallowedmonitoringofintracellular pre-cursor pool and suggested strategies to improve the productyield[29].
MetabolicengineeringofB.subtilis
In addition to L. lactis, B. subtilis has been extensively exploitedas amicrobial cellfactoryfor the overproduc-tionofvariousindustriallyrelevantproductsinthefields of food, pharma, and biotechnology. In the past few decades, numerous studies have been performed in attempts to develop this production host into a highly adaptablechassiswithbothhighyieldsandawiderange ofproducts[30].
Conventional approaches for improving the production capacityofB.subtilisincludemodifyingtheelementsof syntheticpathways, thatis genecopy numbers, promo-ters, RBSs (ribosome binding sites), CDS (coding sequences),andterminators[31]orvaryingthe availabil-ityofrate-limitingcomponents,thatisthecompositionof secretion machinery [32] (Figure 1a). However, these efforts, that are based on the rational modification of specificpathwayorfactors,alwaysrequirea comprehen-siveunderstandingofthetargetmetabolicnetworksand havelimitedsuccessonstrainimprovement[33,34].
Metabolic engineering strategies that integrate newly developed systems and synthetic biology approaches havegreatlyfacilitatedtheunlockingofphenotypeswith
desiredcellularpropertiesinB.subtilis[34]. Combinato-rialpathwayoptimization,whichenablestovarymultiple criticalpathwayelements,canstreamlinemetabolic engi-neeringbyreducingexperimentaleffortsandtheamount ofaprioriknowledge[35](Figure1b).Thecombinatorial engineeringof promotersand RBSs[31],and non-con-served sequences [36] demonstrated great potential in increasing reporter gene expression levels in B. subtilis.Thedividedpartsofcomplexmulti-geneproduct synthetic pathways have beenmodularly optimized for generatingrecombinantstrainswithimprovedproduction of N-acetylglucosamine [37]. Moreover, simultaneous engineeringofthecellsurfaceandtheexpressedtargets leadtoafurtherenhancedsecretionefficiencyof a-amy-lasesinB.subtilis[38].
Global transcription machinery engineering (gTME), which allows multiple and simultaneous perturbations of the whole transcriptome, enables the increase of end-productsbyreroutingmetabolicfluxesatatoplayer ofregulatorynetworks[1](Figure1c). Thisglobal opti-mization strategy can substantially simplify the strain enhancement design even without a complete picture oftheunderlyingmetabolicregulatorymechanisms[39]. Avarietyofglobaltranscriptionfactorsinmultiple micro-organisms have been successfully engineered to elicit variants with improved metabolic capacity or chemical tolerance [40]. In a recent study, the gTME-based approachwasappliedforeffectivelyandquickly unlock-ing B. subtilis variants by randomly mutagenizing the global N-regulator and C-regulator CodY and CcpA, respectively.Theselectedbestphenotype,carrying cru-cialmutationsamonghelix-turn-helixdomains,reacheda twofoldincreasedoverproductionofb-galactosidase. Fur-thermore, this improvement was demonstrated by the significantly enhanced overexpression of green fluores-centprotein, axylanaseand apeptidase [41].
SyntheticbiologytoolsforB.subtilis
B.subtilislendsitselfwelltogeneticmanipulationdueto itsabilitytobecomenaturallycompetentandtotakeup bothcircularand linearformsof DNA.In addition,the genomecaneasilybechangedforvariablepurposes(e.g. deletions,pointmutations,insertions)byemployingthe mechanismofhomologousrecombination.Nevertheless, advancesintechnologiesandincreasingknowledgeabout the characteristics of a successful bacterial production host havedriventhecontinuousdevelopmentof useful B.subtilisstrainsandgenomeeditingtools.ThismakesB. subtilisa very attractive bacterial hostfor academic and biotechnologicalpurposes.
In addition to protease deficient B. subtilis strains (reviewedinRef.[42]),whichshowedimproved produc-tion yields of heterologous proteins (Table 2), the genomeof B.subtilishas beenreducedfurther in order toobtainamoresimplifiedmicrobialchassisthatpossibly
displays evenhigher production yields [43].To date, a genomereductionof36%hasbeenachievedinB.subtilis [2].Thisledtotwoindependentgenome-minimizedB. subtilisstrainswhichhavebeensubjectedtomulti-omics analyses and still display robust growth in complex medium. To copewith thegradual decreasein genetic competence as a consequenceof genomereduction, an induciblecompetencesystem[44]wasintroducedinthe minimalgenomeleadingtoa20-foldincreasein transfor-mation efficiency compared to the reference strain B. subtilis168.This,incombinationwiththepreservationof commonlyusedlociforgenomicintegration,makesthese MiniBacillusstrainsattractivemicrobialhostsfor heterol-ogousexpression.Currently,theMiniBacillusstrainPG10 isexploitedfortheheterologousproductionoflantibiotic peptides [AY van Tilburg et al., unpublished data]. A majoradvantageof thislantibioticproductionsystemin PG10is thelackofextracellular serineproteases which resultsintheproductionofantimicrobialinactive precur-sorpeptideswhichcanbeactivatedinvitroatalaterstage. OtherusefulB.subtilisstrainscanbefoundinthe essen-tialgeneknockdownlibrary[45]andintwoorderedand barcodednon-essentialgeneknockoutlibraries[46].By providing information about gene functions, networks, and pathways in B. subtilis, these single-gene deletion strainscanfacilitatethedesignoftailor-made biological systems.
Inthelastdecade,thegenetictoolboxforgenome engi-neeringinB.subtilishasexpandedsignificantlywiththe development of new tools as well as improvements of existingtools.Multipletoolboxescontainingavarietyof (standardized)elementsfor fine-tuning geneexpression are nowadays available [47,48]. In addition, effort has beendevotedtothegenerationofmoretightlycontrolled expression systems (e.g. the subtilin-regulated gene
expression (SURE) system [49]), as well as expression systemsthatareinducer-free[50]orwhichdonotleave selectable markers or other scarsbehind [51].Also, the CRISPR-Cas9 system has enriched thepossibilities for genome editing in B. subtilis and other Bacillus species [52].ByusingtheCRISPR-Cas9system,disadvantages ofmarkerlesssystemscanbeovercome,whiledeletions, mutationsorinsertionscaneasilybeachievedatanyplace inthegenome.Furthermore,variousdatabaseshavebeen generatedthatprovideavastextentofinformationonthe levelofDNA,proteins,regulators,andmetabolitesinB. subtilis[53,54].
Conclusions
and
outlook
Thelowlevelofproteolyticactivity,theavailabilityofan extensive engineering toolkit, including strictly con-trolled promoters, and the great fermentative capacity ofL.lactismakeitanattractivehostforflavor, antimicro-bial peptide and metabolite production,since heterolo-gousenzymesneededinpathwayengineeringwill com-monlybestablyproduced.B.subtiliscangrowtohighcell densities and is a great host for enzyme production, particularly in view of recent advances to minimize its genome reducing adverse proteolytic degradation and undesirable phenotypessuchas sporulationand biofilm formation. A major advantage of these bacteria is also theirfood-gradestatus,althoughrestrictiveregulationson useofGMOsinfoodandenvironmentstillprecludesome excitingapplications,suchasgutmicrobiota-modulating cultures, engineered probiotic strains or nutraceutical-producing cultures, and oral vaccines. Recentadvances inCRISPR-Cas9useandsomeforeseenmedical applica-tions,ofengineeredmicrobesmightchangethissituation in thenear future.
Conflict
of
interest
statement
Nothingdeclared.
Table2
ExistingandnovelsyntheticbiologytoolsforB.subtilis
Purpose Syntheticbiologytool Reference
Improvedexpressionhost Protease-deficientB.subtilisstrains [42]
Genome-minimizedB.subtilisstrains [2]
Essentialgeneknockdownandnon-essentialgeneknockoutlibraries [45,46] Improvedgeneticcompetence Induciblecompetencesystemtoimprovetransformationefficiency [44] Expressionelements BacillusBioBrickBoxcontainingstandardizedvectors,reporters,promoters,epitopetags,
andoptimizedfluorescentproteins
[47] Acharacterizedphase-dependentendogenouspromoterlibrary [48] Controlledgeneexpression Subtilin-regulatedgeneexpressionsystem(SURE) [49]
Inducer-freeexpressionsystems [50]
Genomeengineering Markerlessgenedeletionsystem [51]
CRISPR-Cas9system(pJOE8999) [52]
Databases SubtiWiki.Databaseofgenes,proteins,metabolicandregulatorypathways. [53]
Acknowledgements
TheauthorsthankAukevanHeelandJakobVielforproductiveinitial discussions.SBvdMissupportedbyagrantfromTKIChemistryandNWO (projectnumber731.014.206),AvTissupportedbyEUH2020granton Rafts4Biotech,H.C.issupportedbyagrantfromChinaScholarshipCouncil (CSC),whileASwassupportedbyH2020grantBachBerry.
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