Perspectives on biotechnological applications of archaea - Hindawi

13 Pages·2014·1.71 MB·English

Archaea1, 75–86
© 2002 Heron Publishing—Victoria, Canada
Perspectives on biotechnological applications of archaea
1 1 1,2
CHIARASCHIRALDI, MARIATERESAGIULIANO and MARIO DE ROSA
1 Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Faculty of Medicine, II University of Naples, via
Costantinopoli16, 80138 Naples, Italy
2 Author to whom correspondence should be addressed ([email protected])
Received October 15, 2001; accepted May 6, 2002; published online May 31, 2002
Summary Many archaea colonize extreme environments. knowntobemuchbroaderthanpreviouslythought,andthis
Theyincludehyperthermophiles,sulfur-metabolizingthermo- hasledtotheexplorationofmanyhithertouninvestigatedhab-
philes,extremehalophilesandmethanogens.Becauseextrem- itats.Second,itisnowrecognizedthatattributesoforganisms
ophilic microorganisms have unusual properties, they are a adaptedtoextremeenvironmentshavethepotentialtoservea
potentiallyvaluableresourceinthedevelopmentofnovelbio- wide array of industrial purposes.
technologicalprocesses.Despiteextensiveresearch,however, Archaea possess genes with recognizable counterparts in
therearefewexistingindustrialapplicationsofeitherarchaeal theBacteria,showingthatthetwogroupshavefunctionalsim-
biomassorarchaealenzymes.Thisreviewsummarizescurrent ilarities.Archaeaalsopossessgenesotherwisefoundonlyin
knowledge about the biotechnological uses of archaea and Eukarya. Nevertheless, most signature genes present in
archaealenzymeswithspecialattentiontopotentialapplica- archaealgenomeshavenoknownfunctionandnohomologs
tions that are the subject of current experimental evaluation. outsideoftheArchaea(Grahametal.2000).Becausemuchof
Topics covered include cultivation methods, recent achieve- the genetic patrimony of the Archaea appears to be unique,
mentsingenomics,whichareofkeyimportanceforthedevel- many genomic studies are currently in progress (Table 1) to
opmentofnewbiotechnologicaltools,andtheapplicationof identify the functions of archaea-specific genes and thereby
wild-type biomasses, engineered microorganisms, enzymes gain a better understanding of cellular processes inarchaea.
andspecificmetabolitesinparticularbioprocessesofindustrial In addition to unique amino acidsequences,extrinsic fac-
interest.
tors(e.g.,proteinfoldingsystems)maybeinvolvedinextrem-
Keywords:biotechnology,extremozymes,highdensitycultiva- ophilicadaptation.Increasingthesizeofthegenomicdataset
tion, recombinant DNA technology. willindicatewhethersomeadaptationsaredependentonspe-
cificaminoacidsequencesorstructuralcontexts(Haneyetal.
1999).Tothisend,comparativestudiesofproteinsequences
Introduction frommesophilicandextremelythermophilichomologousmi-
croorganismsareneeded.Haneyetal.(1999)conductedsuch
Woeseetal.(1977)recognizedthattheProkaryacomprisetwo
astudyonMethanococcusspecies.Comparativestudiesatthe
distinct domains, namely the Bacteria and the Archaea. The
molecularlevel(e.g.,G+Ccontent)orofaminoacidalign-
originalclassificationbasedon16SrRNAhasbeenconfirmed
ment are also needed to identify highly conserved residues.
by extensive phylogenetic studies on a variety of macro-
Manyinterestingenzymeshavebeenisolatedfromextrem-
moleculesfoundinthepatrimonyofthesemicrobes.System-
ophilic microbes (sometimes called extremozymes) (Hough
aticclassificationoftheArchaea,whicharegenerallybelieved
andDanson1999,Schumacheretal.2001).Specificarchaeal
tooriginatefromasingleancestor,hasidentifiedseveralmajor
metabolites have also been purified and characterized and
clusters based on physiological characteristics: hyperther-
mophiles (optimal growth temperatures higher than 70 °C); someofthemhavepotentialindustrialuses.However,several
methanogens (anaerobes with growth temperatures ranging technicaldifficultieshavepreventedthelarge-scaleindustrial
from0to100°C);extremehalophilesorEuryarchaeota(re- applicationofenzymesandspecialmetabolitesfromextrem-
quiringhighsalinesolutions,upto20%sodiumchloride,for ophilic sources, the most important being the availability of
growth); and sulfur-metabolizing thermophilic bacteria or thesecompounds.Large-scalearchaealculturesaredifficultto
Crenarchaeota. setup: medium evaporation hasto betakeninto accountfor
Manyarchaeaareextremophiles,microbesthatnotonlytol- hyperthermophilic cultures, and fermenters must be built of
erate,butgrowoptimallyinhabitatsnormallyconsideredtoo materials resistant to corrosion by the media required for
severe for life. Although several extremophilic microorgan- growth of alkaliphiles, acidophiles and, particularly, halo-
isms have been known for 40 years, the search for extrem- philes.Inaddition,itiswellestablishedthatthebasalexpres-
ophileshasintensifiedinthelastdecadefortwomainreasons. sion level of archaeal enzymes, although sufficient for their
First,therangeofconditionsunderwhichlifecanexistisnow purificationandcharacterizationonalaboratoryscale,islow
76 SCHIRALDI, GIULIANO AND DEROSA
Table 1. List of completed genome sequences (sources: The Institute for Genomic Research (TIGR, Rockville, MD) and PUBMED).
Microorganisms Size (Mb) Reference
Order Species
Crenarchaeota
Desulfurococcales Aeropyrum pernix K1 1.6 Kawarabayasi et al. 1999
Sulfolobales Sulfolobus solfataricus P2 2.9 She et al. 2001
Sulfolobus tokodaii 2.7 Kawarabayasi et al. 2001
Euryarchaeota
Archaeoglobales Archaeoglobus fulgidus DSM 4304 2.1 Klenk et al. 1997
Halobacteriales Halobacterium sp. NRC-1 2.0 Ng et al. 2000
Methanobacteriales Methanobacterium thermoautotrophicum H 1.7 Smith et al. 1997
Methanococcales Methanococcus jannaschii DSM 2661 1.6 Bult et al. 1996
Methanosarcina acetivoransC2A 5.7 Galagan et al. 2002
Methanopyrales Methanopyrus kandleri 1.7 Slesarev et al. 2002
Thermococcales Pyrococcus horikoshii (shinkaj) OT3 1.7 Kawarabayasi et al. 1998
Pyrococcus abyssi GE5 1.7 unpublished
Thermoplasmales Thermoplasma acidophilum DSM 1728 1.5 Ruepp et al. 2000
Thermoplasma volcanium GSS 1 1.5 Kawashima et al. 2000
with respect to that required for industrial purposes (Kelly calinterestandondesigningbioreactorsandbioprocessesthat
andDeming1988,Cowan1992,Herbert1992,Madiganand increaseproductivity.Becauseoftheunconventionalenviron-
Marrs1997).Asaconsequence,scientificinteresthasfocused mentalconditionsneededforthecultivationofextremophiles,
bothonthedesignofinnovativebioreactorstoachievehigher contaminationproblemsareminimal.Therefore,acontinuous
biomassesandmetaboliteproductionrates,andonthedevel- culture approach has been widely adopted.
opmentoftechniquesforcloningandexpressingarchaealen- Continuouscultureexperimentshaveprovidedinsightsinto
zymesinmesophiliccellfactories.Thesuccessfulcouplingof thesignificanceofspecificenzymes,inadditiontoclarifying
thesestrategieshasbeendemonstratedinseveralapplications stress responses and unusual physiological characteristics of
(NordbergKarlssonet al. 2000,Schiraldiet al. 2000, 2001). hyperthermophilicandextremelythermoacidophilicmicroor-
Inadditiontothedevelopmentofindustrialproductionpro- ganisms.Toimprovebiomassyieldofthehyperthermophilic
cesses, basic research is also needed to determine how en- archaeonPyrococcusfuriosus,Ravenetal.(1992)usedcon-
zymes have adapted to extreme conditions at the molecular tinuouscultureswithdefinedandminimalmediaandachieved
level. It has been shown that the modifications used by en- amaximalcelldensityof108cellsml–1whenP.furiosuswas
zymesofpsychrophilestopermitconformationalflexibilityat grownonstarch(RavenandSharp1997).Rinkeretal.(1999)
low temperatures are opposite to those that confer thermo- cultivatedthehyperthermophilicheterotrophsMetallosphera
stability to proteins of thermophiles (Russell 2000). Corre- sedula,P.furiosusandThermococcuslitoralisinachemostat
lating the genetic code with specific chemico-physical (Table2)andobservedtheproductionofexopolysaccharides.
characteristicsprovidesapromisingnewapproachtothede- ContinuousculturesofMethanobacteriumthermoautotroph-
velopment of customized industrial biocatalysts. icumwereperformedatdifferentgassingratesusingcontinu-
Becauseofthediversityofresearchrelatedtothedevelop- ous measurements of the growth-limiting factor H, and a
2
ment of biotechnological processes employing archaea, we simple unstructured mathematical model was developed and
havelimitedthisreviewtoadiscussionofnovelachievements verified(Schilletal.1999).Anotherextremelythermophilic
inthreefields:(1)innovativefermentationprocessesforpro- methanogen,Methanococcusjannaschii,wasgrownincontin-
duction of archaeal biomass, enzymes and metabolites; (2) uousculturetoevaluatethecorrelationbetweenhydrogenase
cloning and expressionstrategiesfor archaeal enzymes,asa activityandparameterssuchasgrowthrateandpressure(Tsao
toolfortheirlarge-scaleproduction;and(3)theindustrialpo- etal.1994).Optimizationofthegrowthmediumisalsoofkey
tential of archaea and their enzymes or specific metabolites. importanceintheproductionofextremophilicbiomassesfor
subsequent exploitation in industry.
Particularinteresthasfocusedonthecultivationofanaero-
Innovative fermentation processes for the production of
bicarchaea,becausetheirsensitivitytooxygenisamajorob-
archaeal biomass, enzymes and metabolites
stacle to cultivation and to the development of genetic
Amajorlimitationtotheindustrialapplicationofarchaealen- exchange systems in archaea. Rothe and Thomm (2000) re-
zymesandmetabolitesisthelowproductivityofthefermenta- portedthereplacementofNaSwithNaSO toprotectthecul-
2 2 3
tionprocessesasaresultoflowgrowthratesandlowbiomass ture from oxygen, even outside the anaerobic chamber, thus
yields.Toovercometheselimitations,attentionhasbeenfo- simplifyingthecultivationmethod.Godfroyetal.(2000)ex-
cusedonstudyingthephysiologyofarchaeaofbiotechnologi- ploited a gas-lift bioreactor for the continuous culture of
ARCHAEA VOLUME 1, 2002
BIOTECHNOLOGICAL APPLICATIONS OF ARCHAEA 77
Table2.Bioreactorscalecultivationofarchaea:continuousandhighcelldensityfermentation.Abbreviation:T =optimalgrowthtemperature.
opt
Microorganism T (°C) Cultivation mode Reference
opt
Metallosphera sedula 74 Continuous Rinker et al. 1999
Methanobacterium thermoautotrophicum 65 Continuous growth-limiting factor H Schills et al. 1996
2
Methanococcus jannaschii 85 Medium optimization CSTR Mukhopadhyay et al. 1999
Methanococcus jannaschii 80–85 Continuous Tsao et al. 1994
Pyrococcus abyssiST549 95 Continuous Godfroy et al. 2000
Pyrococcus furiosus 90 Dialysis Krahe et al. 1996
Pyrococcus furiosus 90 Continuous Raven et al. 1992
Pyrococcus furiosus 90 Batch on starch-based medium Raven and Sharp 1997
Pyrococcus furiosus 98 Continuous Rinker et al. 1999
Sulfolobus shibatae 75 Dialysis Krahe et al. 1996
Sulfolobus solfataricusGθ 75 Microfiltration Schiraldi et al. 1999
Sulfolobus solfataricusMT4 87 Batch on whey-based medium Romano et al. 1992
Thermococcusbarosii 82.5 Duffaud et al. 1998
Thermococcus litoralis 85–88 Batch and continuous Rinker and Kelly 2000
Pyrococcus abyssi ST549 on carbohydrates, proteinaceous archaeainsufficientquantitiestopermitphysiologicalstudies
substrates or amino acids and found that the disaccharides andtheisolationofnumerousenzymesofindustrialinterest,it
maltoseandcellobiosedidnotsupportgrowth,whereaspro- hasnot beenpossibleto scale-upthesetechniquesfor large-
teinaceous materials, such as peptone or amino acids in the scaleproduction.Toovercomethislimitation,severalgroups
presence of sulfur, were good substrates. havefocusedonbioreactordesigninanattempttocouplefer-
The use of waste products as substrates for extremophilic mentation with membrane separationprocesses,and thereby
biomassproduction is an attractive option for producing en- overcome problems associated with the toxicity of accumu-
zymes and metabolites for commercial exploitation. Sulfo- lated metabolites. A novel dialysis bioreactor was designed
lobussolfataricusMT4grownat87°Cina100-lfermenteron and developed at the Technical University of Hamburg and
wheysupplieddirectlyfromdairyfarmsyielded80gofdry usedtostudytheinfluenceofparameterssuchasgassingrate,
biomass per mole of lactose (Romano et al. 1992). substrate supplementation and agitation on growth rate of
Researchattentionhasalsofocusedonthedevelopmentof Pyrococcuswoesei(Rüdigeretal.1992).Kraheetal.(1996)
culture techniques that result in high cell densities. To date, used a dialysis process to obtain high cell densities of three
successful approaches for improving biomass yield and en- thermophilic strains, two of which were from the domain
zymeormetaboliteproductionhaveinvolvedeitherfed-batch Archaea.Afinalcelldensity(drybiomass)of110gl–1was
culturesorthedevelopmentofspecialbioreactorsthatallow achievedin350hforSulfolobusshibatae,whereascultivation
easy removal of toxic metabolites and replacement of nutri- of Pyrococcus furiosus yielded a maximal cell density of
ents. Efficient production of Sulfolobus solfataricus DSM 3×1010cellsml–1,correspondingtoa20-foldincreasecom-
1617 was achieved by a fed-batch technique (Park and Lee pared with the batch process.
1997, 1999). In these studies, maximum cell yield (dry bio- We used a novel microfiltration (MF) technique for the
mass)was23gl–1,whenconstantvolumewasmaintainedby high-density cultivation of thermoacidophilic archaea. The
continuousreplacementofevaporatedwater.Thekineticpa- MF bioreactor consisted of a traditional fermentation vessel
rametersandamathematicalmodelofthefermentationpro- modifiednexttothebafflesbytheinsertionoftwoMFmod-
cess were evaluated. The effects of yeast extract and other ulesmadeofpolypropylenecapillaries(seeFigure1).Topro-
complex components on metabolite formation (e.g., ammo- long the growth phase of the microorganisms, the ferment-
nia) and cell yield were also reported. ationprocesswasdividedintothreeconsecutivephases:batch,
A novel corrosion-resistant bioreactor composed of poly- fed-batchandMF.Thebioreactorhasbeenusedforabout100
etheretherketone,techglass(borosilicateglass),siliciumni- runsovermorethanayearwithoutlossoffiltrationcapacity.
trate ceramics and silicon nitrite ceramics was used for the WeobtainedhighyieldsofS.solfataricusGθ,corresponding
cultivationoftworecentlyisolatedextremehalophilicarchaea to a 15-fold increase in biomass (maximal cell dry biomass
thatproducepoly-γ-glutamicacidandpoly-β-hydroxybutyric of38gl–1)comparedwithbatchculture.Thecellsmaintained
acid,respectively(Hezayenetal.2000).Batchfermentations activesynthesisofreporterenzymesofindustrialinterest,in-
onn-butyricacidascarbonsourceyieldedacelldensity(dry cluding α-glucosidase,β-glycosidase and alcohol dehydrog-
biomass) of 2.3 g l–1, with the accumulation of poly-β-hy- enase (Schiraldi et al. 1999, 2001). Although we obtained
droxybutyric acid comprising up to 53% of the dry biomass. improvedbiomassyieldwiththeMFbioreactor,productionof
Although the application of fed-batch techniques coupled theenzymesofinterestwaslowbecauseofthelongdoubling
with media optimization have facilitated in vitro culture of time(t )ofSulfolobus (7<t <18h)andthelowexpression
d d
ARCHAEA ONLINE at http://archaea.ws
78 SCHIRALDI, GIULIANO AND DEROSA
Figure 1. Schematic view of the
microfiltration bioreactor (adapted
from Schiraldi et al. 1999).
levels of these enzymes. The most promising approach to pressionofP2ribonucleasefromS.solfataricusinwhichthe
overcomingtheselimitationsmaybetocombinefermentation codonofthegenewasmodifiedtoincludethosetripletsthat
techniques with molecular biology techniques. arewidelyusedinE.colicellstoenhanceproteinexpression.
Thismodificationwasachievedbyreversetranslationofthe
amino acid sequence into a degenerate nucleotide sequence
Strategies for cloning and expression of archaeal enzymes and synthesis by PCR amplification of overlapping primers.
and their industrial use To overcome the limitations of different codon usage for
heterologousexpressionofarchaealenzymes,Purcareaetal.
Because archaeal enzymes are adapted to extreme environ-
(2001) successfullycloned and expressedin E. coli a carba-
ments,theyareunusuallystable.Theyaretherefore suitable
moyl phosphatase synthetase (CPSase) from Pyrococcus
candidatesforapplicationsinindustrialprocessesthatareper-
abyssi.OverexpressionoftheCPSasegenewasachievedby
formedunderharshconditions,suchashightemperaturesorin
cotransformation of the host cells with a plasmid encoding
thepresenceoforganicsolventsorhighionicstrength.Forthe
samereasons,theyarealsoidealmodelmoleculesforinvesti- tRNAsynthetasesforlow-frequencyE.colicodons.Ishidaet
gations aimed at elucidating the mechanisms of chemico- al.(1997)wereabletoobtainefficientproductionofathermo-
physical stability of proteins. Biologists already use the ex- philic3-isopropylmalatedehydrogenasewithaGC-richcod-
tremophilicenzymeTaqpolymerasefromThermusaquaticus. inggeneinE.coli,byprovidingaleaderopenreadingframe.
Inadditiontotheisolationofnewenzymes,thespectrumof DePascaleetal.(2001)haveclonedandexpressedinE.coli
available enzyme capabilities could be expanded by random (Rb791) genes encoding the trehalosyl dextrin forming en-
mutagenesis and directed evolution. zyme (TDFE) and the trehalose-forming enzyme (TFE) of
Genesencodingseveralenzymesfromextremophileshave S. solfataricus MT4. More recently de Pascale et al. (2002)
beenclonedinmesophilichosts,withtheobjectiveofoverpro- fusedTFEandTDFEcodingsequencestoobtainanewchi-
ducingtheenzymeandalteringitspropertiestosuitcommer- mericproteinthatconvertsdextrinstotrehaloseathightem-
cial applications (see review by Ciaramella et al. 1995).
perature (75 °C)bysequentialenzymaticsteps.Genefusion
Escherichiacoli,Bacillussubtilisandyeastshavebeenused
wascarriedoutsothattheC-terminusofTFE,modifiedbythe
successfully as mesophilic hosts for several archaeal genes
substitutionofthestopcodonwithaGlyresidue,wasinframe
(Cannioetal.1994,Moraccietal.1994,Ito1997,Jorgensenet
withtheN-terminusofTDFE.Alinkerwasdesignedtofacili-
al.1997,Walshetal.1998,Miuraetal.1999,Niehausetal.
tate recombinant DNA construction without deleting amino
1999, Duffner et al. 2000, Lim 2001).
acidsofeitherproteinmoiety,resultingintheinsertionofthe
The expression system has to be chosen based on the in-
four amino acids Ser-Gly-Ser-Gly.
tended use of the expressed protein. To obtain a sufficient
To facilitate biochemical characterization of unusual
amount of a recombinant enzyme for basic characterization,
archaeal enzymes, production can be increased by using
E.coliexpressionisrecommended,becausethishostiseasily
extremophilic microorganisms as hosts for autologous gene
growninthelaboratoryandthevectorsarewellcharacterized.
expression(Gardneretal.1999).ForMethanococcusspecies,
Expressioninyeastshasadvantagesforlarge-scaleindustrial
production.Furthermore,becauseyeastisa“generallyrecog- integrative and shuttle vectors have been developed to over-
nizedassafe”organism,itrepresentsanidealpilotsystemfor expressspecificenzymeswithcomplexprostheticgroupsthat
the production of enzymes for the food technology industry. areinactiveifexpressedinE.coli(Gardneretal.1999).Ithas
Recombinantenzymescanbeobtainedbycloningandex- also been tested as a host for heterologous proteins such as
pressingasyntheticgene.Fusietal.(1995) reportedtheex- β-galactosidase fromE. coli.
ARCHAEA VOLUME 1, 2002
BIOTECHNOLOGICAL APPLICATIONS OF ARCHAEA 79
Thewell-characterizedE.colimodelhasgreatlyfacilitated expressionproductmaintainsitsnaturalpropertiesincluding
thedevelopmentofmoleculargenetictechniques,e.g.,genetic itsthermophilicityandthermostability.(3)Therigidstructure
markers and gene transfer, in halophilic members of the ofthermophilicproteinsatlowtemperatureappearstoprevent
Archaea(Holmes1990,Holmesetal.1991).Thefindingthat their degradation by hostproteases,leadingto anaccumula-
HalomonasmeridianaandH.elongatacansecretethetherm- tion of recombinant enzyme.Recombinantextremophile en-
ostable α-amylase from Bacillus licheniformisindicatesthat zymes are generally inactive under optimal host growth
membersoftheHalomonasgenuscouldbegoodcandidatesas conditions, and the difference in observed biomass between
cell factories for producing heterologous extracellular en- differentrecombinantsmaybeexplainedbyspecificinterfer-
zymes(Coronadoetal.2000,Frillingsetal.2000).Molecular ence in the replication cycle of the host by the heterologous
tools for thermophilic archaea are still being developed be- geneexpressed.(4)Thermostableenzymescaneasilybepuri-
causesomeofthetoolsdevelopedforbacterialsystemsdonot fiedfrommesophilicproteincontaminantsbysequentialther-
function in these archaeal systems (Keeling et al. 1994, mal precipitation steps. After thermal denaturation, the only
Keeling and Doolittle 1995). remainingenzymaticactivityisthatofthethermophilicpro-
Genetic elements such as viruses and plasmids represent teinexpressed,whichgenerallyhasapuritysufficientforin-
powerfultoolswithwhichtoelucidatethemoleculargenetics dustrialapplications(RossiandDeRosa1994,Schiraldietal.
oftheArchaea.Moreover,newexpressionvectorscanbeob- 2001).
tainedbymanipulatingexistingelements.Ofthese,themost Geneticengineeringtechniquesarevaluabletoolsforcreat-
interestingarethegeneticsystemsthatworkinmultiplemi- ingnovelbiocatalyststhatcanimprovebioprocessesandfacil-
croorganisms.Cannioetal.(1998)constructedashuttlevec- itatetherealizationofinnovativebiotransformations.Molecu-
tor,pEXSs,basedonaSulfolobusshibataenaturalvirusand larbiologyhasthepotentialbothtoovercomethelimitations
anE.coliplasmid.Theplasmidisabletotransformandcanbe onenzymeavailabilityandtodesignnewbiocatalystsspecific
stablymaintainedinbothS.solfataricusandE.coli,becauseit toaparticularindustrialpurpose.Wehaveproposedindustrial
contains the SSV1 viral autonomously replicating sequence applicationsforseveralrecombinantarchaealenzymes.New
(ARS)(Palmetal.1991),thepGEM5Zf(-)E.coliplasmidse- industrialusesofnaturalandrecombinantarchaealenzymes
quence and an antibiotic resistance gene marker for S. sol- will depend on the coupling of genetic strategies and bio-
fataricus, obtained by selection of a suitable mutant of the reactor design.
hygromycin phosphotransferase gene derived from E. coli
(Gritz and Davies 1983). The same group also explored the
Industrial potential of archaeal enzymes and metabolites
feasibilityofusingS.solfataricusasahostfor amesophilic
protein,hygromycinBphosphotransferase(hph)fromE.coli. Mostofthearchaealbioproductsandbioprocessesthathave
Thehphproteinwasisolatedbydirectedevolutionat75°C, beendevelopedinindustrysofararebasedontheunconven-
using a library of random mutant versions of the hph gene tional properties of archaeal biomasses or of their enzymes
(Cannioetal.2001).UseofthepEXSsshuttlevectorallowed andmetabolites.Herewedescribeseveralpromisingapplica-
selectionof amutant versionof thehph genethat contained tionsthathavebeendevelopedinthepastdecade,andhigh-
twopointmutations,butremainedcorrectlyfoldedandfunc- light the few applications that have already reached the
tionallikethewild-typeprotein.Moreover,theconstructwas market.
highly stable in more thermophilic strains at temperatures
Biotechnological applications of archaeal biomasses
from75to82°C,andhenceisexpectedtobeagoodcandidate
asageneticmarkeratevenhighertemperaturesfollowingfur- Despite the potential of microorganisms from the domain
ther thermoadaptation (Cannio et al. 2001). Archaea, their industrial application has not been exploited,
Another vector (pKMSD48) containing both SSV1 and exceptinmicrobiologicalwastetreatmentprocesses(Kandler
pBluescript DNA is completely stable in both E. coli and 1986).Biologicalmethanogenesisisappliedtotheanaerobic
S.solfataricus(Stedmanetal.1999).Unlikeothershuttlevec- treatment of sewage sludge, and agricultural, municipal and
tors,pKMSD48isstableinE.colievenathighcopynumbers. industrial wastes, where the maintenance of a desired meth-
Moreover,becauseitspromotercanbecontrolledbyUVirra- anogenicfloraisachievedbyinnoculation.Methanogensarea
diation,itcanbeusedforinvivooverexpressionofgenesin taxonomically and phylogenetically diverse group of micro-
S.solfataricus(Stedmanetal.1999).Althoughtheseemerg- organisms that obtain energy for growth from the reaction
inggeneticengineeringtoolsmayhavenodirectindustrialap- leadingtomethaneproduction.Methanogensuseonlyasmall
plication,theywillplayacentralroleinthedevelopmentof number of compounds, H or one-carbon atom compounds,
2
methods for the large-scale production of archaeal enzymes andthisspecializationmakesthemdependentonotherorgan-
with potential applications in industry. ismsforasupplyofsubstratesinmostanaerobicenvironments
The method chosen to express an extremophilic enzyme (Boone et al. 1993).
needstotakeintoaccountthedownstreamprocessesusedfor Manybioreactorconfigurationshavebeenexploitedtoin-
enzyme purification. The major findings of studies on the creasetheefficiencyofanaerobicdigestion,suchastherotat-
cloning and expression of archaeal enzymes can be summa- ingbiologicalcontactor,theanaerobicbafflereactorandthe
rizedasfollows.(1)Archaeautilizetheuniversalgeneticcode upflow anaerobic sludge blanket reactor, and several large-
andtheirgenesaretranslatedbyEukaryaandBacteria.(2)The scaleplantsareinoperation(RehmandReed1999).Recently,
ARCHAEA ONLINE at http://archaea.ws
80 SCHIRALDI, GIULIANO AND DEROSA
amethanogenicprocesshasbeenestablishedonalab-scalefor to the knowledge acquired from molecular sequences and
the anaerobic digestion of the organic fraction of municipal crystallographicstudies.Suchknowledgewillpermittherede-
gray-wastebasedonamixedarchaealpopulation;thereactors signofconventionalenzymesforuseasbiocatalystsinharsh
havebeenfeddailyandheldinsteadystateforover2years industrial conditions. Widespread use of DNA polymerases
(Scherer etal.2000).Theseexamplesillustratethepotential fromhyperthermophilicarchaeamayportendfuturecommon
forcouplingwastetreatmentprocessestobiogasproduction, useofextremophileenzymesinbiotechnology,althoughnone
thereby recovering energy from an unavoidable process. of the novel enzymes from these microorganisms has yet
Halophilic archaea have also been evaluated for bio- achievedsignificantindustrialapplicationbecauseofdifficul-
remediationinharshenvironments,forthedegradationofor- tiesencounteredintheirlarge-scaleproduction.Effortstore-
ganic pollutants and in the treatment of concentrated textile solve or ameliorate these difficulties are likely in the near
wastewaters,particularlythefirstdyebathliquorfromthedy- future (Cowan et al. 1992).
ing process, which contains a high salt load (Margesin and Manyarchaealenzymesinvolvedincarbohydratemetabo-
Schinner 2001). Biosurfactant-producing halophilic bacteria lism,particularlythosefromtheglycosylhydrolasefamily,are
can play a significant role in the accelerated remediation of ofspecialinteresttotheindustrialbiotechnologysector.The
oil-pollutedsalineenvironments.Banatetal.(2000)obtained starch processing industry, which converts starch into more
encouragingresultsforhydrocarbonpollutioncontrolatma- valuable products such as dextrins, glucose, fructose and
rinesitesthatrepresentself-sufficientsystems.Lovley(2001) trehalose,canprofitfromtheexploitationofthermostableen-
examined the degradation of hydrocarbons by archaeal mi- zymes.Inallstarchprocessingconversions,hightemperatures
crobesunderanoxicconditions.Sulfate-reducingmicroorgan- arerequiredtoliquefystarchandmakeitaccessibletoenzy-
ismsbelongingtothedomainArchaeahavebeenisolatedfor matic attack. Enzymes from mesophilic organisms are cur-
theircapacitytodegradealkanesanaerobically.Inaquaticsed- rentlyemployedintheseprocesses,butthisislikelytochange
iments, the anaerobic oxidation of methane with sulfate as inthenearfuturewhentheadvantagesoftheintegratedutiliza-
electronacceptorhasbeenreportedandisassumedtoinvolve tion of amylases, pullulanases and α-glucosidases recently
thereversalofmethanogenesiscatalyzedbyarchaeawiththe isolatedfromhyperthermophilicsourcesareharnessed(Sunna
scavenging of an electron-carrying metabolite by sulfate-re- etal.1997,DiLerniaetal.1998,Miuraetal.1999,Levequeet
ducing bacteria (Sporman and Widdel 2000). al.2000,BertoldoandAntranikian2001,Martinoetal.2001,
Becausetheapplicationofacidophilesinbioreactormineral Savchenko et al. 2001). Similarly, an α-amylase produced
processinghasbeenreviewedrecentlybyNorrisetal.(2000), extracellularly by the haloalkaliphilic archaeon Natronococ-
itwillbementionedonlybrieflyhere.Severalindustrialappli- cus sp. strain Ah-36 awaits industrial exploitation. The en-
cationsoftheCrenarcheotaarerelatedtotheiruniquesulfur zyme exhibits maximal activity at 55 °C, pH 8.7 and 2.5 M
metabolism. A variety of mineral sulfide-oxidizing thermo- NaCl and hydrolyzes soluble starch, amylose, amylopectin
philicarchaeahavebeenisolatedfrombothnaturalandindus- and also glycogen, producing mainly maltotriose, with glu-
trialenvironments.Thesemicroorganismshavegainedatten- cose and maltose as side products in negligible amounts
tionbecauseoftheirpotentialintheextractionofmetalsfrom (Kobayashi et al. 1994). Most of the known archaeal pullu-
sulfideoresthatarerecalcitranttodissolution(Norris1990). lanasesareTypeIIenzymes:theyattacktheα-1,6glycosidic
Comparisonofchalcopyriteoxidationbymesophiles,thermo- linkageinpullulan,producingmaltotrioseor,byα-1,4glyco-
philesandthermoacidophilicarchaeashowedthatextraction sidiclinkage,panoseandisopanose,buttheyareinhibitedby
efficiency increased proportionally with increasing tempera- cyclodextrins. However, a Type II archaeal pullulanase with
tureupto80°C(NorrisandOwen1993).Sulfolobusmetal- fairly broad substrate specificity was recently detected in
licushasbeenexploitedformineralbiomining:inapilot-scale crudecellextractsofDesulfurococcusmucosusandwassuc-
bioreactor, it tolerated copper concentrations of 40 g l–1 cessfullyclonedinBacillussubtilis(Duffneretal.2000).The
(Norrisetal.2000).Thesubdivisionofthermoacidophilesinto discoveryofthisthermostabledebranchingenzyme(maximal
major phylogenetic groups is noteworthy, because it reflects activityat85°C)coulddeliverenormousbenefittothestarch
thevariedpotentialofstrainsforefficientmineralsulfideoxi- bioconversion process when applied in the saccharification
dation.Alsorelatedtotheuniquecharacteristicsofthesulfur step.
metabolismofarchaeaistheapplicationofP.furiosusinthe Anotherpromisingapplicationofhyperthermophilicarch-
recyclingoftirerubberbydesulfurizationandsubsequentvul- aealenzymesisintrehaloseproduction.Trehalose,anon-re-
canizationreportedbyBredbergetal.(2001).Ethanol-leached ducing α-1,1-diglucosyl disaccharide, has been shown to
cryo-ground tire rubber treated with P. furiosus for 10 days stabilize enzymes, antibodies, vaccines and hormones, and
wasvulcanizedtogetherwithvirginrubber,leadingtoaprod- alsohasawidevarietyofapplicationsinfoodpreparationsasa
uct with good mechanical properties. preservative as well as a moisture retainer (Arguelles 2000,
Guoetal.2000,Simolaetal.2000).Currently,trehalosepro-
Archaeal enzymes of biotechnological interest
ductionisperformedat45°Cbythesequentialactionoftwo
Inadditiontothepotentialusesofarchaealbiomasses,natural enzymes from the mesophilic bacterium Arthrobacter sp.
andmodifiedarchaealenzymesalsopresenthugepossibilities Q36, namely the trehalosyl dextrin forming enzyme, which
forindustrialapplications.Thebiotechnologicalvalueofthese convertsadextrintothecorrespondingtrehalosyldextrin,and
enzymesisrelatednotonlytotheirdirectapplications,butalso the trehalose-forming enzyme, which further transforms the
ARCHAEA VOLUME 1, 2002
BIOTECHNOLOGICAL APPLICATIONS OF ARCHAEA 81
trehalosyldextrin to trehalose and a low molecular weight ferent xylans but showed no activity toward carbohydrates
dextrin.Thealternateactionoftheseenzymespermitsthetotal (UhlandDaniel1999).Adetailedreviewofxylanases,includ-
conversion of dextrins to trehalose. The use of thermophilic ing those isolated from hyperthermophiles, is presented by
enzymesinthisprocesswouldrepresentanimprovementby Bergquist et al. (2001). Among the few cellulase activities
eliminating problems associated with viscosity and sterility. foundinthedomainArchaea,themostinterestingincludethe
Thetrehalosebiosyntheticpathwaywasfoundinthearchaeal cellulaseCelSfromS.solfataricus(Limauroetal.2001)anda
enzymaticpatrimonyofSulfolobales(Kobayashietal.1996, β-endoglucanase fromP. furiosus(Cady et al. 2001).
Di Lernia et al. 1998). These enzymes were cloned and ex- Hyperthermophilic proteases are important as degrading
pressed with high yield in E. coli Rb-791 (de Pascale et al. agents in detergent formulations (Schumacher et al. 2001).
2001),andtherecombinantstrainsweregrownathighdensity Proteasesandproteasomesplayakeyroleinthecellularme-
inamicrofiltrationbioreactor(Schiraldietal.2001).Totest tabolismofarchaea.Theadaptationofthesemicroorganisms
whetherthesebiocatalystscouldbeusedinanindustrialset- toextremehabitatsismediatedbyaninterplaybetweenpro-
ting, we assembled a plug flow reactor containing recombi- teinfoldingandhydrolysis(Iidaetal.2000,Maupin-Furlowet
nant E. coli cells expressing the necessary enzymatic al.2000,deVosetal.2001,Wadsworthetal.2001). Lipases
activities.Trehalosewassuccessfullyproducedfromdextrins and esterases from Archaeoglobus fulgidus (Manco et al.
at 75 °C in a continuous system, with a final conversion of 2000)andSulfolobusshibatae(Huddlestonetal.1995)have
90% (Di Lernia et al. 2001). beenpurifiedandcharacterized,andarealsoofinterestinde-
Anotherrecentlyisolatedenzyme,cyclomaltodextringluc- tergent formulations and the dairy industry. Hydrolases and
anotransferase, from the newly isolated hyperthermophilic isomerasesfromextremelyhalophilicarchaeahavepotential
archaeon Thermococcus sp. (Tachibana et al. 1999), shows applicationsinseveralbiotransformationsintheproductionof
optimumstarchdegradingactivityandcyclodextrinsynthesis foodsupplements;themicroorganismsthemselvescanbeex-
attemperaturesabove90°C.Thus,itcouldenhancethestarch ploited in the production of fermented food (Margesin and
bioconversionprocessbyloweringtheviscosityandreducing Schinner 2001).
contamination. An alkaline cyclomaltodextrin glucanotrans-
ferase is already used in the industrial production of cyclo- Archaeal metabolites for biotechnology
dextrins.Thisenzymehasreducedproductioncostsandpaved Somearchaealmetabolites,suchasproteins,osmoticallyac-
thewayforcyclodextrinuseinlargequantitiesinfoodstuffs, tive substances (so-called compatible solutes), exopolysac-
chemicals and pharmaceuticals (Horikoshi 1999). charides and special lipids have potential industrial applica-
Degradation of cellulose to its monomer, glucose, can be tions. Halophilic archaea offer a multitude of actual or
achieved through the synergistic action of three enzymes: potential biotechnological applications. Certain strains con-
endoglucanases,exoglucanasesandβ-glucosidases.Thelatter tain membrane-bound retinal pigments, bacteriorhodopsin
twoenzymeshavebeenisolated,purifiedandclonedfromsev- (BR)andhalorhodopsin,whichenablemicroorganismstouse
eralSulfolobalesspecies(Cubellisetal.1990,Grogan1991, lightenergytodrivebioenergeticprocesses(Oren1994,Lanyi
Moraccietal.1992,Nuccietal.1993,Moraccietal.1995)as 1995).ThethermodynamicandphotochemicalstabilityofBR
well as from Pyrococcus furiosus (celB) (Voorhorst et al. hasledtomanyusesintechnicalapplications,suchasinho-
1995, Lebbnik et al. 2001), and Pouwels et al. (2000) com- lography,spatiallightmodulators,artificialretina,volumetric
pared their activity and stability. An ultra high temperature andassociativeopticalmemories.Bacteriorhodopsin,isolated
processfortheenzymatichydrolysisoflactosefortheproduc- from Halobacterium salinarum, is currently being commer-
tionofnoveloligosaccharideswasdevelopedbyPetzelbauer cialized by COBEL (Barcelona, Spain) and Munich Innova-
et al. (2000) using β-glycosidases from S. solfataricus and tiveBiomaterials(Munich,Germany)(MargesinandShinner
P. furiosus. D’Auria et al. (1996) developed a continuous 2001). Furthermore, BR can be exploited for the renewal of
cellobiose hydrolysis bioreactor system for glucose produc- biochemical energy such as the back conversion of ADP to
tionbyimmobilizingaβ-glycosidaseisolatedfromS.solfat- ATP.AdevicebasedonBRandATPsynthasehasbeendevel-
aricus and expressed in Saccharomyces cerevisiae. The oped and patented (Saito et al. 1992).
systemcanberunatahighflowrateandhasahighdegreeof Halophilic bacteria produce compatible solutesthat main-
conversion,productivityandoperationalstability(D’Auriaet tain a positive water balance in the cell and are compatible
al. 1996). withthecellularmetabolism.Thesecompatiblesolutesareex-
Several other polymer-degrading enzymes isolated from cellent stabilizers for biomolecules (da Costa et al. 1998,
archaea,suchasxylanasesandcellulases,couldplayimpor- Welsh2000,SantosanddaCosta2001).Ectoineandderiva-
tantrolesinthechemical,pharmaceutical,paper,pulporwaste tiveshavebeenpatentedasmoisturizersincosmetics(Mon-
treatment industries.Commercial interestin the applications titscheetal.2000),althoughthemostpromisingapplication
ofxylanases,whichdegradexylans,themostabundantform maybeasstabilizersinthepolymerasechainreaction(Sauer
ofhemicellulose,isgrowing.Xylanasescanplayakeyroleas and Galinski 1998). Compatible solutes produced by halo-
quality-oryield-improvingagentsinthefood,feed,andpulp philic archaea include mannosylglycerate (MG) and di-
andpaperindustries(Viikarietal.1994).Recently,axylanase glycerol phosphate, and they have also beeen identified in
thatisactiveattemperaturesupto100°Cwascharacterizedin P.furiosus,P.woeseiandArchaeoglobusfulgidus.Thestabi-
ThermococcuszilligiistrainAN1.Thisenzymecanattackdif- lizingeffectofMGonseveralenzymessubjectedtoheatingor
ARCHAEA ONLINE at http://archaea.ws
82 SCHIRALDI, GIULIANO AND DEROSA
freeze-dryingwassuperiortothatofotherstabilizers(Lamosa costsavings,therebyenablingindustrialapplicationsofthese
et al. 2000). unique biomaterials.
Archaeallipidsareuniqueinrelationtotheirtopologyand
function in archaeal membranes and represent an important
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