- Email: [email protected]
Biotechnology of the Archaea
14 KilhlPr, K.. Ljungqrist, C., Kondo, A., Vride, A. wd Ndrwn, H. (I 991) Rio/‘fcr~umlqpir9. 642-h 15 Morimoto, K.. Tirsirrcs, A. and Ccorgopoulor, C. (1991)) Smw Pmfcim irr&oio.~ydadJMrdiiirc, Cold Spring Harbor 16 Coff, S. A., Voclhny. R. and Goldberg, A. L. (1988) in L%iqrririu(Rcchsteiner. M., cd.), pp. 207-238, Ylenus~ Prm 17 ;pencc, J., Cegielska, A. and Grorgopoulos, C. (1990) j. Borferiof.172.7157-7166 18 Blom, A., Harder. W. and Matan, A. (I YY2).4~yl. Emiron. Mimbid. 58.331-334
19 Goti, S. A. and Goldberg. A. L. (1985) Ccl/ 41.587-593 20 Shcnnm, M. Y. 2nd Goldberg, A. L. (1992) EAfBOj. 1 I. 71-77 21 Goldberg, A. L. and SCJohn, A. C. (1976) .+m. Rw. Biorlrm. 45, 747-m 22 Kitam, K. Fujimoro. S., Nakao. M., Watanabe. T. and Nakao. Y. (I 987)J. B,orcc/rnol.5, 77-86
Biotechnology of the Archaea Don A. Cowan The Archaea, designated sjnce 1979 as a separate Super-Kingdom (the highest taxonomic order), are a highly novel group of microorganisms which look much like bacteria but have many molecular and genetic characteristics that are more typical of eukaryotes. These unusual organisms can be conveniently divided according to their ‘extreme’ environmental niche, into three broad phenotypes: the thermophiles, methanogens and extreme halophiles. Each group has unique biochemicai features which can be exploited for use in the biotechnological industries. The extreme molecular stability of thermophije enzymes, the novel C, pathways of the methanogens and the synthesis of organic polymers by some halophiles are all currently or potentially valuable examples of the biotechnology of the Archaea.
Before the late lWOs, the Archaea were not recognized as a discrete and identifiable grouping. Howhalophilic and mcthanogenic 65%X, numerous ‘bacteria’ which showed some uncharacteristic biochemical properties had been isolated and studied over the preceding half century. The grouping ofthese organisms, together with certain thermophiles, into a defined and cohcrcnt taxonomic entity, merely awaited the development of the molecular techniques appropriate for the determination of fundamental phylogeny. Two of the three phenotypes which comprise the Archaea are well known, and the biology and applications of these organisms - the methanogens and the
Mophilcs - have been gradually established over many decades. The USCof ana-robic mcthanogcnic culture for the production of methane (‘biogas’) is an established technolo&y, while the appcarauce of halophilic bacteria in association tvvith salted food products can bc tither deleterious or advantageous. The biotcchnoloby &the third archaeal phenotype the thrrmophiles - is less well developed, reflecting the more-recent discovery ofthis group. However. in each instant-. the biotechnological applications ofthe Archaea, either real or anticipated, stem Tom the unusual metabolism ofthese organisms. This review cannot possibly cc’:cr the bioloa ofthe Archaea in more than a cursory manner. For more detail, the reader is directed to a number ofcscel!enr and comp~hensive reviews on this rapidly espandiiig field’-“. _.__--__-._
8 1992. ElsewrScience PublishersLtd (UK)
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fiBTECHSEPTEMBER I992 &‘OL1Ct
a
ARCtf.4KIBACTERlA
prokaryotes and the eukaryotes) were restructured into three discrete groups, named by the :aththors as the Eukaryotes, the Eubacteria and the Archaebactcria (Fig. la)“. A modified system ofnomenclature which emphasizes the fundamental evolutionary ditTerences between the bacteria (i.e. the Eubacteria) and the Archaebacteria was recently presented by Wocsc’s group (Fig. 3b)“. The three groups (which are to be termed Domains) thus become the Bacteria, the Eucarya and the Archaea. Further divisions within the archaeal domain reflect phenotypic and genotypic differences, roughly comprising the hyperthermophilic Archaea, the methanogenic Archaea and the extremely halophilic Archaea (Fig. 1b). For rhe purposes of this article, it is more convenient to discuss the biotechnology of the Archaca in tcmls of these phenotypic groupings rather than the phylogenetic units. Thus the thrrmophiies (encompassing thermophilic, extremely thcrmophillc and hypcrthcrmophilic representatives; see Table I for an explanation of these terms) include the crenotes (see Glossary) and certain thermophilic members of the euryotes. The h;lophihc Archaca and the methanogens (both thmnophilic and non-thermophiiicj are discussed separately for convenience.
I
Hypx. thennophilcs
Therimcoccale~ Mahanccoccalcs
Me!hanogens
Mcthanomicrabiales I Extreme holophiles
1 Halophiles
1 ElJCAKYA ]
Figure1 (aa]The universal phylogenetic tree. After Ref. 11. (b) The tkec domains of life. This is a ‘rooted’ tree, indicating the relative position of the progenote or ancestral cell type. The three archaeal phenoepes are indicated. Redrawn, with permission, from ‘Ref. 12.
Archaed pk+geny In a paper published in ‘977l”, Carl Worse and Gecrgc Fox reported the use of 16S- and f 8S-rRNA sequence data in realigning the fundamental phylogeny oflife. On the basis ofscquence similarities and diffcrenccs, the two traditional primary kingdoms (the
Table 1. Classificationof organismsby growthtemperatures Temperature i”Cj
~Term
Minimum’ Klh1
Optimumb U&t1
ik%ximum’ K,,J
Thermophile
‘30
‘50
‘60
Extreme thermophile Hyperthermophile
‘40 ‘70
‘65 ‘90
‘70 Cl15
‘Minimum temperature at which growth will occur. “Temperature required for shortest doubling time. ‘Maximum temperature at which growth will occur. -_.--__l,.-_---.__ TiBTECtl SEPTEM3ER 1992 NOI. 10)
The thermophilie Archaea Diversity The thermophilic representatives of the Archaea comprise some 19 genera but fewer than 40 species, some of the better kndlvn of which are listed in Table 2. This is a diverse collection of organisms with a wide variation of morphological, physiological and biochemical charactcriscics. -However, ail possess the ability to survive and Q~OWat high temperatures (i.e. the characteristic of thcrmophily).
Thermophilic habitats (or biotopes) in the tempcrature range 40”-70°C are extremely coinmcm (examples include solar-heated soil, decaying vegetation, industrial waste heat, self-heating coai tips and arc tailings). However, biotopes with temperatures ofgreater than 70°C, where most thermophilic Archaca are found, arc relatively rare. The most common ‘hightsmpcraturc’ thermal habitats arc of geothermal origin, usually associated with tectonically active zones. These are widely, though sparsely, distributed over the earth’s surface. The alkaline thermal spring diffca from the marine equivalent only in that the water erupting in the laitcr is saline, and that tompzracures of >lilO°C can be reached because ofthe added hydrostatic pressure. A special version of the marine biotopc is found ac great depths in ocean rifts. Because of the reactivity oi super-heated saline water, the waters issuing from these deep-sea hydrothcmlal vents arc very highly mineralized, and the precipitation of insoluble metal salts from the issuing vents has given rise to the term ‘black smokers’. Temperatures in the region of
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reviews
1
Table 2. Diversity of thermophilic Archaea
PH
---I Energy metabolism
65
2.5
Heterotrophic
Anaerobic
82
6.5
Heterotrophic
Jhermococcus ceder
Anaerobic
82
6.5
Heterotrophic
Pyrococcus furiosusa
Anaerobic
98
6.5
Heterotrophic
Pyrodictium brockii
Anaerobic
105
6.5
Autotrophic
Jhermoplasma aridophilum
Anaerobic
55
6.5
Heterotrophic
Hyperthermus bl.fylicus
Anaerobic
86
6.5
Fermentative
Methanothermus fervidus
Anaerobic
95
6.5
Methanogenic
Methanococcus jannaschii
Anaerobic
95
6.5
Methanogenic
Methanopyrus kandferi
Anaerobic
100
6.5
Methanogenic
Organism
Culture conditions
Sulfolobus acidocaldarius
Aerobic
Desuffurococcus mobilis
“Since the isolation of I? furiosus:3, rhis organismhas become the ‘E. coli’ of the thermophilic Archaea.
35O-400°C have been recorded in the vent waters although microbial populations are apparently restricted to cooler regions ofless than 1XPC. Etxymes$om thermophilic Archaea In general, any enzyme isolated fr>rn an extreme thermophile can be assumed to be more thermostablc than the homologous enzyme isolated from a lessthermophilic source. This trend is clear from a com-
parison of proteo!ptic enzyme5 from various sources (Table 3)““; many comparisons ofthis type with different enzymes are no;v published. One apparent conscqtience of protein themlostability is the observed high degree of resistance to other potential denaturants: detergents. chaotropic agents such as urea and ~~anic!L;.ium hydrochloride, organic solvents and oxidizing agents. There is evidcncc that thcrmostability also klparts resistance to cnzymic
Table 3. The thermostability of proteases from mesophiies, thermophiies and hyperthermophilesa Half-lie (time at given temperature)
Topt Protease
Ref.
Organism
1°C)
El.subtifis
+27
Subtitisin
ii.a.
i(: iiiin Q
60%
14
B. stearothermopf%s
55
Alkaline protease
n.a.
15 min 8
87°C
14
B. thefmoproteofyticus
60
Thermoiysin
86
60 mind
80°C
14
J. aquaticus
72
Caldoiysin
92
‘30 h @ 15min@
80°C 95%
T. ce!er
88
Protease
n.a.
40 min @
95%
16
D. mucosus
85
Archaelysin
9%
80 min @I 95%
17
S. solfataricus
87
Hetitral protease
n.a.
40 min Q
95%
16
P. furiosus
94
66 kDa prctease
n.a.
98%
18
Pyroiysiwcomp!ex .-
‘115
3 7 min @ 105%
19
‘33 h
@
aAbbreviatioos:Top,,temperature at which cells doubie in the shortest time; TM, meking ‘%zperi;ure (at which denaturation occursi: n.a.. not available.
------TIBTECII
--WTEMBER
1992 WOL10)
318 reviews
i cleavage (proteolysis) and to chemical reactions causing irreversible protein degradation at high tcmperaturesZQ_ The resistance of thermostable proteins to denaturation by organic solvents appears to bc a gcnera1 characteristic”. A common misconception is that thermophiiicity allows very rapid reaction rates to be achieved by carrying out reactions at high temperatures. This misconception is based on incorrectly extrapolating Arrhenius’ law* from the properties of mesophilic enzymes, presuming that
’ the reaction rate of a hyprrthennophilic enzyme should be about X-fold higher at 90°C than char of the equivalent mesophilic enzyme at 37%’ WC now know this to be incorrect in almost all instances and, for sound physical reasons, thermophilic enzymes at their ‘optimum’ temperatures (see Table 1; footnote b) exhibit turnover rates of the same order of magnitude as do mesophilic enzymes at their optimum temperatures. The mechanism of this apparent limitation ofactivity is, at least in part, the restraint in conformational flexibility which is thought to be coin&dent with enhanced thcrmostability~~. Evidcncc supporting this r&cionship is stiil sketchy and there is great potential for dctaifed structure/function/ stability studies. It should not be assumed, for example, that the conformational restraints will be the same in all regions of the folded protein structure, In some instances, however, very large increases in reaction rates can be achieved by using thcrmophilic enzymes at high temprraturcs; for example where, at higher temperatures, the substrate is more susceptible to catalysis. The best examples are the hydrolytic degradations of polymeric substrates, particularly protcolytic degradation of protein and amylolytic dcgradation of starch. Industry has taken full advantage of the latter, particularly in the high-temperature saccharitication proccsscs currently employed in most enzymic starch hydrolysis plant:. A problem that is generally associated with the largescale USCof enzymes from thermophilic Archaea is that of low-biomass yields. Typically, archaeal fermentations yield 0.1-l .0 g wet weight of biomass per litre (-lV-lo” cells per millihtre) compared with the 5-30 g wet weight of biomass per litre yield for common industriai bacterial strains. While this is a major impediment to commercial application of thrrmophilic Archaea (whether as whole-ccl1 biocatalysts, as sources ofcnzymes or as sources of other biomolcculcs), recent developments in fermentation stratcges have led to cell densities approaching those of more “classical’ organiims (up to 5 x IW cells per mi!fiIitrc; K. Sharp, pcrs. commun.),
An alternative option, wl:ose feasibility is demon;tratcd by the growing number of examples reported in the literature, is the cloning and over-expressior, of the genes that encode the hyperthcnnophilic proteins.
,_l___i--.__
TIETECH SEPTEMBER 1992 IVOL
Applications
of enzymes
from
thermophik Archaea Current applications
The commercial importance of cnzymcs from thcrmophilic Archaca is illustrated by a single application: the polymerase chain reaction (PCR), a rapid and efiicient exponential m!?thod of amplitjring specific DNA sequences. The automation of PCR involved replacing the Klenow fragment of DNA polymcrase with the thcrmostablr Tag polymcrase’” from the thcnnophilic eubacteriuti Thenmu aqtmtiws - a major step forward in the technology. Howcvcr. the thermostability of Taq polymerase is only just sufficient, and a further recent development is the use of extremely thcrmostable DNA polymerascs from two diffcrcnt hypcrthcnnophilic Archara (Y?II?IECIIOCOCCM /itt~mii and Pyryrorocc~ firriosm). In addition to cnzymc thermostability: su&cssful application of DNA polymerases in PCR needs processivity and proof-reading (3’5’ cxonuclcase) activity. The proof-reading capacity of the P. firrimrrs DNA polymcrase is reported to r&cc the error frequency by a t&tor of 14, compared with Taq polymcrase?J. Future applications To date, the number of enzymes from thermophilic Archaebacteria which have been studied in any detail barely exceeds the total number of named species. these organisms probably contain as Neverthe&,, wide a range ofdifferent activitic s as any other diverse group of microorganisms. In addition, certain activities and enzyme systems will be unique to this group of organisms, an excellent example being the undoubtedly novel, but as yet uncharactcrizcd, enzymes ofthc biphytanyl tctrarthcr lipid bio:ynthetic pathways’“, The success of thernrophilic enzymes in PCR may give us a clue to the future role of the Archaea in biotechnology. This is an excellent example of a ‘niche’ application, where the specilic properties ofthe enzyme are complementary to the highly specific requircmcnts of a process.
In considering future commercial and industrial applications of thcrmophilic enzymes, one inevitably turns to the current: areas of industrial enzymology. Over 75% of the world usage of enzymes (estimated value at over UKL900 million in 1990)z’Xis 1~ the detergent, starch and dairy industries. Although, in many instances, a thermostable alternative could replace the etizymc in current use, doing so may not be commercially viable. For example: (1) some industrial biocatalytic processes are incompatible with hightemperature operation (e.g. cheese-making); (2) commercial trends favom low-temperature operation (e.g. domestic detergents); (3) cnzymcs of sufficient thennostability are already in USC(e.g. in the saccharification of starch); (4) there is no obvious process advantage in increasing the reaction tcmpcraturc (e.g. biosynthetic production of fine chemicals such as
319 reviews
amino acids and penicillins); and (5) many ofthe major industrial biocatalyst users are committed to existing low-temperature operation through investment in specific yla?t 2nd equipment, and the advantages of higher-temperature operation would be insufficient to offset the costs of replacing that equipment. In summary, the conditions under which a thermophilic enzyme would be a serious contender as a replacement for any ‘bulk’ commercial enzyme might include many (ifnot all) of the following: (1) it must be available in similar (or equivalent) quantity with equally reliable supply; (2) it must have a similar or lower price (per unit of activity); (3) it must exhibit significantly better performance in many respects (not just stability); and (4) there must be no major altcrations to existing plant and equipment (or investment in new plant) required. This assessment indicates that the commercial &ture of thermophilic archaeal enzymes may not be in replacing existing ‘industrial’ enzymes, particulady ir, the high-volume, low-cost enzyme market, but rather in ‘niche’ applications, where the unique properties that these enzymes possess can offer significant process advantages. Although specialized, the commercial success of PCR indicates that such applications could be highly profitable.
Such an example is the development’7 of a ligascdependent DNA-amplification tecE:+*e. The ligase amplification reaction (LAP.) (Fig. 2) has potential applications in the identification of gene defects, ar.;l has already been used on a laboratory scale for the detection of i%globin mutantsZn. The requirements for a thermophilic and thermostable DNA ligase m LAR are obvious - the ligation must be carried out near the DNA melting temperature (T,J (a minimum of 6YC), and the enzyme must be stable during the subsequent dissociation step (carried out at -92°C). To date, the only commercially available thermostable DNA-ligase is a recombinant derived from the thermophi!ic Eubacterium 77zen1nrsrhen~~opkihrs,and expressed in E. coli. The appearance of a cloned hyperthermophilic DNA ligase is surely only a matter of time.
The secondary alcohol-specific alcohol dehydrogenases, which catalyse the oxidation of secondary alcohols and, less readily, the reverse reaction (the reduction of ketones), have a promising hture in biotechnology. There is considerable interest in the USCof such enzymes in the chemical-synthesis indwties’9,30, particularly the pharmaceutical industries where the biological production of chiral synthons is gaining in importance in the chemicaf synthesis of chirally pure pharmaceutical agents. Although alcohol dehydrogenases show a wide distribution and are well characterized, only two examples of thermostable alcohol dehydrogenases derived Tom hyperthermophilic Archaea are currently known (Table 4). The
Complementary target(T)
J.llli~l A
IIrllLL B
Mismatched target (T*)
__
-_ A
Figure 2 Schematic representationof the ligase amplification reaction UR) (after Ref. 27). TheDNAtemplateis heatdenatured and four complementary oligonuciaotide primers are hybridized to the target at near the mefbng temperature (TM) (Step 11. Thermostable DNA-fig&e is then added, and ligation between adjacent primers proceeds (Step 2); the success of the technique relies on the fact that. at the high tempera::lre, only adjacent and perfectly complementary oligonucleotides will be ligated together. The ligation products are heatdenahired and form new target templates ior the next cycle (Step 31, resulting in an exponential ampliiation. Oligonucleotides with a single base mismatch, however, will not ligate and will amplify in linear and not exponential fashion.
reduction by SrrlfnloBtrs alcohol dchydrogenascJ1 of aromatic ketones to their respective chiral alcohols is a particularly valuable activity. In the reactions catalyscd by alcohol dehydrogenases, both the substrate and product may bc hydrophilic organic molecules (such as alcohols, aidehydes and ketones), and such organic ‘solvents’ are oflen potent denaturants of enzymes. In addition, the high cost of NADH and NADPH ensures that, for all but laboratory-scale operations, a system !or recycling the oxidized cofactor is essmtial. One etfectivc recycling option is to include an alcohol co-substrate which, in being oxidized by the enzyme, regenerates the reduced cofactor, However, this results in an even higher concentration ofpotentially denamring vrganic components. These hctors, therefore. Gocr the use of enzymes which are stable in orgamc solvents. In the biocatalytic reduction of a variety of pure chiral alcohols, a moderately thcrmostablc alcohol dehyd:ogenase isolated Tom a eubactcdal tbermophile ~~entroar~aerohi~rr~~ br&ii3” has been u,;ed. However. TIBTECH SEPTEMBER1992 iVOL 101
320
Table 4. Specificitwa d secondary-alcoholdehydrogenasesfrom hyperthermophilicArchaea Relative a&vii
Topt Organism
PC)
Sulfolobus solfataricus
87
Hyperthermus butykus
96
Cofactor
Ethanol
NADP
Butan-Z-al
(“/I”
Cyclohexanol
Benzyl alcohol
Ref.
100
188
410
x9
31
100
76
174
0
b
“The enzyme’s activity in ethanol is taken as 1011%. 'L.Galabeand D. A. Cowan.tinpublished.
reaction tempcraturcs greater than 45°C and organic solvent concentrations ofmore than about 1 O”/n induce rapid enzyme inactivation (A. Plant and D. A. Cowan, unpubiishcd). ADH-cataiysed reduction trizla with S@M~rrs cells”~~suggest that the hyprrthennophilic enzymes may be excellent aiternativcs. Reports of laboratory studies using thcrmophilic alcohol dehydrogenascs for biotramfortnation purposes will undoubtedly incrcasc. and the availability of thcrmostable archacal enzymes in com:nercial quantitics would provide an additional stimulus to such dcveiopments. 0 Bterases Esterases are enzymes that also show a wide distribution, and mesophiiic examples have been used in organic biosynthose9 3o. In aqueous solution, estcrases cataiyse the hydrolytic cleavage of esters to fonn the constituent acid and alcohol. However, in reaction systems where the aqueous component of the solvent is reduced sufficiently to reduce the thermodynamic water activity, non-hydrolytic reactions are pro!noted. Transesterification, not favour-d in aqueous solution because of the prcvalencc of the hydrolytic reaction, can bc greatly enhanced in organic solvents. Both the reactants and products oftranscsterification are usually highly soluble in an organic phase, and the reactant5 may cvcn form the organic phase themselves. Clearly, it is essential that the biocatalyst is stable under the solvent conditions used.
Taking advantage of the intrinsic thcrmostabiiity of archacal enzymes and the enhanced stability of proteins in low-water cnvironincnh, S+lobtfs su!fnlaricus alcohol dehydrogcnase has been used to catalyst ‘gas(Ref. phase’ reactions at tempcraturcs around 1OOT 34). While only in the early stages of development, this ingenious tcchnolo&J has the advantage ofmicimizing the use ofsolvents (a popular objective in these cncrgy- and waste-conscious times) and of reducing substrate and product-diffusional Iimitations.
The extraction ofsoluble metals from suiphide-containing ores can be facilitated by the use of iron- and sulphur-oxidizing thcnnophiiic bacteria (for rcvicw. see Ref. 35). Mcsophiiic Eubactcria have been used
for large-scale (500 tonnes yr-1) leaching of uranium ores, while the use of thennophiles extends only to pilot-scale opcratlons for particularly refractory ores yielding high-value metals. Thermophihc biomining is particularly advantageous where the leac1,ii.g rate is temperature dependent (e.g. the release of copper from chaicopyrite) but these applications are restricted to the USCof finely ground mineral preparations in biareactor systems. The acidophilic Archaea S~lfolobrrs, Aci&urs and b!fcta//@sphnm are candidates for this technology. Non-k? has reported thdt high extraction rates have been obtained with S~r~Zabusstrains but that the cells wcrr sensitive to damage in high-mineraldensity stirred biorractors.
There will undoubtedly bc many other biocatalytic systems where stability and/or function at high temperatures can impart a significant process advautagc. Enzymes such as aldolascs and hydrogenascs (in organic semi-synthesis), glucosidases (in oligosaccharidc synthesis), amidases and peptidascs (in pcptidc synthesis) and phosphohpascs (in phosphoiipid synthesis) ail represent examples ofcontcndcrs. In particular, the conditions required to reverse normal reaction equilibria may often involve the use of potentially destabilizing coiiditions (such as high substrate concentrations, or the addition of solvents), and many promoted by such reactions may bc more &icientiy the USC‘ of more stable biocatalysts. The halophilic Archzea The organisms Coccoid and rod.-shaped organisms requiring high concentrations of NaCi for growth were isolated in the early 1900s 36.These organisms were recognized as a taxonomic entity some years before the Archaca and, in a recent major revision of the taxonomy)‘, wcrc grouped in a new order Halobactcrialrs. comprising six genera (Table 5). The halophilic Archaea arc widely distributed in hypersaline environments such as solar salterns and j soda lakes. The latter contain high concentrations of Na,CO, and represent a halo-aikaliphilic environment. Halophiiic Archaea can also be obtained from man-made hypcrsalinc environments such as salted food products (including saitcd meats, hides, dried fish and fish pastes). in these examples of ‘traditional’
321 reviews
biotechnology, the role of the halophilic Archaca can be either advantageous or deleterious. Protein hydrolysis by archaeal hydrolases in fish-paste preparation contributes to the characteristic flavours of these preparations, whereas similar processes in other salted food products are considered an uzcceptable putrefaction process.
The occurrence of dense populations of halophilic microorganisms (both bacterial and archaeal) in natural saltems has raised the prospecr of using such environments for the production of single cell protein (SCP). Exploiting alkaline saline lakes for open-pond halophle ‘farming’is an attractive low-cost option for producing dietary protein. in arid areas where malnutrition is often endemic. However, the economics of such processes are marginal, and higher-biomass yie!ding cyanobacteria such as S@ttlina, and eukaryotes such as Drrnaliellaare more viable candidates.
Several of the halophilic Archaea, in particular Hafoferax r~~editerrmei,produce quantities of Intracelhtfar poly-@-hydroxybutyrate (PHB) and poly-@hydroxyvalerate (PHV)3s. Copolymers of PHB and PHV are currently produced by ICI as a biodegradable bioplastic, and are an attractive alternative to the oil-derived thermoplastics. H. ntediterrarteibiomass yields about 0.3 g of biopolymer (either PHB or PHV, depending on the substrate provided) per gram dry weight, which is considerably less than the commercially used Eubacteda Aicaligenes erctropkus. However, she use of H. rttediterranei may offer some advantages, pa&rularly relating co greater biomass production. The range of potential microbial contaminants in a hypenaline environment is very low. As a consequence, very cheap large-scale growth options such as open-pond production are available, H. trrediterrattei has other characteristics which make it of possible industrial value in countries with a suitable climate and associated saline environments. These properties include rapid growth on inexpensive, readily available carbon substrates, the production of different polyhydroxyalkanoates (PHA) in response to organic acid substrate supply, spontaneous cell lysis in water (which simplifies recovery of the PHA), and high genomic stabilitv3s.
For the few enzymes from halophilic Archaea which Lave been characterized in detail, the obligate requirement for a high-ionic-strength environment to maintain structural integrity is remarkable, but hardly surprising, since the intrarellular environment may contain K+ concentrations of up to 5 M (Ref. 39). While many halophile enzymes require NaCl concentrations >l M for optimum stability and activity, others are inhibited by high salt concentrations. The hypetxline requirement ofsomc halophile proteins imposes serious limitations on the purification
&
Table 5. Examples of the hafophik
Archa&
Natronobacterium
37
9.5
3.545
M
NaCl
Nationococcus
37
9.5
3.5-4.5
M
NaCl
Nalobacterium
37
7-8
3.545
MNaCl
l-falococcos
37
7-8
?.545
M
I-falaferax
37
6-8
2.0-3.0 M NaCI, 5.0-50.0 mMMg2+
Haloarcula
37
6-8
2.045
and handling of these enzymes and is incompatible with ionic-adsorption chromatography (the basis of most purification strategies). Alternative chromatographic procedures such as affinity, hydrophobicinteraction and gel-permeation chromatography are usually used on a laboratory scale. On a process scale, the need to avoid the use of the most common largescale purification methods, together with the continual requirement for high salt concentrations represent considerable disadvantages. Although I am not aware of any current industrial process operating under hypersaline conditions such that halophile enzymes could be exploited, sncrialized applications in reactions requiring high ionic strength (e.g. in chemical processing) may well emerge.
The purple membrane of Halobarte&!ttrI&&t* contains the integrai membrane protein bacteriorhodopsiG. This is a light-driven proten translocator whose function is to gensrate a transmxubrane electrochemical gradient which drives the ?:yathesis of ATP. The biotechnological potential of bacteriorhodopsin lies in the synth&s of artificial membranes which would be caprblc of using sunlight Lx the production of electricity. To achieve this, the clectrochemical gradient, rather than ATP, would bc tapped to produce an electrical currer@‘. The bacteriorhodopsin system has also attracted corxlderablc attention as a target for biochip desigt+, in optical information storage processes such as hoiography’“, and as a riteans ofsynthesizing large quantities of AT?‘. While the technological problems associated with the design of artificial light-harvesting biomembranes are considerable, some of the properties of bactxiorhodopsin are par&xlarly advantageous. Purpl. I, ;.:r‘branes are particularly stable and will fully renatxc afeer complete denaturation. Using an electric field, bacteriorhcdopsin molecules can also be coordinately orientated, an important requirement for unidirectional proton pumping. ?? Clirricidapplicatim
The type II DNA topoisomcrascs of the halophilic Archaea are sensitive to some antiturnour drugs that
NaCl
WI NaCl
322 reviews
..
Table 6.Some representative methanogenicArchaea Genus Mithanomicrobium Methanogenium Methanospirillum Methanosarcina &thanococcoides Methanofobus Methanococcus Halomethanccoccus Methanothrix Methanobacterium Methanothermus Methanopyrus
Representative species
Topt WI
Mm. mobile Mg. limicola Mg. tbermophilicum Ms. hungatei Ms. barkeri
40 40
MC. mefhylutens
Ml. tindarius MC. halophilus MC. igneus tl. mahi Mt. soehngenii Mb. formicicum M. thermoautotrophicum Mt fervidus Mp. kandleri
3E7 :; 26-36 88 33: 37 :z 98-100
PH optimum 6.1-6.9 ::“o 6*?-;.4 7.0-7.5 6.E 4 6.5’ 7.& 5:: 6.5 6.5
Substratesa H,, formate H,, formate HZ, formate Hz, formate H,, Me, MeNH,, AC Me, MeNH, Me, MeNH, “: Mz:H2 Me,%eN%i, AC
Formate H,, COz H,, CO2 H,, CO,
aAbbreviations:Me, methanol;MeNH,, methylamine;AC,a&ate.
are active against the eukaryotic DNA topoisomerase II. This raises the possibility 7;: using the halophilic enzyme in screerling for cytostatic agenri. A simple screening assay using negatively supercoiled Hal&~teriwt piasmids
Glossary Crenotes- One lineage of the archaeal phylogenetic tree. Comprising only thermophilic and hyperthermophilic organisms, many of which were formerly referred to as ‘S-dependent Archaeobacteria’ and/or ‘thermoacidophiles’. Euryotes-The second lineage of the Archaea, comprising the most primitive of the thermophilic isolates, the methanogens and the extreme halophls. Factor,,, - So called because of a characteristic absorbance at 420 nm, this coenzyme is largely responsible for the characteristic autofluorescence of methanogens. The structure of FdzOcomprises a deazaffavin-ribitol phosphate linked to two or more glutamate residues. Methanopterin- A blue fluorescent compound found in methanogens such as M. thermoautotrophicum.‘This methanogen cofactor is a novel 7-methylpterin derivative, and is structurally related to folic acid (the equivalent compound in bacterial and eukaryal C1 metabolism).
.. ._~-.-.__.___._ __.^..._.___ TIETECH SEPTEMBER
1992 @/OL 10)
The metbafiogens The mcthanogens have come to recent public prominence as the source of potentially explosive methane accumulations in landfill sites and as the primary source of one of the gases implicated in the putative depletion of atmospheric ozone. They are the most-widely distributed of all the Archaea, being fotmd in most common anaerobic habitats, p.miculady in aquatic and marine sediments, sewage sludge and the rumcn and gut of animals and ofinsects. The methanogens, of which over Xl genera have been described, arz pl,cnotypically the most diverse group of the Archaea, with mcsophilic, thermophilic and halophilic representatives (Table 5). They are all characterized by the possession sf the highly complex and novei mcthanogenzsis pathway for the reduction ofsimplc organic compounds (such as carbon dioxide, metha:iol, acetate, formate and carbon monoxide) to methane (for review, see Ref. 45). All arc obligate anaerobes and no alternative energy sources have been demonstrated. The methanogcncsis pathways are unique to the Archaea in that nti other organisms couple methane synthesis to energy generation. Many of the components (including many of the cofactos [e.g. Factor,,, and Methanopterin; see Glossary] and a number ofthe enzymes) of the r.aethanogcnesis pathways are also unique to the methanogenic Archaea. Biotechnology
of the rnethanopns
Of all the Archaea, the methanogens have been exploited most sucrcssfully for biotechnological purposes through their ability to generate methane46 In many countries, and particularly in India and China, the production of ‘biogas’ from human, animal and municipal waste is an economically viable process. The advantages of industrial methanogencsis are considcrable: the technology can be both simple and cheap, the source material is often readily available and inexpensive (in the case ofwaste material, use in mcthano-
323 reviews
genesis is an a?ltcmativc to expensive or environmcntally unacccptablc drsptwal methods), and the product is a particularly clean fuel with a high calorific content. Methanogen metabolism offers numerous opportunities for biotechnological development. While the individual molecular components (particular’y the unique enzymes and cofacton) do not have ar.y current biotcchnological application, the potential for UCG* in future biotransformation processes of&s zxciting possibilities. For example, linking hydrogen oxidation to specific reduction processes usingthe methanogenic hydrogcnases or utilizing the cnzymcs of the CO, reduction pathway as a source ofone-carbon units for biosynthesis might prove to be valuable additions to the current caraloguc of bioccnversion options. Condusions Because of the serendipitous nature of scicncc and the changing requirements of industry, Icgislation and commerce, it would bc foolish to make any specific statement on the &cure role ofthe Archaca in biotechnology. Nevertheless, Archaca are a valuable rcsourcc, and their specific characteristics (such as salt-tolerance, thermostability, and methanogencsis) will certainly lead to novel process applications. Exactly what these processes might be, or the rate at which they develop, is a matter for speculation. What we can say with certainty is that the development of the Archaea as a biotcchnological resourec will be strongly dependent on fr!c.,re research efforts. A better understanding of the enzymology, physiology and genetics ofthese unique organisms can only be beneficial in extending the opportunities for biotechnologica1 exploitation.
1 I
/ j I
!
Do YQU wish to make a contribution to, or to comment on any afticle in
XBTECH? Please contact: The Editor. Elsevkr Tremds Journals. 68 Hills Road, ~mbridge, UK CB2 ItA.
19 Goti, S. A. and Goldberg. A. L. (1985) Ccl/ 41.587-593 20 Shcnnm, M. Y. 2nd Goldberg, A. L. (1992) EAfBOj. 1 I. 71-77 21 Goldberg, A. L. and SCJohn, A. C. (1976) .+m. Rw. Biorlrm. 45, 747-m 22 Kitam, K. Fujimoro. S., Nakao. M., Watanabe. T. and Nakao. Y. (I 987)J. B,orcc/rnol.5, 77-86
Biotechnology of the Archaea Don A. Cowan The Archaea, designated sjnce 1979 as a separate Super-Kingdom (the highest taxonomic order), are a highly novel group of microorganisms which look much like bacteria but have many molecular and genetic characteristics that are more typical of eukaryotes. These unusual organisms can be conveniently divided according to their ‘extreme’ environmental niche, into three broad phenotypes: the thermophiles, methanogens and extreme halophiles. Each group has unique biochemicai features which can be exploited for use in the biotechnological industries. The extreme molecular stability of thermophije enzymes, the novel C, pathways of the methanogens and the synthesis of organic polymers by some halophiles are all currently or potentially valuable examples of the biotechnology of the Archaea.
Before the late lWOs, the Archaea were not recognized as a discrete and identifiable grouping. Howhalophilic and mcthanogenic 65%X, numerous ‘bacteria’ which showed some uncharacteristic biochemical properties had been isolated and studied over the preceding half century. The grouping ofthese organisms, together with certain thermophiles, into a defined and cohcrcnt taxonomic entity, merely awaited the development of the molecular techniques appropriate for the determination of fundamental phylogeny. Two of the three phenotypes which comprise the Archaea are well known, and the biology and applications of these organisms - the methanogens and the
Mophilcs - have been gradually established over many decades. The USCof ana-robic mcthanogcnic culture for the production of methane (‘biogas’) is an established technolo&y, while the appcarauce of halophilic bacteria in association tvvith salted food products can bc tither deleterious or advantageous. The biotcchnoloby &the third archaeal phenotype the thrrmophiles - is less well developed, reflecting the more-recent discovery ofthis group. However. in each instant-. the biotechnological applications ofthe Archaea, either real or anticipated, stem Tom the unusual metabolism ofthese organisms. This review cannot possibly cc’:cr the bioloa ofthe Archaea in more than a cursory manner. For more detail, the reader is directed to a number ofcscel!enr and comp~hensive reviews on this rapidly espandiiig field’-“. _.__--__-._
8 1992. ElsewrScience PublishersLtd (UK)
.-.- .-
fiBTECHSEPTEMBER I992 &‘OL1Ct
a
ARCtf.4KIBACTERlA
prokaryotes and the eukaryotes) were restructured into three discrete groups, named by the :aththors as the Eukaryotes, the Eubacteria and the Archaebactcria (Fig. la)“. A modified system ofnomenclature which emphasizes the fundamental evolutionary ditTerences between the bacteria (i.e. the Eubacteria) and the Archaebacteria was recently presented by Wocsc’s group (Fig. 3b)“. The three groups (which are to be termed Domains) thus become the Bacteria, the Eucarya and the Archaea. Further divisions within the archaeal domain reflect phenotypic and genotypic differences, roughly comprising the hyperthermophilic Archaea, the methanogenic Archaea and the extremely halophilic Archaea (Fig. 1b). For rhe purposes of this article, it is more convenient to discuss the biotechnology of the Archaca in tcmls of these phenotypic groupings rather than the phylogenetic units. Thus the thrrmophiies (encompassing thermophilic, extremely thcrmophillc and hypcrthcrmophilic representatives; see Table I for an explanation of these terms) include the crenotes (see Glossary) and certain thermophilic members of the euryotes. The h;lophihc Archaca and the methanogens (both thmnophilic and non-thermophiiicj are discussed separately for convenience.
I
Hypx. thennophilcs
Therimcoccale~ Mahanccoccalcs
Me!hanogens
Mcthanomicrabiales I Extreme holophiles
1 Halophiles
1 ElJCAKYA ]
Figure1 (aa]The universal phylogenetic tree. After Ref. 11. (b) The tkec domains of life. This is a ‘rooted’ tree, indicating the relative position of the progenote or ancestral cell type. The three archaeal phenoepes are indicated. Redrawn, with permission, from ‘Ref. 12.
Archaed pk+geny In a paper published in ‘977l”, Carl Worse and Gecrgc Fox reported the use of 16S- and f 8S-rRNA sequence data in realigning the fundamental phylogeny oflife. On the basis ofscquence similarities and diffcrenccs, the two traditional primary kingdoms (the
Table 1. Classificationof organismsby growthtemperatures Temperature i”Cj
~Term
Minimum’ Klh1
Optimumb U&t1
ik%ximum’ K,,J
Thermophile
‘30
‘50
‘60
Extreme thermophile Hyperthermophile
‘40 ‘70
‘65 ‘90
‘70 Cl15
‘Minimum temperature at which growth will occur. “Temperature required for shortest doubling time. ‘Maximum temperature at which growth will occur. -_.--__l,.-_---.__ TiBTECtl SEPTEM3ER 1992 NOI. 10)
The thermophilie Archaea Diversity The thermophilic representatives of the Archaea comprise some 19 genera but fewer than 40 species, some of the better kndlvn of which are listed in Table 2. This is a diverse collection of organisms with a wide variation of morphological, physiological and biochemical charactcriscics. -However, ail possess the ability to survive and Q~OWat high temperatures (i.e. the characteristic of thcrmophily).
Thermophilic habitats (or biotopes) in the tempcrature range 40”-70°C are extremely coinmcm (examples include solar-heated soil, decaying vegetation, industrial waste heat, self-heating coai tips and arc tailings). However, biotopes with temperatures ofgreater than 70°C, where most thermophilic Archaca are found, arc relatively rare. The most common ‘hightsmpcraturc’ thermal habitats arc of geothermal origin, usually associated with tectonically active zones. These are widely, though sparsely, distributed over the earth’s surface. The alkaline thermal spring diffca from the marine equivalent only in that the water erupting in the laitcr is saline, and that tompzracures of >lilO°C can be reached because ofthe added hydrostatic pressure. A special version of the marine biotopc is found ac great depths in ocean rifts. Because of the reactivity oi super-heated saline water, the waters issuing from these deep-sea hydrothcmlal vents arc very highly mineralized, and the precipitation of insoluble metal salts from the issuing vents has given rise to the term ‘black smokers’. Temperatures in the region of
317
reviews
1
Table 2. Diversity of thermophilic Archaea
PH
---I Energy metabolism
65
2.5
Heterotrophic
Anaerobic
82
6.5
Heterotrophic
Jhermococcus ceder
Anaerobic
82
6.5
Heterotrophic
Pyrococcus furiosusa
Anaerobic
98
6.5
Heterotrophic
Pyrodictium brockii
Anaerobic
105
6.5
Autotrophic
Jhermoplasma aridophilum
Anaerobic
55
6.5
Heterotrophic
Hyperthermus bl.fylicus
Anaerobic
86
6.5
Fermentative
Methanothermus fervidus
Anaerobic
95
6.5
Methanogenic
Methanococcus jannaschii
Anaerobic
95
6.5
Methanogenic
Methanopyrus kandferi
Anaerobic
100
6.5
Methanogenic
Organism
Culture conditions
Sulfolobus acidocaldarius
Aerobic
Desuffurococcus mobilis
“Since the isolation of I? furiosus:3, rhis organismhas become the ‘E. coli’ of the thermophilic Archaea.
35O-400°C have been recorded in the vent waters although microbial populations are apparently restricted to cooler regions ofless than 1XPC. Etxymes$om thermophilic Archaea In general, any enzyme isolated fr>rn an extreme thermophile can be assumed to be more thermostablc than the homologous enzyme isolated from a lessthermophilic source. This trend is clear from a com-
parison of proteo!ptic enzyme5 from various sources (Table 3)““; many comparisons ofthis type with different enzymes are no;v published. One apparent conscqtience of protein themlostability is the observed high degree of resistance to other potential denaturants: detergents. chaotropic agents such as urea and ~~anic!L;.ium hydrochloride, organic solvents and oxidizing agents. There is evidcncc that thcrmostability also klparts resistance to cnzymic
Table 3. The thermostability of proteases from mesophiies, thermophiies and hyperthermophilesa Half-lie (time at given temperature)
Topt Protease
Ref.
Organism
1°C)
El.subtifis
+27
Subtitisin
ii.a.
i(: iiiin Q
60%
14
B. stearothermopf%s
55
Alkaline protease
n.a.
15 min 8
87°C
14
B. thefmoproteofyticus
60
Thermoiysin
86
60 mind
80°C
14
J. aquaticus
72
Caldoiysin
92
‘30 h @ 15min@
80°C 95%
T. ce!er
88
Protease
n.a.
40 min @
95%
16
D. mucosus
85
Archaelysin
9%
80 min @I 95%
17
S. solfataricus
87
Hetitral protease
n.a.
40 min Q
95%
16
P. furiosus
94
66 kDa prctease
n.a.
98%
18
Pyroiysiwcomp!ex .-
‘115
3 7 min @ 105%
19
‘33 h
@
aAbbreviatioos:Top,,temperature at which cells doubie in the shortest time; TM, meking ‘%zperi;ure (at which denaturation occursi: n.a.. not available.
------TIBTECII
--WTEMBER
1992 WOL10)
318 reviews
i cleavage (proteolysis) and to chemical reactions causing irreversible protein degradation at high tcmperaturesZQ_ The resistance of thermostable proteins to denaturation by organic solvents appears to bc a gcnera1 characteristic”. A common misconception is that thermophiiicity allows very rapid reaction rates to be achieved by carrying out reactions at high temperatures. This misconception is based on incorrectly extrapolating Arrhenius’ law* from the properties of mesophilic enzymes, presuming that
’ the reaction rate of a hyprrthennophilic enzyme should be about X-fold higher at 90°C than char of the equivalent mesophilic enzyme at 37%’ WC now know this to be incorrect in almost all instances and, for sound physical reasons, thermophilic enzymes at their ‘optimum’ temperatures (see Table 1; footnote b) exhibit turnover rates of the same order of magnitude as do mesophilic enzymes at their optimum temperatures. The mechanism of this apparent limitation ofactivity is, at least in part, the restraint in conformational flexibility which is thought to be coin&dent with enhanced thcrmostability~~. Evidcncc supporting this r&cionship is stiil sketchy and there is great potential for dctaifed structure/function/ stability studies. It should not be assumed, for example, that the conformational restraints will be the same in all regions of the folded protein structure, In some instances, however, very large increases in reaction rates can be achieved by using thcrmophilic enzymes at high temprraturcs; for example where, at higher temperatures, the substrate is more susceptible to catalysis. The best examples are the hydrolytic degradations of polymeric substrates, particularly protcolytic degradation of protein and amylolytic dcgradation of starch. Industry has taken full advantage of the latter, particularly in the high-temperature saccharitication proccsscs currently employed in most enzymic starch hydrolysis plant:. A problem that is generally associated with the largescale USCof enzymes from thermophilic Archaea is that of low-biomass yields. Typically, archaeal fermentations yield 0.1-l .0 g wet weight of biomass per litre (-lV-lo” cells per millihtre) compared with the 5-30 g wet weight of biomass per litre yield for common industriai bacterial strains. While this is a major impediment to commercial application of thrrmophilic Archaea (whether as whole-ccl1 biocatalysts, as sources ofcnzymes or as sources of other biomolcculcs), recent developments in fermentation stratcges have led to cell densities approaching those of more “classical’ organiims (up to 5 x IW cells per mi!fiIitrc; K. Sharp, pcrs. commun.),
An alternative option, wl:ose feasibility is demon;tratcd by the growing number of examples reported in the literature, is the cloning and over-expressior, of the genes that encode the hyperthcnnophilic proteins.
,_l___i--.__
TIETECH SEPTEMBER 1992 IVOL
Applications
of enzymes
from
thermophik Archaea Current applications
The commercial importance of cnzymcs from thcrmophilic Archaca is illustrated by a single application: the polymerase chain reaction (PCR), a rapid and efiicient exponential m!?thod of amplitjring specific DNA sequences. The automation of PCR involved replacing the Klenow fragment of DNA polymcrase with the thcrmostablr Tag polymcrase’” from the thcnnophilic eubacteriuti Thenmu aqtmtiws - a major step forward in the technology. Howcvcr. the thermostability of Taq polymerase is only just sufficient, and a further recent development is the use of extremely thcrmostable DNA polymerascs from two diffcrcnt hypcrthcnnophilic Archara (Y?II?IECIIOCOCCM /itt~mii and Pyryrorocc~ firriosm). In addition to cnzymc thermostability: su&cssful application of DNA polymerases in PCR needs processivity and proof-reading (3’5’ cxonuclcase) activity. The proof-reading capacity of the P. firrimrrs DNA polymcrase is reported to r&cc the error frequency by a t&tor of 14, compared with Taq polymcrase?J. Future applications To date, the number of enzymes from thermophilic Archaebacteria which have been studied in any detail barely exceeds the total number of named species. these organisms probably contain as Neverthe&,, wide a range ofdifferent activitic s as any other diverse group of microorganisms. In addition, certain activities and enzyme systems will be unique to this group of organisms, an excellent example being the undoubtedly novel, but as yet uncharactcrizcd, enzymes ofthc biphytanyl tctrarthcr lipid bio:ynthetic pathways’“, The success of thernrophilic enzymes in PCR may give us a clue to the future role of the Archaea in biotechnology. This is an excellent example of a ‘niche’ application, where the specilic properties ofthe enzyme are complementary to the highly specific requircmcnts of a process.
In considering future commercial and industrial applications of thcrmophilic enzymes, one inevitably turns to the current: areas of industrial enzymology. Over 75% of the world usage of enzymes (estimated value at over UKL900 million in 1990)z’Xis 1~ the detergent, starch and dairy industries. Although, in many instances, a thermostable alternative could replace the etizymc in current use, doing so may not be commercially viable. For example: (1) some industrial biocatalytic processes are incompatible with hightemperature operation (e.g. cheese-making); (2) commercial trends favom low-temperature operation (e.g. domestic detergents); (3) cnzymcs of sufficient thennostability are already in USC(e.g. in the saccharification of starch); (4) there is no obvious process advantage in increasing the reaction tcmpcraturc (e.g. biosynthetic production of fine chemicals such as
319 reviews
amino acids and penicillins); and (5) many ofthe major industrial biocatalyst users are committed to existing low-temperature operation through investment in specific yla?t 2nd equipment, and the advantages of higher-temperature operation would be insufficient to offset the costs of replacing that equipment. In summary, the conditions under which a thermophilic enzyme would be a serious contender as a replacement for any ‘bulk’ commercial enzyme might include many (ifnot all) of the following: (1) it must be available in similar (or equivalent) quantity with equally reliable supply; (2) it must have a similar or lower price (per unit of activity); (3) it must exhibit significantly better performance in many respects (not just stability); and (4) there must be no major altcrations to existing plant and equipment (or investment in new plant) required. This assessment indicates that the commercial &ture of thermophilic archaeal enzymes may not be in replacing existing ‘industrial’ enzymes, particulady ir, the high-volume, low-cost enzyme market, but rather in ‘niche’ applications, where the unique properties that these enzymes possess can offer significant process advantages. Although specialized, the commercial success of PCR indicates that such applications could be highly profitable.
Such an example is the development’7 of a ligascdependent DNA-amplification tecE:+*e. The ligase amplification reaction (LAP.) (Fig. 2) has potential applications in the identification of gene defects, ar.;l has already been used on a laboratory scale for the detection of i%globin mutantsZn. The requirements for a thermophilic and thermostable DNA ligase m LAR are obvious - the ligation must be carried out near the DNA melting temperature (T,J (a minimum of 6YC), and the enzyme must be stable during the subsequent dissociation step (carried out at -92°C). To date, the only commercially available thermostable DNA-ligase is a recombinant derived from the thermophi!ic Eubacterium 77zen1nrsrhen~~opkihrs,and expressed in E. coli. The appearance of a cloned hyperthermophilic DNA ligase is surely only a matter of time.
The secondary alcohol-specific alcohol dehydrogenases, which catalyse the oxidation of secondary alcohols and, less readily, the reverse reaction (the reduction of ketones), have a promising hture in biotechnology. There is considerable interest in the USCof such enzymes in the chemical-synthesis indwties’9,30, particularly the pharmaceutical industries where the biological production of chiral synthons is gaining in importance in the chemicaf synthesis of chirally pure pharmaceutical agents. Although alcohol dehydrogenases show a wide distribution and are well characterized, only two examples of thermostable alcohol dehydrogenases derived Tom hyperthermophilic Archaea are currently known (Table 4). The
Complementary target(T)
J.llli~l A
IIrllLL B
Mismatched target (T*)
__
-_ A
Figure 2 Schematic representationof the ligase amplification reaction UR) (after Ref. 27). TheDNAtemplateis heatdenatured and four complementary oligonuciaotide primers are hybridized to the target at near the mefbng temperature (TM) (Step 11. Thermostable DNA-fig&e is then added, and ligation between adjacent primers proceeds (Step 2); the success of the technique relies on the fact that. at the high tempera::lre, only adjacent and perfectly complementary oligonucleotides will be ligated together. The ligation products are heatdenahired and form new target templates ior the next cycle (Step 31, resulting in an exponential ampliiation. Oligonucleotides with a single base mismatch, however, will not ligate and will amplify in linear and not exponential fashion.
reduction by SrrlfnloBtrs alcohol dchydrogenascJ1 of aromatic ketones to their respective chiral alcohols is a particularly valuable activity. In the reactions catalyscd by alcohol dehydrogenases, both the substrate and product may bc hydrophilic organic molecules (such as alcohols, aidehydes and ketones), and such organic ‘solvents’ are oflen potent denaturants of enzymes. In addition, the high cost of NADH and NADPH ensures that, for all but laboratory-scale operations, a system !or recycling the oxidized cofactor is essmtial. One etfectivc recycling option is to include an alcohol co-substrate which, in being oxidized by the enzyme, regenerates the reduced cofactor, However, this results in an even higher concentration ofpotentially denamring vrganic components. These hctors, therefore. Gocr the use of enzymes which are stable in orgamc solvents. In the biocatalytic reduction of a variety of pure chiral alcohols, a moderately thcrmostablc alcohol dehyd:ogenase isolated Tom a eubactcdal tbermophile ~~entroar~aerohi~rr~~ br&ii3” has been u,;ed. However. TIBTECH SEPTEMBER1992 iVOL 101
320
Table 4. Specificitwa d secondary-alcoholdehydrogenasesfrom hyperthermophilicArchaea Relative a&vii
Topt Organism
PC)
Sulfolobus solfataricus
87
Hyperthermus butykus
96
Cofactor
Ethanol
NADP
Butan-Z-al
(“/I”
Cyclohexanol
Benzyl alcohol
Ref.
100
188
410
x9
31
100
76
174
0
b
“The enzyme’s activity in ethanol is taken as 1011%. 'L.Galabeand D. A. Cowan.tinpublished.
reaction tempcraturcs greater than 45°C and organic solvent concentrations ofmore than about 1 O”/n induce rapid enzyme inactivation (A. Plant and D. A. Cowan, unpubiishcd). ADH-cataiysed reduction trizla with S@M~rrs cells”~~suggest that the hyprrthennophilic enzymes may be excellent aiternativcs. Reports of laboratory studies using thcrmophilic alcohol dehydrogenascs for biotramfortnation purposes will undoubtedly incrcasc. and the availability of thcrmostable archacal enzymes in com:nercial quantitics would provide an additional stimulus to such dcveiopments. 0 Bterases Esterases are enzymes that also show a wide distribution, and mesophiiic examples have been used in organic biosynthose9 3o. In aqueous solution, estcrases cataiyse the hydrolytic cleavage of esters to fonn the constituent acid and alcohol. However, in reaction systems where the aqueous component of the solvent is reduced sufficiently to reduce the thermodynamic water activity, non-hydrolytic reactions are pro!noted. Transesterification, not favour-d in aqueous solution because of the prcvalencc of the hydrolytic reaction, can bc greatly enhanced in organic solvents. Both the reactants and products oftranscsterification are usually highly soluble in an organic phase, and the reactant5 may cvcn form the organic phase themselves. Clearly, it is essential that the biocatalyst is stable under the solvent conditions used.
Taking advantage of the intrinsic thcrmostabiiity of archacal enzymes and the enhanced stability of proteins in low-water cnvironincnh, S+lobtfs su!fnlaricus alcohol dehydrogcnase has been used to catalyst ‘gas(Ref. phase’ reactions at tempcraturcs around 1OOT 34). While only in the early stages of development, this ingenious tcchnolo&J has the advantage ofmicimizing the use ofsolvents (a popular objective in these cncrgy- and waste-conscious times) and of reducing substrate and product-diffusional Iimitations.
The extraction ofsoluble metals from suiphide-containing ores can be facilitated by the use of iron- and sulphur-oxidizing thcnnophiiic bacteria (for rcvicw. see Ref. 35). Mcsophiiic Eubactcria have been used
for large-scale (500 tonnes yr-1) leaching of uranium ores, while the use of thennophiles extends only to pilot-scale opcratlons for particularly refractory ores yielding high-value metals. Thermophihc biomining is particularly advantageous where the leac1,ii.g rate is temperature dependent (e.g. the release of copper from chaicopyrite) but these applications are restricted to the USCof finely ground mineral preparations in biareactor systems. The acidophilic Archaea S~lfolobrrs, Aci&urs and b!fcta//@sphnm are candidates for this technology. Non-k? has reported thdt high extraction rates have been obtained with S~r~Zabusstrains but that the cells wcrr sensitive to damage in high-mineraldensity stirred biorractors.
There will undoubtedly bc many other biocatalytic systems where stability and/or function at high temperatures can impart a significant process advautagc. Enzymes such as aldolascs and hydrogenascs (in organic semi-synthesis), glucosidases (in oligosaccharidc synthesis), amidases and peptidascs (in pcptidc synthesis) and phosphohpascs (in phosphoiipid synthesis) ail represent examples ofcontcndcrs. In particular, the conditions required to reverse normal reaction equilibria may often involve the use of potentially destabilizing coiiditions (such as high substrate concentrations, or the addition of solvents), and many promoted by such reactions may bc more &icientiy the USC‘ of more stable biocatalysts. The halophilic Archzea The organisms Coccoid and rod.-shaped organisms requiring high concentrations of NaCi for growth were isolated in the early 1900s 36.These organisms were recognized as a taxonomic entity some years before the Archaca and, in a recent major revision of the taxonomy)‘, wcrc grouped in a new order Halobactcrialrs. comprising six genera (Table 5). The halophilic Archaea arc widely distributed in hypersaline environments such as solar salterns and j soda lakes. The latter contain high concentrations of Na,CO, and represent a halo-aikaliphilic environment. Halophiiic Archaea can also be obtained from man-made hypcrsalinc environments such as salted food products (including saitcd meats, hides, dried fish and fish pastes). in these examples of ‘traditional’
321 reviews
biotechnology, the role of the halophilic Archaca can be either advantageous or deleterious. Protein hydrolysis by archaeal hydrolases in fish-paste preparation contributes to the characteristic flavours of these preparations, whereas similar processes in other salted food products are considered an uzcceptable putrefaction process.
The occurrence of dense populations of halophilic microorganisms (both bacterial and archaeal) in natural saltems has raised the prospecr of using such environments for the production of single cell protein (SCP). Exploiting alkaline saline lakes for open-pond halophle ‘farming’is an attractive low-cost option for producing dietary protein. in arid areas where malnutrition is often endemic. However, the economics of such processes are marginal, and higher-biomass yie!ding cyanobacteria such as S@ttlina, and eukaryotes such as Drrnaliellaare more viable candidates.
Several of the halophilic Archaea, in particular Hafoferax r~~editerrmei,produce quantities of Intracelhtfar poly-@-hydroxybutyrate (PHB) and poly-@hydroxyvalerate (PHV)3s. Copolymers of PHB and PHV are currently produced by ICI as a biodegradable bioplastic, and are an attractive alternative to the oil-derived thermoplastics. H. ntediterrarteibiomass yields about 0.3 g of biopolymer (either PHB or PHV, depending on the substrate provided) per gram dry weight, which is considerably less than the commercially used Eubacteda Aicaligenes erctropkus. However, she use of H. rttediterranei may offer some advantages, pa&rularly relating co greater biomass production. The range of potential microbial contaminants in a hypenaline environment is very low. As a consequence, very cheap large-scale growth options such as open-pond production are available, H. trrediterrattei has other characteristics which make it of possible industrial value in countries with a suitable climate and associated saline environments. These properties include rapid growth on inexpensive, readily available carbon substrates, the production of different polyhydroxyalkanoates (PHA) in response to organic acid substrate supply, spontaneous cell lysis in water (which simplifies recovery of the PHA), and high genomic stabilitv3s.
For the few enzymes from halophilic Archaea which Lave been characterized in detail, the obligate requirement for a high-ionic-strength environment to maintain structural integrity is remarkable, but hardly surprising, since the intrarellular environment may contain K+ concentrations of up to 5 M (Ref. 39). While many halophile enzymes require NaCl concentrations >l M for optimum stability and activity, others are inhibited by high salt concentrations. The hypetxline requirement ofsomc halophile proteins imposes serious limitations on the purification
&
Table 5. Examples of the hafophik
Archa&
Natronobacterium
37
9.5
3.545
M
NaCl
Nationococcus
37
9.5
3.5-4.5
M
NaCl
Nalobacterium
37
7-8
3.545
MNaCl
l-falococcos
37
7-8
?.545
M
I-falaferax
37
6-8
2.0-3.0 M NaCI, 5.0-50.0 mMMg2+
Haloarcula
37
6-8
2.045
and handling of these enzymes and is incompatible with ionic-adsorption chromatography (the basis of most purification strategies). Alternative chromatographic procedures such as affinity, hydrophobicinteraction and gel-permeation chromatography are usually used on a laboratory scale. On a process scale, the need to avoid the use of the most common largescale purification methods, together with the continual requirement for high salt concentrations represent considerable disadvantages. Although I am not aware of any current industrial process operating under hypersaline conditions such that halophile enzymes could be exploited, sncrialized applications in reactions requiring high ionic strength (e.g. in chemical processing) may well emerge.
The purple membrane of Halobarte&!ttrI&&t* contains the integrai membrane protein bacteriorhodopsiG. This is a light-driven proten translocator whose function is to gensrate a transmxubrane electrochemical gradient which drives the ?:yathesis of ATP. The biotechnological potential of bacteriorhodopsin lies in the synth&s of artificial membranes which would be caprblc of using sunlight Lx the production of electricity. To achieve this, the clectrochemical gradient, rather than ATP, would bc tapped to produce an electrical currer@‘. The bacteriorhodopsin system has also attracted corxlderablc attention as a target for biochip desigt+, in optical information storage processes such as hoiography’“, and as a riteans ofsynthesizing large quantities of AT?‘. While the technological problems associated with the design of artificial light-harvesting biomembranes are considerable, some of the properties of bactxiorhodopsin are par&xlarly advantageous. Purpl. I, ;.:r‘branes are particularly stable and will fully renatxc afeer complete denaturation. Using an electric field, bacteriorhcdopsin molecules can also be coordinately orientated, an important requirement for unidirectional proton pumping. ?? Clirricidapplicatim
The type II DNA topoisomcrascs of the halophilic Archaea are sensitive to some antiturnour drugs that
NaCl
WI NaCl
322 reviews
..
Table 6.Some representative methanogenicArchaea Genus Mithanomicrobium Methanogenium Methanospirillum Methanosarcina &thanococcoides Methanofobus Methanococcus Halomethanccoccus Methanothrix Methanobacterium Methanothermus Methanopyrus
Representative species
Topt WI
Mm. mobile Mg. limicola Mg. tbermophilicum Ms. hungatei Ms. barkeri
40 40
MC. mefhylutens
Ml. tindarius MC. halophilus MC. igneus tl. mahi Mt. soehngenii Mb. formicicum M. thermoautotrophicum Mt fervidus Mp. kandleri
3E7 :; 26-36 88 33: 37 :z 98-100
PH optimum 6.1-6.9 ::“o 6*?-;.4 7.0-7.5 6.E 4 6.5’ 7.& 5:: 6.5 6.5
Substratesa H,, formate H,, formate HZ, formate Hz, formate H,, Me, MeNH,, AC Me, MeNH, Me, MeNH, “: Mz:H2 Me,%eN%i, AC
Formate H,, COz H,, CO2 H,, CO,
aAbbreviations:Me, methanol;MeNH,, methylamine;AC,a&ate.
are active against the eukaryotic DNA topoisomerase II. This raises the possibility 7;: using the halophilic enzyme in screerling for cytostatic agenri. A simple screening assay using negatively supercoiled Hal&~teriwt piasmids
Glossary Crenotes- One lineage of the archaeal phylogenetic tree. Comprising only thermophilic and hyperthermophilic organisms, many of which were formerly referred to as ‘S-dependent Archaeobacteria’ and/or ‘thermoacidophiles’. Euryotes-The second lineage of the Archaea, comprising the most primitive of the thermophilic isolates, the methanogens and the extreme halophls. Factor,,, - So called because of a characteristic absorbance at 420 nm, this coenzyme is largely responsible for the characteristic autofluorescence of methanogens. The structure of FdzOcomprises a deazaffavin-ribitol phosphate linked to two or more glutamate residues. Methanopterin- A blue fluorescent compound found in methanogens such as M. thermoautotrophicum.‘This methanogen cofactor is a novel 7-methylpterin derivative, and is structurally related to folic acid (the equivalent compound in bacterial and eukaryal C1 metabolism).
.. ._~-.-.__.___._ __.^..._.___ TIETECH SEPTEMBER
1992 @/OL 10)
The metbafiogens The mcthanogens have come to recent public prominence as the source of potentially explosive methane accumulations in landfill sites and as the primary source of one of the gases implicated in the putative depletion of atmospheric ozone. They are the most-widely distributed of all the Archaea, being fotmd in most common anaerobic habitats, p.miculady in aquatic and marine sediments, sewage sludge and the rumcn and gut of animals and ofinsects. The methanogens, of which over Xl genera have been described, arz pl,cnotypically the most diverse group of the Archaea, with mcsophilic, thermophilic and halophilic representatives (Table 5). They are all characterized by the possession sf the highly complex and novei mcthanogenzsis pathway for the reduction ofsimplc organic compounds (such as carbon dioxide, metha:iol, acetate, formate and carbon monoxide) to methane (for review, see Ref. 45). All arc obligate anaerobes and no alternative energy sources have been demonstrated. The methanogcncsis pathways are unique to the Archaea in that nti other organisms couple methane synthesis to energy generation. Many of the components (including many of the cofactos [e.g. Factor,,, and Methanopterin; see Glossary] and a number ofthe enzymes) of the r.aethanogcnesis pathways are also unique to the methanogenic Archaea. Biotechnology
of the rnethanopns
Of all the Archaea, the methanogens have been exploited most sucrcssfully for biotechnological purposes through their ability to generate methane46 In many countries, and particularly in India and China, the production of ‘biogas’ from human, animal and municipal waste is an economically viable process. The advantages of industrial methanogencsis are considcrable: the technology can be both simple and cheap, the source material is often readily available and inexpensive (in the case ofwaste material, use in mcthano-
323 reviews
genesis is an a?ltcmativc to expensive or environmcntally unacccptablc drsptwal methods), and the product is a particularly clean fuel with a high calorific content. Methanogen metabolism offers numerous opportunities for biotechnological development. While the individual molecular components (particular’y the unique enzymes and cofacton) do not have ar.y current biotcchnological application, the potential for UCG* in future biotransformation processes of&s zxciting possibilities. For example, linking hydrogen oxidation to specific reduction processes usingthe methanogenic hydrogcnases or utilizing the cnzymcs of the CO, reduction pathway as a source ofone-carbon units for biosynthesis might prove to be valuable additions to the current caraloguc of bioccnversion options. Condusions Because of the serendipitous nature of scicncc and the changing requirements of industry, Icgislation and commerce, it would bc foolish to make any specific statement on the &cure role ofthe Archaca in biotechnology. Nevertheless, Archaca are a valuable rcsourcc, and their specific characteristics (such as salt-tolerance, thermostability, and methanogencsis) will certainly lead to novel process applications. Exactly what these processes might be, or the rate at which they develop, is a matter for speculation. What we can say with certainty is that the development of the Archaea as a biotcchnological resourec will be strongly dependent on fr!c.,re research efforts. A better understanding of the enzymology, physiology and genetics ofthese unique organisms can only be beneficial in extending the opportunities for biotechnologica1 exploitation.
1 I
/ j I
!
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