cellulase, clostridia, and ethanol†decreasing urban air pollution), and is particularly...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2005, p. 124–154 Vol. 69, No. 11092-2172/05/$08.00�0 doi:10.1128/MMBR.69.1.124–154.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cellulase, Clostridia, and Ethanol†Arnold L. Demain,1* Michael Newcomb,2 and J. H. David Wu2

Charles A. Dana Research Institute for Scientists Emeriti, Drew University, Madison, New Jersey,1 and Department of ChemicalEngineering, University of Rochester, Rochester, New York2

INTRODUCTION .......................................................................................................................................................124Need for Alternative Energy Source.....................................................................................................................124

THE CLOSTRIDIUM THERMOCELLUM CELLULASE SYSTEM.....................................................................126Strains and Media ..................................................................................................................................................126Properties of the Cellulase System.......................................................................................................................127

THE CLOSTRIDIUM THERMOCELLUM CELLULOSOME...............................................................................128Components SL and SS ..........................................................................................................................................128Cellulosome Structure............................................................................................................................................130Cohesins and Dockerins ........................................................................................................................................130Cellulose-Binding Domain.....................................................................................................................................131Attachment of Cellulosomes to the Cell Surface................................................................................................132Assembly of the Cellulosome.................................................................................................................................133Genes and Enzymes................................................................................................................................................136Dockerin-Containing Proteins...............................................................................................................................137

CARBON SOURCE NUTRITION............................................................................................................................137REGULATION OF CELLULASE PRODUCTION................................................................................................140

Carbon Source Regulation.....................................................................................................................................140Cellulase Gene Clusters.........................................................................................................................................141Negative Regulation................................................................................................................................................142

CLOSTRIDIAL COCULTURES...............................................................................................................................143APPLICATION OF RECOMBINANT DNA TECHNOLOGY TO THE SELECTIVITY PROBLEM ............145CLOSING COMMENTS ...........................................................................................................................................147ACKNOWLEDGMENTS ...........................................................................................................................................147REFERENCES ............................................................................................................................................................147

INTRODUCTION

Need for Alternative Energy Source

Of the total amount of gasoline used in the United States asa transportation fuel and by industry, about half has to beimported (358). The amount of solar energy received at theearth’s surface is 2.5 � 1021 Btu/year, which far exceeds thepresent human usage of 2.0 � 1017 Btu/year. The amount ofenergy from the sun which is stored as carbon via photosyn-thesis is 10 times the world usage. Lignocellulose is the mostabundant renewable natural resource and substrate availablefor conversion to fuels. It is inexpensive, plentiful, and renew-able. On a worldwide basis, terrestrial plants produce 1.3 �1010 metric tons (dry weight basis) of wood per year, which isequivalent to 7 � 109 metric tons of coal or about two-thirds ofthe world’s energy requirement. Available cellulosic feedstocksfrom agriculture and other sources are about 180 million tonsper year (203). Furthermore, tremendous amounts of celluloseare available as municipal and industrial wastes which todaycontribute to our pollution problems. Thus, there is great in-

terest in the use of cellulosic biomass as a renewable source ofenergy via breakdown to sugars that can then be converted toliquid fuel.

With the hike in oil prices around the world in the 1970s andthe realization that the world’s oil supply is finite, the quest foralternative fuels began in 1975, and researchers looked foreconomical ways to produce ethanol, preferably from abun-dantly available, biodegradable, and renewable raw materials.Ethanol is an excellent transportation fuel, in some respectssuperior to gasoline (199, 200). In particular, with respect togasoline, neat (unblended) ethanol burns more cleanly, has ahigher octane rating, can be burned with greater efficiency, isthought to produce smaller amounts of ozone precursors (thusdecreasing urban air pollution), and is particularly beneficialwith respect to low net CO2 put into the atmosphere. Further-more, ethanol by fermentation offers a more favorable tradebalance, enhanced energy security, and a major new crop for adepressed agricultural economy. Ethanol is considerably lesstoxic to humans than is gasoline (or methanol). Ethanol alsoreduces smog formation because of low volatility; its photo-chemical reactivity and that of its combustion products are low,and only low levels of smog-producing compounds are formedby its combustion (359). Its high heat of vaporization, highoctane rating, and low flame temperature yield good engineperformance. Cellulosics, at $42 per dry ton, cost the same aspetroleum, at 6 to $7 per barrel based on equivalent mass or 12to $13 per barrel based on equivalent energy content (203,

* Corresponding author. Mailing address: Charles A. Dana Re-search Institute for Scientists Emeriti, HS-330, Drew University, Mad-ison, NJ 07940. Phone: (973) 408-3937. Fax: (973) 408-3504. E-mail:[email protected].

† This review is dedicated to the late Marek Romaniec, who broughtthe light of molecular biology to our M.I.T. laboratory but whose ownlight went out much too soon.

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356); today, the price of petroleum is much higher. It appearsprobable that ethanol from cellulose could become competi-tive with gasoline if the processing costs for the former couldbe lowered. Ethanol is also used as an oxygenate to reduceautomobile emissions. With the current and impending phase-out of methyl tert-butyl ether as an oxygenate in many states inthe United States, ethanol will fill the void. Useful reviews onthe biological conversion of lignocellulosic biomass to ethanolhave been published (183, 196, 198, 200, 201, 203, 204, 206,224, 356).

The potential quantity of ethanol that could be producedfrom cellulose is over an order of magnitude larger than thatproducible from corn. In contrast to the corn-to-ethanol con-version, the cellulose-to-ethanol route involves little or no con-tribution to the greenhouse effect and has a clearly positive netenergy balance (five times better). As a result of such consid-erations, microorganisms that metabolize cellulose havegained prominence in recent years (38, 329).

Lignocellulose is difficult to hydrolyze because (i) it is asso-ciated with hemicellulose, (ii) it is surrounded by a lignin sealwhich has a limited covalent association with hemicellulose,and (iii) much of it has a crystalline structure with a potentialformation of six hydrogen bonds, four intramolecular and twointermolecular, giving it a highly ordered, tightly packed struc-ture (344). Pretreatments aim at increasing the surface area ofcellulose by (i) removing the lignin seal, (ii) solubilizing hemi-cellulose, (iii) disrupting crystallinity, and/or (iv) increasingpore volume. The value of a cellulase system that attacks crys-talline cellulose lies in the observation that many of the pre-treatments which increase surface area also increase crystal-linity. These include dilute sulfuric acid, alkali, andethylenediamine.

The rate-limiting step in the conversion of cellulose to fuelsis its hydrolysis, especially the initial attack on the highly or-dered, insoluble structure of crystalline cellulose, since theproducts of this attack are readily solubilized and converted tosugars. A great deal of effort has gone into the development ofmethods for conversion of cellulose to sugars. Most of thiswork has emphasized the biochemistry, genetics, and processdevelopment of fungi (especially Trichoderma reesei) coupledto the further conversion of the sugars produced to ethanol by

yeast (Saccharomyces cerevisiae). After many years of study, itis becoming apparent that such a process is not the only po-tential solution.

The large amounts of attention and resources devoted overthe past 30 years to the Trichoderma-Saccharomyces concepthave perhaps interfered in the industrial development of thepotential of the cellulolytic, thermophilic anaerobic bacteriasuch as Clostridium thermocellum (49, 51, 66, 81, 216) and theircellulases and hemicellulases (28, 32, 78, 195, 204, 274, 301,315). C. thermocellum breaks down cellulose, with the forma-tion of cellobiose and cellodextrins as main products. Cellobi-ose, a disaccharide of two glucose moieties held together by a�-1,4 linkage, can be further utilized by the organism, and thefinal end products are ethanol, acetic acid, lactic acid, hydro-gen, and carbon dioxide (181) (Fig. 1). Small cellodextrins canalso be taken into the cell, broken down further, and metab-olized (203). The interest in this organism is due to severalfactors (Table 1). First, C. thermocellum can utilize lignocellu-losic waste and generate ethanol, a rare property among livingorganisms. Indeed, the cellulase system of C. thermocellummediates hydrolysis of lignin-containing materials such ashardwood pretreated with dilute acid as well as model sub-strates not containing lignin (196, 198). Second, for large-scaleculture, anaerobiosis is an advantage because one of the mostexpensive steps in industrial fermentations is that of providingadequate oxygen transfer, e.g., for cellulase production. Third,since the optimum temperature for the cultivation of the or-

FIG. 1. The clostridial coculture process in which C. thermocellum serves as the cellulase and hemicellulase producer. The hemicellulose-derived pentoses can be utilized by C. thermosaccharolyticum but not C. thermocellum. C. thermosaccharolyticum uses cellobiose faster and is abetter ethanol producer. In addition to cellobiose, cellodextrins are also produced from cellulose and can be utilized directly.

TABLE 1. Advantages of using C. thermocellum for ethanolfermentation from biomass

The cellulolytic and ethanogenic nature, allowing saccharificationand fermentation in a single step

The anaerobic nature, negating the need for expensive oxygentransfer

Low cell growth yield, favoring ethanol conversionThe thermophilic nature, facilitating ethanol removal and recoveryThe thermophilic nature, reducing cooling costThermophilic fermentation being less prone to contaminationThermophilic biomass-degrading enzymes enhancing protein stabilityAmenability to coculture with other ethanol-producing and pentose-

fermenting organisms

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ganism is 60°C, the problems of contamination are lessenedand the cooling of large fermentors is much simplified. Fourth,growth at a high temperature facilitates the recovery of etha-nol. Fifth, thermophiles are thought to be robust microorgan-isms and contain stable enzymes. Sixth, anaerobes generallyhave a low cell yield, and hence more of the substrate isconverted to ethanol. Experience suggests that even more ad-vantageous are in situ cellulase production and the high ratesof growth on and metabolism of cellulose and hemicellulose.Since the carbon dioxide produced during fermentation andfuel use of ethanol is recycled by growth of plants, ethanol doesnot contribute to carbon dioxide accumulation in the atmo-sphere and possible global warming (359).

In 1990, 90% of new cars in Brazil used neat ethanol, whilethe remainder utilized a blend of 20 to 22% ethanol and 80 to78% gasoline. Three to four billion gallons are produced inBrazil from sugar per year. The United States in 1987 wasusing 8.6 metric tons (340 million bushels) of corn to make 850million gallons of anhydrous ethanol for blending with gasolineat a level of 10%. The ethanol-gasoline blend was supplyingabout 8% of the U.S. gasoline market. Although small, thisfigure had risen from less than 1% in 1981 (355). In 1990, theUnited States consumed 112 billion gallons of gasoline. By1993, the United States was producing 1.0 billion gallons ofethanol from corn (358); by 1996, the amount had risen to 1.3billion gallons (197, 247); by 2000, it was 1.6 billion gallons; andby 2001, it was 1.8 billion gallons (84, 349). If all cellulosicfeedstocks in the United States could be used for ethanol, 20billion gallons could be produced (203, 224), which would bemore than enough to add 10% ethanol to all gasoline used inthe United States.

Cellulosic biomass in the early 1980s, at 20 to $30/dry ton,was much cheaper than corn, at $110/dry ton, yet cellulosehydrolysis was not competitive with starch hydrolysis to pro-duce sugar. The problems included cellulose crystallinity andlignin content, requiring expensive pretreatment. Also, cornusage provided oil and protein by-products, which were ofmuch higher value than lignin (247). By 1990, the cost ofethanol production from cellulose (by Trichoderma plus yeast)was thought to be approaching that of production from corn(by yeast) on an unsubsidized basis (130, 196, 200). However,neither ethanol produced from corn nor ethanol producedfrom cellulose is currently competitive with conventional liquidfuels. A state-of-the-art process designed for ethanol produc-tion from cellulose had a selling price of U.S. $1.35 per gallonin 1988 (200), $1.18 per gallon in 1996 (201), and $1.20 in 2003(356). For production from corn, the price in 1994 was $1.22per gallon and that in 2001 was $1.65 (355, 357). However, aprice of 50 cents per gallon would have been required forethanol to compete with gasoline in the early 1990s, 67 centsper gallon would have been required in 1994, and 70 cents pergallon would have been required in 2000. With improved tech-nology applied to the microbial biomass-to-ethanol technol-ogy, it was projected that the selling price of pure ethanolcould be 50 cents per gallon and in the best case could be aslow as 34 cents per gallon (201). Thus, substantial cost reduc-tions are possible and could make biological ethanol a com-petitive neat transportation fuel (206).

The cost of the cellulase in the Trichoderma-yeast process isstill prohibitively high, whereas for a direct clostridial coculture

process, the enzymes cost very little because they are producedby the fermenting organism in the course of ethanol produc-tion. The direct fermentation of cellulose to ethanol could save50 cents per gallon compared to a state-of-the-art Trichoder-ma-yeast simultaneous saccharification and fermentation pro-cess, since the former process combines cellulase production,hydrolysis, and fermentation in a single bioreactor (130). Con-version of mixed hardwood flour to ethanol in a continuousfermentor was 2.5 times higher with C. thermocellum than withthe simultaneous saccharification and fermentation process us-ing Trichoderma cellulase, �-glucosidase, and S. cerevisiae(310); furthermore, the rate of conversion was four timeshigher. Mixed cultures of thermophilic anaerobic bacteria offerthe further potential of decreasing the production costs oflignocellulose conversion to ethanol by twofold (198). Withresources dedicated to the exploitation of these bacteria, theconversion of agricultural, forest, and urban resources intoethanol could become an economic substitute for petroleumfuels when oil prices are about U.S. $30 or more per barrel.This new technology could provide a profitable outlet for re-newable resources sooner than would occur by waiting for theTrichoderma-yeast process to become economical by gradualincreases in the price of petroleum. The primary advantages ofa direct clostridial conversion include elimination of capital oroperating costs for enzyme production, greatly reduced diver-sion of substrate for enzyme production, and compatible en-zyme and fermentation systems. Moreover, with the increaseduse of ethanol as an octane enhancer replacing tetraethyl leadand as an oxygenate replacing methyl tert-butyl ether, the de-velopment of an anaerobic bacterial process using waste feed-stocks is likely to be justified at current petroleum prices.

THE CLOSTRIDIUM THERMOCELLUM CELLULASESYSTEM

Strains and Media

Because of the need to develop alternative forms of liquidenergy, in the late 1970s we began studies at the MassachusettsInstitute of Technology (M.I.T.) involving C. thermocellumstrain ATCC 27405. This microbe had been isolated by Viljoenet al. (336) from manure and later described by McBee (218).The Japanese strain F1 and Russian strain F7 are very similarto ATCC 27405 DSM 1237, NCIB 10682) (110). Our groupwas chiefly concerned with its nutrition, regulation of enzymesynthesis, and enzymology. We found that at a low yeast extractconcentration (0.2%), only cellulose and cellobiose werereadily utilized, out of approximately 50 carbon sources. How-ever, upon increasing yeast extract to 0.6%, the organism as-similated glucose (304). We developed a chemically definedmedium in which the yeast extract was replaced by para-ami-nobenzoic acid, vitamin B12, pyridoxamine, and biotin (140)and found that C. thermocellum could use ammonium, nitrate,urea, or certain amino acids as major nitrogen sources (93).The organism was found to break down xylan but could not usethe resulting xylose or xylose oligomers. Another strain of C.thermocellum (YM4) requires para-aminobenzoic acid, vitaminB12, vitamin B6, and folic acid (237).

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Properties of the Cellulase System

Our initial enzymatic studies employed a cellulase assay withthe chromogenic substrate trinitrophenyl-carboxymethylcellu-lose (TNP-CMC), which measured endo-�-glucanase activity.We found the TNP-CMCase activity of C. thermocellum to beconstitutive, neither repressed nor inhibited by cellobiose orglucose (304), and over 95% of the extracellular enzymaticactivity on TNP-CMC appeared to be unaffected by oxygen,thiol compounds, thiol reagents, and mono- or divalent cat-ions.

Unlike fungal cellulases, the C. thermocellum cellulase com-plex has very high activity on crystalline cellulose (140); i.e., ithas “true cellulase activity” (also called Avicelase) which ischaracterized by its ability to completely solubilize crystallineforms of cellulose such as cotton and Avicel (141). This uniquecellulase system has been studied by a number of groups bio-chemically, immunologically, and via molecular biologicaltechniques. The complex is comprised of: (i) numerous endo-�-glucanases which are responsible for the random breakdownof amorphous types of cellulose, including CMC and TNP-CMC (4, 30, 31, 41, 62, 80, 93, 122, 143–145, 162, 221, 239, 240,249, 267, 268, 276, 295, 296, 304, 307, 312); (ii) at least fourexoglucanases (152, 222, 234, 308, 342, 372); (iii) a cellobiosephosphorylase that breaks down cellobiose to glucose and glu-cose-1-phosphate (5); (iv) a cellodextrin phosphorylase thatphosphorylyzes �-1,4-oligoglucans (300); and (v) two �-gluco-sidases that hydrolyze cellobiose to glucose (3, 4, 104). C.thermocellum also possesses at least six xylanases (107, 232),two lichenases (367), two laminarinases (332), and minor ac-tivities of �-xylosidase, �-galactosidase, and �-mannosidase(164). A strain of C. thermocellum has been shown to degradepectin and probably produces pectin lyase, polygalacturonatehydrolase, and pectin methylesterase (314). We found the ac-tivity responsible for hydrolysis of crystalline cellulose, unlikethe endoglucanases, to be inhibited by cellobiose (141); glu-cose had no such inhibitory effect.

In our experiments (141), filter paper was found to be thepreferred substrate for Trichoderma cellulase, whereas cottonwas the best substrate for the clostridial enzyme complex. Thishigh activity on a highly ordered substrate reflects C. thermo-cellum’s ability to proliferate under thermophilic, anaerobicconditions on partially digested plant tissues. As an anaerobedepending solely on glycolysis for its cellular energy, C. ther-mocellum cannot afford to produce large quantities of extra-cellular proteins. Thus, it makes a cellulase complex with about50-fold-higher specific activity than that of Trichoderma underthe assay conditions used; the latter is excreted at a high ex-tracellular concentration but is rather weak in activity.

For a long time, it was puzzling that C. thermocellum couldgrow well on crystalline cellulose whereas its extracellular fluidonly poorly degraded crystalline cellulose (e.g., Avicel). Whenthe organism was grown on agar containing Avicel, we foundclear zones around the colonies, indicating production of anactive, extracellular enzyme. We finally were able to solve thisproblem. We found the extracellular cellulase activity from C.thermocellum to have three unusual properties that had notbeen previously seen with cellulase preparations from otherbacteria or fungi and which made it a uniquely active enzymecomplex. (i) This thermophilic cellulase complex contains sulf-

hydryl groups that are essential either for the saccharificationof crystalline celluloses such as cotton fibers, filter paper, orAvicel or for its structural stability (142). This property re-quires that the cellulase complex be used under reducing con-ditions and renders the activity susceptible to oxidation andsulfhydryl inactivation. When the cellulase activity is protectedby reducing agents such as dithiothreitol (DTT), cysteine, so-dium dithionate, glutathione, or mercaptoethanol, it displayshigh specific activity. (ii) The complex has a requirement forCa2�. Cotton, Avicel, and filter paper are completely solubi-lized under reducing conditions in the presence of Ca2�. Al-though its activity was stimulated by 10 mM DTT, it was in-hibited by a lower DTT concentration (0.1 to 1 mM). Wefound this to be due to autooxidation of low DTT producingH2O2, which inactivates the enzyme under aerobic or anaero-bic conditions (139). The inactivation was prevented by cata-lase but not by superoxide dismutase or hydroxyl radical scav-engers. As expected, sulfhydryl oxidizing agents [e.g.,o-iodosobenzoate and 5,5�-dithio-bis-(2 nitrobenzoic acid)] in-hibited the activity, and the inhibition was reversed by 10 mMDTT. Furthermore, nonoxidizing SH reagents such as N-eth-ylmaleimide, iodoacetate, and p-chloromercuribenzoate werealso inhibitory. Since oxidation of thiol groups involves metals,it was not surprising that a low concentration of EDTA (1 mM)inhibited inactivation by low DTT concentration whereasH2O2 and Cu2� stimulated inactivation. The component of thecellulase complex that was susceptible to sulfhydryl inactiva-tion appeared not to be an endoglucanase, since endoglu-canase activity is unaffected by oxidation or thiol reagents. Itprobably is exoglucanase CelS (also known as SS, equivalent tocomponent S8 of the complex) whose stability is increased byCa2� and thiols and whose activity is inhibited by cellobiose(234). Another exoglucanase (CBH3) has a molecular mass (78kDa) similar to that of CelS but some different properties, suchas substrate specificity and less inhibition by cellobiose; it alsois protected by Ca2� and DTT from high temperature inacti-vation (308). It should be noted, however, that some endoglu-canases (e.g., CelD) are stimulated and stabilized at high tem-perature by Ca2� (55). (iii) The third unusual property of theC. thermocellum true cellulase system is the possible involve-ment of a transition metal ion, presumably iron (139). Cellu-lose saccharification under anaerobic (reducing) conditionswas inhibited by the chelators o-phenanthroline and dipyridyl;this was reversed by preincubation with Fe2� plus Fe3�. Thepresence of a catalytic metal ion and/or sulfhydryl groups couldprovide an explanation for the high specific activity of clostrid-ial cellulase on crystalline cellulose.

Breakdown products of cellulose are both cellodextrins andcellobiose. When these enter the cell, they can be broken downextracytoplasmically via phosphorolytic cleavage by cellodex-trin phosphorylase and/or cellobiose phosphorylase or via hy-drolytic cleavage by �-glucosidase. Two intracellular �-gluco-sidases have been purified, characterized, and cloned (3, 103,148, 153, 294). Rates of phosphorolytic cleavage have beenfound to be 20-fold higher than those of hydrolytic cleavage(365). From a bioenergetic standpoint, phosphorolytic cleav-age is more beneficial because it provides a route to ATPsynthesis. Cellodextrins appear to be more favorable to phos-phorolysis than hydrolysis; the apparent Km for cellodextrinphosphorylase (measured with cellopentaose) was found to be



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considerably lower (0.61 mM) than that for cellobiose phos-phorylase (3.3 mM). It appears that the mean cellodextrinlength assimilated during growth on cellulose is equal to orlarger than four hexose units (Y.-H. P. Zhang, and L. R. Lynd,Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. I-002,2002).

C. thermocellum cells as well as its extracellular cellulasecomplex completely solubilize the model crystalline substrateAvicel and a more realistic substrate, dilute-acid-pretreatedmixed hardwoods (198, 202). With either cells or extracellularfiltrate, the performances on the two substrates were similar,indicating that insoluble lignin does not interfere with growthor hydrolysis. The pretreated hardwood supports growth notonly in batch culture but also in continuous culture. A newprocedure, an enzyme-linked immunosorbent assay, is nowavailable for quantitation of cell and cellulose mass concentra-tions during batch fermentations of C. thermocellum (364).

THE CLOSTRIDIUM THERMOCELLUM CELLULOSOME

Components SL and SS

Early studies in our laboratory on purification of the cellu-lase complex from C. thermocellum (137) indicated that thetrue cellulase activity was part of a large aggregate with amolecular weight of over 1.5 � 106. Workers in Israel (16, 21,179) purified a multisubunit complex from the culture super-natant that they named the cellulosome (18, 20, 174, 175).

The highly ordered arrangement of the cellulosome gives itstability. This resistance to environmental insults correlateswith its resistance to dissociation into individual componentseven in the presence of urea or nonionic detergent; hence,purification of individual proteins was extremely difficult (174,175). However, we accomplished a breakthrough in the puri-fication of this complex aggregate of cellulolytic proteins (353,354). Using Avicel breakdown as a turbidimetric assay for truecellulase activity and CMC hydrolysis as an assay for endoglu-canases, we found the aggregate to contain at least four endo-glucanases of different molecular weights accompanying truecellulase activity. We dissociated the aggregate by mild sodiumdodecyl sulfate (SDS) treatment plus EDTA and DTT, but theresulting individual fractions exhibited only endoglucanase ac-tivity, the true cellulase activity being lost. However, we wereable to reconstitute true cellulase activity by combining two ofthe major components, which we called SS (Mr � 82,000) andSL (Mr � 250,000). SS and SL are more abundant than anyother cellulosomal components (179, 351). They were purifiedby gel filtration chromatography and by elution from an SDS-polyacrylamide gel, respectively. The reconstituted true cellu-lase activity yielded cellobiose as the predominant product ofhydrolysis, was inhibited by cellobiose, required Ca2� and re-ducing conditions, and thus behaved like the crude cell-freesupernatant. In 1984, we proposed that an exoglucanase wasthe component subject to oxidation (139). SS, an exoglucanase,appeared to be that component responsible for cellobiose in-hibition, calcium dependency, and oxidation sensitivity of thetrue cellulase activity (169, 170, 231, 234). These results indi-cate that SS plays an important role in the cellulolytic activityof the cellulosome and that it may be the rate-limiting cellu-losomal component. SL is glycosylated, a rare modification for

a bacterial extracellular protein. As many as 13 cellulosomalproteins may be glycosylated (164, 236, 249), but SL has themajor part of the sugar, with about 40% of this componentbeing carbohydrate (97, 99). The oligosaccharides that havebeen characterized are O-glycosidically attached via galacto-pyranose to threonine residues of SL (98). These threonineresidues are in the Thr/Pro-rich regions which link the cellu-lose-binding domain (CBD) to the enzyme receptor regions(i.e., cohesins; see below) of SL. The major carbohydrate has(i) a basic tetrasaccharide structure containing two galactoseunits, one galactitol unit, and one 3-O-methyl-N-acetylglu-cosamine unit and (ii) a disaaccharide structure containingD-galactose.

SS alone acted on CMC, but SL alone had little to no enzy-matic activity (179, 354). The enzymatic activity of SS on CMCwas not enhanced by SL, but its adsorption to crystalline cel-lulose was (354). We hypothesized that the cooperative degra-dation of crystalline cellulose involves an interaction betweenSS (and presumably other cellulases), SL, and the insolublesubstrate. SL (an anchorage subunit) would function to bind SS

(and other catalytic proteins of the complex) to the cellulosesurface in a manner optimal for hydrolysis (351, 353). As dis-cussed below, the DNA sequence of the SL gene (cipA) revealsthat SL indeed contains a CBD and multiple enzyme receptordomains, consistent with the anchor-enzyme hypothesis. Theanchor-enzyme model has been further confirmed by usingrecombinant forms of SS and SL (168). The anchorage functionof SL is the basis of our current understanding of the cellulo-some structure (see next section).

We and others devoted considerable efforts to the isolationand purification of a number of proteins of the C. thermocellumsystem as well as the cloning, expression, and sequencing oftheir relevant genes in Escherichia coli and other hosts (80, 96,148, 163, 276, 277). Of particular interest was our gene se-quencing of SL (96, 277) and the gene sequencing of SS (341).

Protein SL, which is equivalent to component S1 describedby Lamed et al. (179), is now called the cellulosome-integratingprotein (CipA), the scaffolding protein, or scaffoldin (18). Itcontains approximately 1,850 amino acid residues and is themost important protein of the cellulosome. In addition to itsfunction of binding other members of the cellulase complex toitself, it also binds to cellulose (33). Our first cloning andsequencing of cipA involved a truncated 5� region (277). Later,the remainder of the gene was cloned by chromosome walkingand the entire sequence was determined (96). Its nucleotidesequence revealed a deduced protein size of 196,800 Da, aCBD, and nine domains of about 150 to 166 amino acid resi-dues each. The CBD is of type 3 (28, 96). The nine repeatedsequences, called cohesins by Bayer et al. (18), are quite similarto each other, i.e., exhibiting between 60 and 100% identity,with six of the nine domains being at least 90% identical. Theyare the receptors that bind the individual cellulases, xylanases,and other enzymes to CipA (88, 96, 327) (Fig. 2).

Protein SS, also called CelS (343), is the major catalyticsubunit of the cellulosome and is equivalent to component S8described by the Israeli group (231). It is an exoglucanase(168–170, 231, 234). Sequencing of celS revealed an open read-ing frame of 2,241 bp encoding 741 amino acid residues with apredicted molecular weight of 80,670 (341, 343). The sequencerevealed that CelS belonged to a new cellulase family (341). It



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was later classified as a member of family 48 glycosyl hydro-lases. Although it is the most abundant catalytic subunit of thecellulosome, its low or complete lack of activities on CMC andother synthetic substrates explains why it had been difficult toclone its gene. In fact, relatively few enzymes in this familyhave been identified since we reported the celS sequence. Fam-ily 48 enzymes are found mostly in bacterial cellulase systems(P. M. Coutinho and B. Henrissat, 1999, Carbohydrate-ActiveEnzymes server at http://afmb.cnrs-mrs.fr/CAZY/index.html).They are now considered a key component in the bacterialscheme for breaking down cellulosic materials (133). A recentstudy on Ruminococcus albus mutants that are defective incellulose degradation found that all three mutants failed toproduce family 48 and family 9 enzymes (67), again indicatingthat the family 48 enzymes are crucial for bacterial cellulase sys-tems. CelS is the only family 48 enzyme found in the C. thermo-cellum genome (http://genome.jgi-psf.org/draft_microbes/cloth/cloth.home.html).

CelS contains a duplicated 24-amino-acid-residue dockerin,the site of binding to scaffoldin. The recombinant enzymeproduced in E. coli behaves like an exoglucanase, hydrolyzingphosphoric acid-swollen cellulose faster than Avicel and hy-drolyzing Avicel more rapidly than CMC (169, 342). Like thecellulosome itself, recombinant CelS is inhibited by cellobioseand only marginally so by glucose (167). Inhibition by cellobi-ose was found to be competitive.

In collaboration with Alzari’s group in the Pasteur Institute,we determined the crystal structure of CelS (111). The overallstructure resembles that of C. cellulolyticum CelF (262, 263,271). The protein folds into an (�/�)6 barrel with a tunnel-shaped substrate-binding region. The most salient feature of

the CelS structure is that its tunnel-shaped substrate-bindingsite, which is capable of binding seven glucose moieties, isadjacent to an open cleft that accommodates two glucose moi-eties. It is proposed that the cellulose chain threads throughthe tunnel and that the glycosidic bond between the secondand third glucose residues is hydrolyzed to produce cellobioseas the product. Upon the release of cellobiose from the opencleft, the cellulose chain slides forward through the tunnel witha distance of two glucose units. This “processivity,” consistingof alternating steps of hydrolysis and sliding-threading, ex-plains the cellobiohydrolase nature of CelS. Structural com-parisons with other (�/�)6 barrel glycosidases indicate thatCelS and endoglucanase CelA, a family 8 glycosidase whosesequence is unrelated to that of CelS and which has a groove-shaped substrate-binding region, use the same catalytic ma-chinery to hydrolyze the glycosidic linkage, despite a low se-quence similarity and a different endo-exo mode of action.CelS and CelA can therefore be classified in a new clan ofglycoside hydrolases. For the substrate-binding site, CelA hasan open cleft while CelS has a closed tunnel, explaining theirdifferent endo-exo modes of action, as also observed in otherendo-exo pairs (see reference 324 for a review).

Thus, an association is formed by a synergistic cassette ofcatalytic proteins, which is optimal for hydrolysis of insolublepolymers to the level of soluble oligosaccharides. Synergismbetween two cloned C. thermocellum endoglucanases and onecloned exoglucanase has been observed in vitro (333). Theproximity of these synergistic enzymes to their cellulosic sub-strate as mediated by the scaffolding protein CipA may providethe structural basis for the high specific activity of the cellulo-some.

FIG. 2. A schematic diagram of the C. thermocellum cellulosome. The type I dockerins mediate attachment of the catalytic subunits to thescaffoldin, which is comprised of nine cohesins, a CBD, a hydrophilic domain of unknown function (X) and a type II dockerin. The scaffoldinlikewise binds through its type II dockerin domain to a type II cohesin-containing protein on the bacterial cell surface that is thought to anchorthe complex through a series of three SLH domains (see Fig. 3 for details). Reproduced from reference 337 with permission of John Wiley & Sons.



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Cellulosome Structure

Cellulosomes are crucially important for the efficient break-down and utilization of crystalline cellulose (22, 23, 29, 73, 174,175, 293, 305). The cellulosome is a macromolecular machine(multienzyme complex) which, like a ribosome, is dedicated toorganized, concerted, synergistic, and efficient catalysis of cel-lular activities (22). Cellulosomes are unique in that no otherextracellular protein complexes with the size and complexity ofthe cellulosomes have been reported. They have molecularweights of 2 � 106 to 6 � 106, have diameters of about 18 nm,and contain 14 to 50 polypeptides ranging in size from 37 to210 kDa (164, 174, 175, 215). Over 95% of the endoglucanaseactivity of C. thermocellum is associated with the cellulosome(37). Cellulosomes contain 5 to 7% carbohydrate (99).

Researchers at the University of Georgia (81, 131) foundeven larger aggregates, of ca. 108 � 106 Da (polycellulosomes).Such protuberances covering the surface of the cell are packedwith (poly)cellulosomes; each protuberance seems to containseveral hundred cellulosomes (176). A mutant which did notbind cellulose was found to lack cellulosomes and protuber-ances (21). When cells are grown on cellobiose, cellulosomecomplexes are packed into discrete exocellular structures.When grown on cellulose, these polycellulosome-containingorganelles (protuberances with diameters of 60 to 200 nm)undergo extensive structural modification (17). After attach-ment to the insoluble substrate, the protuberances rapidly ag-gregate into “contact corridors” that physically mediate be-tween the cellulosome, which is attached to the cellulose, andthe bacterial cell surface. Protuberances are not producedwhen the organism is grown under cellulase-repressing condi-tions (178). The proteins of the cellulosome are arranged in ahighly ordered chain-like array (215). The cellulose-bound cel-lulosome clusters appear to be the sites of active cellulolysis,and the products may be channeled down the fibrous structuresto the cell. Cellulosomes also contain lipids with a high con-centration of unsaturated fatty acids (44). The lipid material isthought to be localized mainly at the contact point betweencellulosomes and crystalline cellulose. Both the cellulosomesdescribed by the Israeli group (177) and the polycellulosomesdescribed by the Georgia group display the same requirement forreducing agents and Ca2� that we had found for true cellulaseactivity of the C thermocellum culture supernatant (139).

The most important component of the cellulosome is thenonenzymatic scaffoldin (96, 233). It is a unique scaffoldingprotein subunit, which assembles cellulases and related en-zyme subunits (Fig. 2). The catalytic subunits, on the otherhand, contain different modules (dockerins) which are respon-sible for their attachment to the scaffold (96, 327). Importantin this relationship are (i) cohesin domains on scaffoldin, (ii)dockerin domains on the enzymes, and (iii) a CBD on thescaffoldin, binding the complex to cellulose. Cellulosomes andscaffoldin have been found in many bacteria, such as Clostrid-ium cellulovorans (306), Clostridium cellulolyticum (258), Clos-tridium josui (149), Clostridium acetobutylicum (254, 279), Ace-tovibrio cellulolyticus (360), Bacteroides cellulosolvens, R. albus,Ruminococcus flavefaciens (273), Vibrio sp., and the anaerobicfungal genera Neocallimastix, Piromyces, and Orpinomyces(305). At least eight scaffoldin genes from cellulolytic bacteriahave been sequenced (74). These include cipA from C. ther-

mocellum, cbpA from C. cellulovorans (306), cipC from C. cel-lulolyticum (258), cipA from C. josui (149), cipA from C. ace-tobutylicum (254, 280), scaB from R. flavefaciens (71), cipBcfrom B. cellulosolvens (70), and cipV from A. cellulolyticus (69).The last two named organisms are closely related to the clos-tridia (191). Quite similar to the C. thermocellum cellulosomalcomplex are those of the mesophilic anaerobes C. cellulovorans(72) and C. cellulolyticum (90). In C. cellulovorans, the cellu-losome contains a large, nonenzymatic scaffoldin, called CbpA,which has a signal peptide, a CBD, a hydrophilic domain(HLD) present four times, and a hydrophobic domain presentnine times. The hydrophobic domains are the cohesins of thisspecies. Although C. acetobutylicum is not known to degradecellulose, the genome sequence reveals the presence of a largecellulosome gene cluster (254). This cluster contains the genesencoding the scaffolding protein CipA, the processive endocel-lulase Cel48A, several endoglucanases of families 5 and 9, themannanase Man5G, and a hydrophobic protein, OrfXp. Thegenetic organization of this large cluster is very similar to thatof C. cellulolyticum. An inactive cellulosome with an apparentmolecular mass of 665 kDa has been subsequently reported(279). Recently, the entire scaffoldin (CipA) of this bacteriumhas been cloned and successfully expressed in E. coli (280).Chimeric miniscaffoldins consisting of domains derived fromC. cellulolyticum and C. thermocellum have been expressed inC. acetobutylicum (266). In C. papyrosolvens, a mesophilicanaerobe, the cellulase system (53) can be fractionated byion-exchange chromatography into seven high-molecular-weightmultiprotein complexes, with the molecular weights ranging from500,000 to 600,000. Each complex has a different ultrastructure(269) and a unique profile of enzymatic activities (270). Thecommon protein appearing in each fraction is a glycoprotein (Mr

� 125,000) that lacks any enzymatic activity. Whether this proteinserves as the scaffoldin of the complexes and how these complexesare assembled remain to be investigated.

A number of studies on the dissociation of the cellulosomehave been done. We used a mild SDS-EDTA-DTT treatment(352, 354) to separate the components of the cellulosome.Morag and coworkers (235) found that cellulosomes were dis-sociated under nondenaturing conditions by incubation at 60°Cin the presence of EDTA and crystalline cellulose. During thisdissociation, scaffoldin remained tightly bound to the cellulosebut the enzyme subunits were released. A mixture of the dis-sociated free subunits minus scaffoldin had activity equal tothat of undissociated cellulosomes on soluble or acid-swollencellulose but had only 25 to 30% of the activity on crystallinecellulose. Bhat and Bhat (39) reported that cellulosomes canbe disassociated without much loss of the ability to degradecrystalline cellulose by use of 50 mM Na acetate buffer (pH5.0) containing 10 mM DTT, 10 mM EDTA, and 0.2% SDS at30°C for 25 min.

Cohesins and Dockerins

Two types of cohesin exist: type I cohesins bind specificallyto type I dockerin domains on the catalytic subunits, and typeII cohesins are on some cell surface proteins which bind thedockerin of scaffoldin to the cell wall.

Cloning and DNA sequencing showed that genes encodingat least nine cellulosomal endoglucanases, one exoglucanase



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(169), one xylanase, and one lichenase (367) from C. thermo-cellum contain a highly conserved, noncatalytic region of 50 to60 residues which is usually found at the carboxy terminus.These duplicated sequences, now called docking sequences ordockerins (18), are not essential for catalytic activity (113) butare responsible for the binding of the respective cellulosomalenzymes, e.g., endoglucanase D (CelD) and xylanase Z(XynZ), to one or more of the nine cohesins of CipA (186).Dockerin domains consist of two very similar segments of 22 to24 residues connected by a peptide containing 8 to 17 aminoacid residues; they are over 65% identical among the differentsubunits of the cellulosome (27, 28, 292, 327). Two types ofdockerin exist: type I (186), which anchors catalytic subunits toscaffoldin, and type II, which anchors scaffoldin and free en-zymes to the cell surface. Both depend on Ca2�. All cellulo-somal enzymes contain dockerin modules. Based on currentunderstanding, if an enzyme does not contain a dockerin, it isnot part of a cellulosome. For example, endoglucanase CelCdoes not contain dockerins, does not bind to scaffoldin, and isnot cellulosomal. When the dockerin of CelD was grafted ontoCelC, it gained the ability to bind to CipA (325).

The anchor-enzyme model that we proposed was verified bydata obtained from recombinant proteins (168), showing that(i) recombinant CelS, via its dockerin, forms a stable complexwith cohesin 3 (also known as R3) of CipA but not with theCBD of CipA and (ii) the attachment of recombinant CelS tocellulose is dependent on the presence of a protein sequencecontaining both cohesin 3 and CBD but not on either alone. Inboth of these cases, the binding of CelS was dependent on itsdockerin, since removal of the dockerin eliminated binding.The binding of endoglucanase CelD to a recombinant “mini-CipA” containing a cohesin and a CBD enhanced catalyticactivity, as did the binding of CelS to CipA (89). CelD is themost active endoglucanase of the C. thermocellum cellulosome(92). Scaffoldin enhanced the activity of CelD by at least 10-fold on Avicel but had no effect on the activity of a truncatedCelD lacking an intact dockerin. Similarly, the activity en-hancement of an endoglucanase from C. thermocellum againstAvicel by the presence of scaffoldin was found to be the resultof the attachment of the enzyme to a structure bearing a CBD(151). The anchorage function of the scaffoldin has also beendemonstrated by using the truncated forms of CipC of C.cellulolyticum (260). In C. cellulovorans, mini-CbpA could helpcellulase components degrade insoluble cellulose but not sol-uble cellulose (243), as would be expected from the anchor-enzyme model. In this work, it was also demonstrated that anendoglucanase initiates the attack on cellulose, followed byExgS, which is homologous to CelS.

The CelS dockerin can bind any of the cohesins of CipA(207). In C. thermocellum, the draft genome sequence indicatesthat there are at least 72 dockerin-containing proteins (see“Dockerin-Containing Proteins” below) but only nine cohesinsper scaffoldin molecule. These cohesins are highly homolo-gous, and five of them have more than 90% identity. Thecohesins of a strain recognize nearly all of the dockerins of thesame strain. These observations indicate that, at least in indi-vidual species, binding between catalytic dockerins and scaf-foldin cohesins is relatively nonspecific (32, 168, 207, 362; P.Beguin, Abstr. Pasteur Symp., p. 37–40, 1995), with the excep-tion of one report on binding efficiency or specificity for the C.

cellulovorans CbpA (261). In C. josui, the affinity of a dockerinfor various cohesins can vary, and up to a 34-fold difference hasbeen observed (136). Although cellulosomes in a particularspecies appear to be heterogeneous and their assembly doesnot appear to follow a single pattern, there is specificity be-tween species. For example, there is no interaction betweenthe cohesin domain of C. thermocellum and dockerins of C.cellulolyticum and vice versa (257) (see details in “Assembly ofthe Cellulosome” below).

Calcium is the main metal of the cellulosome, and the reac-tion between dockerins and cohesins requires calcium in C.thermocellum (59, 362) and C. cellulolyticum (259); dockerinsbind calcium (see “Assembly of the Cellulosome” below). Thisis the reason that the cellulosome can be dissociated by mildconditions if EDTA is present (25, 37, 352–354). EDTA inhib-its cellulosomal activity due to its ability to chelate Ca2� (131,137, 139). Ca2� is tightly bound in the cellulosome once it istaken up (58). If incubated in 50 mM Tris buffer (pH 7.5), 0.1M NaCl, and 5 mM EDTA at 37°C, the cellulosome breaks upinto polypeptides and the Ca2� is released. Bands with massesof 160, 98, 76, and 54 kDa are lost, and new bands of 150, 132,91, 71, 57, and 46 kDa appear. The 91- and 71-kDa polypep-tides represent CelS and truncated CelS, respectively. Cleav-age occurs at asparagine residue 681, eliminating 60 residuesfrom the C terminus. All catalytic subunits examined contain asimilar asparagine residue which is part of their dockerin re-gions. Thus, the dissociated catalytic subunits may be suscep-tible to proteolytic degradation at this position.

In view of the calcium requirement for optimal true cellulaseactivity, it is of interest that endoglucanase D has three bindingsites for calcium and that calcium lowers the dissociation con-stant (KD) for CMC and increases thermal stability (81, 147).This endoglucanase also has one possible binding site for zinc(147). Calcium also increases the thermostability of exoglu-canase CelS, the most abundant enzyme of the cellulosome(170). Finally, protein folding of the dockerin is dependent onCa2�, explaining why Ca2� is essential for the cohesin-dock-erin interaction and hence the structural stability of the cellu-losome (210, 211).

Cellulose-Binding Domain

As mentioned above, the enhancement of activity of CelS,CelD, and an endoglucanase from C. thermocellum againstAvicel by the presence of scaffoldin was found to be the resultof the attachment of the enzymes to a structure bearing a CBD(89, 151, 168). Different CBDs appear to target different siteson crystalline cellulose (50). In comparing the C. thermocellumCipA CBD with three others (two from T. reesei and one fromCellulomonas fimi), the CipA CBD appeared to target manymore sites of the cellulose molecule than did the other three.

Individual domains of CipA, obtained by protease or spon-taneous degradation, bind to cellulose, to cellulosomal en-zymes, or both (288). Since certain fragments as large as 200kDa which failed to bind cellulose were obtained, it was con-cluded that the CBD was at one of the termini of CipA anddistinct from the catalytic domain (301). The experimentalobservation is therefore consistent with the CipA domainstructure deduced from its DNA sequence. Although CBDsare also found in some but not all catalytic subunits, the CBD



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on CipA binds cellulose much more tightly than CBDs onindividual cellulosomal enzymes that have been studied. TheC. thermocellum CipA CBD belongs to family IIIa (19, 329) asdo the CBDs on all other known scaffoldins except ScaB of R.flavefaciens, which does not have a CBD. Purified C. thermo-cellum CipA CBD binds crystalline cellulose with a KD of 0.4�M and a maximum binding capacity of 10 mg of CBD per gof microcrystalline cellulose (0.54 �mol/g) (230). The capacityfor amorphous cellulose is 20-fold higher. The KDs of otherclostridial scaffoldin family IIIa CBDs are as follows: 0.6 �Mfor CbpA of C. cellulovorans (102), 0.038 �M for CipA of C.acetobutylicum (280), and 0.14 �M for C. cellulolyticum (91).

The C. thermocellum CipA CBD has been cloned and over-expressed in E. coli, and its crystal structure has been deter-mined (330). As the cohesin, the CBD assumes a nine-stranded� sandwich with a jelly-roll topology. Cellulose binding likelyinvolves interactions between a planar strip of aromatic aminoacid residues on a surface of the CBD and glucose moieties ona cellulose chain and between polar amino acids and two ad-jacent glucose chains of crystalline cellulose. The CBD binds acalcium ion whose function is unknown. The crystal structureof the C. cellulolyticum CipC CBD has also been determined(302). It is very similar to that of the CBD from the CipA, withminor differences. It includes a well-conserved calcium-bindingsite, a putative cellulose-binding surface, and a conserved shal-low groove of unknown function. It is clear that the function ofthe scaffoldin CBD is to anchor the catalytic subunits to thesubstrate surface. An additional function of the CBD in mod-ifying the cellulose surface to facilitate enzymatic hydrolysishas also been suggested (68, 260).

Each of the known scaffoldins except the R. flavefaciens ScaBhas one internal or N-terminal CBD. The catalytic subunit mayalso have its own CBD. The CBD in a nonscaffoldin subunitmay further enhance binding of the cellulosome to cellulose.However, in some family 9 cellulases, their respective catalyticdomains are immediately adjacent to a family IIIc CBD, ofwhich many aromatic amino acid residues on the planar stripthought to be crucial for binding to cellulose are not conserved(19). Examples include CelI (101, 123, 373), CelN (373), CelQ(9), CelF of C. thermocellum (19), CelG of C. cellulolyticum(90), EngH of C. cellulovorans (194, 321), CelZ of C. sterco-rarium (134), and cellulase E4 of Thermomonospora fusca(132). Family IIIc CBDs are considered to play a very differentrole from that of the family IIIa CBDs. The crystal structure ofcellulase E4 revealed the novel feature of the catalytic domainand adjacent family IIIc CBD interacting with each other(285). Sakon et al. (285) first proposed that the CBD may actby binding to a single cellulose chain and feeding it into theactive-site cleft of the catalytic domain and thus that it partic-ipates directly in the catalytic function of the enzyme (285,348). Thus, family IIIc CBDs are better considered as a “cel-lulose-binding subsite” of the catalytic domain (19, 132). In-deed, deletion of the family IIIc CBD from these enzymesrendered the enzyme almost completely inactive (9, 90, 101,132, 285). It is interesting that each cellulosomal catalytic sub-unit (CelF, CelN, CelG, and EngH) has a family IIIc CBD onlybut each noncellulosomal enzyme (CelI, CelZ, and E4) has anadditional family IIIa CBD. These cellulosomal proteins maythus depend on the scaffoldin’s family IIIa CBD for binding tocellulose. It is also interesting that all except 2 of 13 identified

or putative cellulosomal cellulases containing a family 9 glyco-syl hydrolase domain have a family IIIc CBD (7 enzymes) or animmunoglobulin (Ig)-like domain (4 enzymes) immediately ad-jacent to the catalytic domain (370). The Ig-like domain likelyparticipates in the catalytic function, as does the family IIIcCBD. Bayer et al. (23) have classified family 9 glycosyl hydro-lases into four themes of molecular architecture: (i) the themeA enzymes lack any accessory module, (ii) the theme B en-zymes possess a family IIIc CBD fused to the C-terminal endof the catalytic domain, (iii) the theme C enzymes possess anN-terminal Ig-like domain, and (iv) the theme D enzymescontain both an Ig-like domain and a family 4 CBD. Recently,a new type of CBD (i.e., the family 30 CBD) was found to beN terminal to the family 9 catalytic domain of C. thermocellumCelJ, both being linked by an Ig-like domain (8). The family 30CBD binds to cellulose and is crucial for catalytic activity.Although the involvement of the CBD in the catalytic functionremains to be characterized, it is clear that CelJ belongs to anew theme of family 9 enzymes (8). The family 4 CBD isgenerally not essential for catalytic function, except for C.cellulolyticum CelE (94). The family 4 CBD of the C. thermo-cellum CelK has a binding capacity of �4 mmol/g of cellulose(154), similar to those of the family 3 CBDs which have beencharacterized. It is interesting that LicA, a noncellulosomal C.thermocellum �-1,3-glucanase, has four family 4 CBDs at itsC-terminal end (86).

An interesting application of the CBD is its use in cloningcellulosomal genes in E. coli (242). The recombinant productsof such genes from C. cellulovorans unfortunately are ex-pressed as insoluble inclusion bodies. However, when the cat-alytic domain contained the CBD of the noncellulosomalEngD enzyme, the recombinant protein was produced in E.coli in soluble form.

Attachment of Cellulosomes to the Cell Surface

Cellulosomes are attached to the cell during early log phase,they start to become free in late exponential growth, and mostare in the medium and attached to cellulose in the stationaryphase (20, 64, 215). All cellulosomal proteins are excreted tothe outside of the cell, possessing leader peptides which areremoved during excretion (305). The complex is constructedextracellularly, probably at the cell surface. As protein foldingof the dockerin is dependent on Ca2� (210), it is possible thatassembly of the cellulosome is facilitated by the presence ofCa2� outside the cell (337).

CipA itself contains, at its COOH terminus, a dockerin,which suggested that it may self-associate or play a role inanchoring the cellulosome to the surface of the cell (28, 88, 96).Gel scanning densitometry indicated the cellulosome to con-tain at least two CipA components per 2.1-MDa complex (179,180). Evidence against CipA self-association (287) was ob-tained by finding that the seventh cohesin of CipA did not bindto the dockerin of CipA. Indeed, the dockerin of CipA doesnot bind to any of the CipA cohesins (207).

Immediately downstream from cipA in the C. thermocellumgenome are three open reading frames (ORFs) encoding cellsurface proteins, forming a three-gene cluster on DNA. Thesefour genes form two operons (87). All three ORFs encodeC-terminal domains (three repeats of about 65 amino acid



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residues) responsible for the cell surface layer (S-layer) loca-tion of the encoded proteins. These are called S-layer homol-ogy (SLH) repeats, i.e., modules which are present in polypep-tides associated with bacterial cell surfaces and promote anoncovalent attachment of the protein to peptidoglycan of thecell wall (214). The three genes are called olpA (olp for outerlayer protein; previously known as ORF3p), olpB (previouslyORF1p), and orf2p. A fourth gene, sdbA, encoding another cellsurface protein, is in another part of the genome (186). Theproteins encoded by these genes anchor cellulosomes or freeenzymes to the cell.

The gene furthest from cipA, i.e., olpA, encodes a proteincontaining a cohesin sequence in its NH2-terminal regionwhich can bind the dockerins of the catalytic subunits (88, 286).Protein OlpA has been localized to the cell surface of C.thermocellum (286). It was hypothesized that this region mayanchor the cellulosome to the cell surface (87). However, therewas no binding found between the dockerin of CipA and thereceptor domain of OlpA (287). Thus, CipA does not appearto anchor itself to cell surface protein OlpA. OlpA has a singletype I cohesin molecule binding cellulosomal enzymes via theirtype I dockerins. Since the OlpA receptor binds to the dock-erin of CelD, it has been suggested that the catalytic proteinsbind to OlpA prior to the binding to CipA of the cellulosome.Gene olpB encodes OlpB, which is also located at the cellsurface (189).

Protein SdbA of C. thermocellum has a type II cohesin do-main in its NH2-terminal domain which specifically binds thetype II CipA dockerin domain (187); its carboxy terminuscontains SLH repeats. A model of the attachment of CipA tothe cell involving use of its type II dockerin binding to acohesin domain in the SdbA cell surface protein was proposedby Beguin and Alzari (27). SdbA is anchored via its SLHdomain to the S-layer of the cell, which is external to thepeptidoglycan layer of the cell wall. Actually, the binding be-tween CipA and the cell surface probably occurs between thecarboxy-terminal dockerin domain of CipA and the type IIcohesin domains at the amino-terminal ends of OlpB, ORF2p,and SdbA (32). SdbA has one cohesin domain, Orf2p has two,and OlpB has four cohesins, presumably binding one, two, andfour molecules of scaffoldin, respectively. The binding is a typeII interaction (186), which involves calcium, as does the type Iinteraction. Binding of CipA dockerin to SdbA as a function ofCa2� concentration is sigmoid, corresponding to a Hill coeffi-cient of 2, suggesting the presence of two cooperatively boundCa2� ions in the cohesin-dockerin complex. Western blottingof C. thermocellum subcellular fractions and electron micros-copy of immunocytochemically labeled cells indicated thatSdbA is not only a cell surface protein but also a cellulosomecomponent (187). It has been determined that OlpB binds tothe C. thermocellum cell wall with a dissociation constant onthe order of 107 M (56).

Figure 3 summarizes our current understanding of how thecellulosome is attached to the cell surface in C. thermocellum.Some noncellulosomal enzymes, may bind directly to the cellsurface, e.g., xylanase XynX. It contains no dockerin domainbut does have SLH segments (32). Similarly, LicA containsthree SLH domains at its N-terminal end that have been shownto mediate its association with cells (86). Many scaffoldins,including that of C. cellulovorans (CbpA), do not have a dock-

erin. It has been postulated that the four HLDs of CbpA, eachhaving partial homology with the SLH domain, play a role inbinding the cellulosome to the cell surface (320). Furthermore,EngE, one of the three major subunits of the C. cellulovoranscellulosome, has three SLH domains at the N-terminal half, afamily 5 glycosyl hydrolase domain, and a dockerin at the Cterminus (320). It has been shown that EngE bridges the cel-lulosome and the cell by binding to the cellulosome via itsdockerin and to the cell surface via its SLH domains (166).Thus, a cellulosomal enzyme may serve as an anchor for thecellulosome on the cell surface. Most scaffoldins carry HLDswith copy numbers ranging from one to six (74). The HLDs aresometimes called the X domains (19). The solution structure ofone of such domain, the X2-1 domain of C. cellulolyticum, hasbeen determined (238). It has an immunoglobulin-like foldwith two �-sheets packed against each other.

Assembly of the Cellulosome

The three-dimensional structures of two type I cohesin do-mains from C. thermocellum and one from C. cellulolyticumhave been solved by X-ray crystallography (303, 313, 323).Cohesin 2 of C. thermocellum CipA forms a nine-stranded�-sandwich with an overall jelly-roll topology, similar to that ofa bacterial cellulose-binding domain (303, 330). The structureof cohesin 7 of CipA is similar to that of cohesin 2, althoughminor differences were observed. These differences may be dueto amino acid sequence deviations, since the cohesin domainsat both ends of CipA (in particular, cohesins 1, 2, and 9) areless conserved than the central domains, which have 95%identity with one another (136, 207, 323). Despite low se-quence identity (32%), the C. cellulolyticum cohesin domain(cohesin 1 of CipC) has a fold similar to those of the C.thermocellum cohesins. However, the putative dockerin bind-ing surface is notably much less polar. This difference mayexplain the cross-species specificity of cohesin in recognizingthe cognate dockerin (313). Structures of these three cohesinssuggest that all cohesins may be similar in their overall folding.

The type I cohesin-dockerin interaction is extremely strong,with dissociation constants on the order of 1010 M in thepresence of calcium (82, 136, 219, 290). This high affinity ex-plains the stability of the quaternary structure of the cellulo-some in the extracellular environment. Our study (208) andthat of Fierobe and coworkers (82) showed that both dupli-cated segments of the dockerin domain are required for bind-ing to cohesin. As mentioned, it has also been demonstratedthat within a given species, the interaction between the variousdockerins and cohesins is nonselective (207, 362). However,the interaction displays species specificity between dockerinsand cohesins from C. thermocellum and the mesophile C. cel-lulolyticum (257). Based on sequence comparisons of dockerinsfrom these two species, two residues in each duplicated seg-ment, i.e., positions 10 and 11 of each calcium-binding loop,were pinpointed as being likely recognition determinants in thebinding of dockerin to cohesin (257). This prediction has beencorroborated by experiments in which the species specificity ofthe interaction was altered through site-directed mutagenesisof these residues (219, 220). However, the results also indi-cated that additional residues are involved in the interaction,



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FIG. 3. Schematic drawing depicting cellulosome attachment to the cell surface through the type II dockerin-cohesin interaction with SLHdomain-containing cell surface proteins, SdbA, Orf2p, and OlpB (22). On the other hand, OlpA, which contains a type I cohesin, is presumed toanchor an enzyme or protein containing a type I dockerin. Adapted from reference 337 with permission of John Wiley & Sons.



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since binding between the mutated dockerins and the comple-mentary cohesins from the same species was not disrupted.

As a further step toward understanding the cohesin-dock-erin interaction, we undertook a solution nuclear magneticresonance (NMR) study to determine the structure of thedockerin domain from the C. thermocellum CelS. To this date,CelS is the only cellulosomal protein with three-dimensionalstructures of both the catalytic and dockerin domains deter-mined. Interestingly, two-dimensional 15N-1H heteronuclearsingle quantum correlation NMR spectroscopy revealed thatCa2� induces folding of the dockerin into its tertiary structure(210). The calcium-dependent folding may be a mechanismthat evolved to prevent folding into an active form in thecytoplasm, where the free Ca2� concentration is only on theorder of 1 mM. The Ca2� requirement could potentially safe-guard against binding of the catalytic subunits to scaffoldinprior to secretion (210). It is remarkable that the unfoldeddockerin remains soluble at the concentration typically usedfor NMR analysis (�1 mM). The solution structure of thefolded CelS dockerin in the presence of Ca2� has been deter-mined by protein NMR spectroscopy (209, 337). Although thehighly conserved dockerin domain is characterized by twoCa2�-binding sites with sequence similarity to the EF-handmotif, the dockerin domain assumes a completely differentstructure. The structure consists of two Ca2�-binding loop-helix motifs connected by a linker; the E helices entering eachloop of the classical EF-hand motif are absent from the dock-erin domain. This result agrees with the secondary structureprediction reported by Pages et al. (257). Each dockerin Ca2�-binding subdomain is stabilized by a cluster of buried hydro-phobic side chains. Structural comparisons reveal that, in itsnoncomplexed state, the dockerin fold displays a dramatic de-parture from that of Ca2�-bound EF-hand domains and rep-resents a novel Ca2�-binding domain.

The dockerin structure is symmetric, as expected from itsduplicated primary sequence. How the symmetric dockerindocks to the nonsymmetric cohesin had been a puzzle. Whileexperimental data indicate that the type I cohesin and dockerinform a 1:1 complex, results of site-directed mutagenesis onpositions 10 and 11 of the dockerin on either segment suggestthat either segment of the dockerin is capable of binding to thecohesin (290). The notion that only one of the two halves of thesymmetric dockerin is used to bind to cohesin was confirmedwhen the cohesin-dockerin complex structure was determinedby coexpressing cohesin 2 of CipA and the dockerin domainof a xylanase (Xyn 10B) in E. coli (52). While the cohesinstructure remains essentially unchanged in the complex, thedockerin undergoes conformational change and orderingcompared with its solution structure to display a near-per-fect internal twofold symmetry. On the other hand, theclassical 12-residue EF-hand coordination to two calciumions is maintained. Significantly, the cohesin binds predom-inantly to the second segment of the dockerin, suggestingthat the first segment of the dockerin may provide the sec-ond binding site, enabling the formation of a 1:2 complex(one dockerin to two cohesins) and thus a higher order ofthe cellulosome structure. On the other hand, a few aminoacid residues on the first segment also participate in thecomplex formation, through either hydrophobic interactionsor hydrogen bonding. Therefore, both segments of the dock-

erin participate in binding even in a 1:1 complex. The resultsagree with a report demonstrating that the two segments(subdomains) of the CelS dockerin are both required forinteraction with a cohesin (208). Similar results were ob-tained for the C. cellulolyticum dockerin, the two segmentsof which are homologous enough to replace each other (82).The structure of the complex further revealed that protein-protein recognition is mediated by hydrophobic interactionsbetween one face of the cohesin and the helices of thedockerin. Many of these hydrophobic amino acids of thedockerin have been predicted to be involved in proteindocking, since they are solvent accessible in the dockerinsolution structure (209). Isothermal titration calorimetryalso showed that the cohesin-dockerin binding is mainlyhydrophobic (290). Five hydrogen bonds between the twoproteins are found, which are dominated by a Ser-Thr pair(positions 10 and 11 of the Ca2�-binding loop). Notably, theSer-Thr pairs are strictly conserved in the C. thermocellumdockerins but not in the C. cellulolyticum dockerins. Otheramino acid residues forming direct hydrogen bonds with thecohesin, such as Arg (or Lys) and Leu (or Ile), are also wellconserved. Conservation of the amino acid residues involvedin hydrophobic interactions and hydrogen bonding in boththe cohesin and dockerin explains the lack of binding selec-tivity in cohesin-docking binding in C. thermocellum (207,362). On the other hand, replacements of the hydrogenbonding residues (Ser, Thr, and Arg) with other residues(Ala, Ile, and Lys) in C. cellulolyticum dockerins provide apartial explanation for the cross-species specificity of thedockerin in recognizing the cognate cohesin. Bioinformaticanalysis and site-specific mutagenesis have previously indi-cated the important roles of the dockerin’s Ser-Thr pair andArg (position 22 based on numbering of the Ca2�-bindingloop) in complex formation (219, 220, 257). The cohesinamino acids previously identified to be involved in dockerin-cohesin interaction by site-directed mutagenesis (226) werefound to be present in the interface of the complex (52).

The structures of the type I dockerin, cohesin, and theircomplex shed light on how the cellulosome is assembled andprovide a blueprint for reengineering the cellulosome for bio-technological explorations. Unfortunately, to date, no three-di-mensional structures of type II components have been reported.The dissociation constant for a type II cohesin-dockerin pair (i.e.,the CipA dockerin and the SdbA cohesin) is on the order of 109

M (135). Secondary-structure assignments of the SdbA cohesinhave been made by 1H, 13C, and 15N protein NMR (309). It wasfound that the positioning of the secondary structural elements isvery similar to that observed in the type I cohesin except that an�-helix was present between �-strands 6 and 7 of SdbA, whichmay contribute to the type I-type II specificity. Further elucida-tion of the structures of type II components is essential for un-derstanding the molecular basis of this specificity.

Although species specificity is generally observed, exceptionshave been reported. For example, in C. thermocellum, thedockerin of Cel9D-Cel44A (formerly CelJ [1]) does not seemto bind to cohesins 1, 4, and 7 (136) or to cohesins 2 and 3 (362)of CipA. Its binding specificity remains to be determined. Fur-thermore, in C. josui, the affinities of binding of a dockerin tovarious cohesins can vary as much as 34-fold (136). Anotherexception to species specificity was reported in the same study,



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revealing that the Xyn11A dockerin of C. thermocellum bindsto various cohesins of C. josui with high affinities (KD of �108

M) (136). Thus, binding specificity and affinity may be influ-enced by subtle differences in the primary or tertiary structuresof the dockerin and cohesin. Further studies are needed tocompletely understand these subtleties.

Genes and Enzymes

Genes cloned from C. thermocellum in E. coli, S. cerevisiae,Bacillus subtilis, Bacillus stearothermophilus, and Lactobacillusplantarum include cipA and genes for over 30 endoglucanases,4 exoglucanases (also known as cellobiohydrolases), 2 cellular�-glucosidases, 6 xylanases, 2 lichenases (�-1,3-1,4-glu-canases), 1 putative xylan esterase, 2 laminarinases (�-1,3-glucanases), 1 mannanase, 1 chitinase, and 1 pectate lyase (15,22, 27, 34, 46, 62, 63, 83, 96, 103, 104, 106, 107, 120, 124, 143,145, 148, 160, 162, 163, 221, 222, 225, 239, 240, 267, 268,275–278, 281, 284, 291, 292, 295–297, 299, 305, 312, 332, 341,343, 366, 367, 369, 371).

Four cellulosomal cellobiohydrolases have been reported inC. thermocellum. They are CbhA (formerly Cbh3; componentS3) (222, 308, 332, 375), CelS (S8) (343), CelK (S5) (152), andCelO (372). Genes cbhA and celK have been cloned and se-quenced. Gene celK is upstream of cbhA with an interveningsequence of 524 bp (374). The two genes are highly homolo-gous, both encoding a family 4 CBD, an Ig-like domain, afamily 9 glycosyl hydrolase, and a dockerin. The only differenceis that CbhA contains two fibronectin-like domains and a fam-ily 3b CBD, both N terminal to the dockerin. These two genesare likely the result of gene duplication and recombination.CelO consists of a family 3b CBD, a family 5 glycosyl hydrolysisdomain, and a dockerin. It produces cellobiose as the onlyproduct from cellulose. The crystal structures of CelS in com-plex with its substrate or inhibitor indicate that CelS hydrolyzesthe cellulose chain from the reducing end (111). CelO activitieson various substrates indicate that it also attacks the cellulosechain from the same end (372). On the other hand, the samestudy revealed that CbhA hydrolyzes the cellulose chain fromthe nonreducing end. The facts that the catalytic domain ofCelK is highly homologous to that of CbhA and that CelK isactive on p-nitrophenyl-�-D-cellobioside indicate that its modeof action is like that of CbpA (13). Why the bacterium needstwo sets of exocellulases remains to be explained. It appearsthat CbhA and CelO are capable of reducing the viscosity of aCMC solution (332, 372), whereas CelS is not (170, 234),indicating that the former two enzymes have some endoglu-canase activity. A recent reexamination of the mode of actionof CbhA showed that it had a very high activity on CMCrelative to that on ball-milled crystalline cellulose, it rapidlyreduced the viscosity of CMC, and it produced 40% insolublereducing sugars from filter paper. Thus, in these tests CbhAbehaved like an endocellulase (C. McGrath and D. B. Wilson,personal communication), and it should be reclassified as such.The results illustrate the importance of using multiple criteriato evaluate an exoglucanase. CelS behaved like an exoglu-canase in these same tests (170, 342). The behaviors of CelKand CelO in these tests remain to be studied. CelF, the CelSequivalent in C. cellulolyticum, also displays significant activityin reducing the viscosity of a CMC solution (271).

Some of the cloned endoglucanase genes are celA (26), celB(105), celC (298), celD (143, 326), celE (113), celF (245), celG(188), celH (361), celI (101, 123, 373), celJ (encoding compo-nent S2 of the complex) (1, 8), celM (162), celN (373), celQ (9),celT (172), and celX (113).

Six of the xylanases present in the C. thermocellum cellulo-some are XynA, XynB, XynC, XynX, XynY, and XynZ (107),the genes of which have all been cloned (121, 146). Despite thelarge number of xylanases, the organism cannot grow on xylanor xylose.

The cellulosomal pectate lyase gene in C. cellulovorans hasan ORF containing 2,742 bp and encoding a protein of 914amino acid residues with a molecular mass of 94,458 Da. Theprotein contains a dockerin at its C terminus. It breaks downpolygalacturonic acid into di- and trigalacturonic acids. This isthe first reported pectate lyase in cellulolytic clostridia (319).The mannanase gene man26B of the C. thermocellum cellulo-some has been cloned and sequenced (171).

Sequencing of celS has been described above (see “Compo-nents SL and SS”). Another important catalytic component isCelJ (2), the largest catalytic subunit, which is an endoglu-canase equivalent to component S2 described by Lamed et al.(179, 180). Like CelS, it has some activity on Avicel, and it alsoattacks CMC, lichenan, and xylan.

In the early 1990s, we reported on the isolation of three newendoglucanases. These included one having a molecular massof 83 kDa, an isoelectric point of 3.55, an optimum pH of 6.6and an optimum temperature of 70°C (80). It hydrolyzed CMCand, at a higher rate, cellotetraose and cellopentaose; Avicel,cellotriose, and cellobiose were not attacked. It was originallythought to be CelS due to its similar size and its reaction withCelS antibodies; however, CelS has been found to be an exo-glucanase (234). Thus, our designation of this protein as CelSwas erroneous. A second new endonuclease had the followingproperties: an Mr of 76,000, an isoelectric point of 5.05, anoptimum pH of 7.0, and an optimum temperature of 70°C(276). Both of these endoglucanases were inhibited by Hg2�

and p-chloromercuribenzoate, suggesting the existence of thiolgroups essential for activity or stability. Ca2� stimulated bothendoglucanases, whereas Mg2� stimulated the 76-kDa enzymebut inhibited the 83-kDa enzyme. NaCl inhibited the largerenzyme but not the smaller one. The substrate specificities ofthe two enzymes were similar. A third new endoglucanase wasCelM (162). Its nucleotide sequence indicated a molecularmass of 35 kDa. The enzyme had an optimum pH of 5.5 to 6.5and a temperature optimum of 60°C. It lacked the dockerinand thus appeared to be noncellulosomal. It attacked CMC butnot Avicel or xylan. It was not affected by Ca2�, Mg2�, orNaCl. Its inhibition by p-chloromercuribenzoate suggested thepresence of thiol groups; the deduced amino acid sequenceindicated the presence of seven cysteine residues.

A simplified cellulosomal fraction which was more active onAvicel (8-fold) and on CMC (15-fold) than the crude extracel-lular protein was isolated from the cellulase complex of C.thermocellum (161); it was called the subcellulosome prepara-tion. The isolation utilized a lectin (Jacalin) preferentiallybinding to galactosyl carbohydrates. Subcellulosome activitywas inactivated by SH reagents and inhibited by the chelatorsEDTA and �-phenanthroline. This subcellulosome was com-posed of six major proteins, including CipA and CelS, ranging



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in molecular mass from 58 to 210 kDa. These were cloned andexpressed in E. coli (163). A similar subpopulation of cellulo-somes was isolated by cellulose affinity chromatography fol-lowed by anion-exchange chromatography (6). Its activity wasslightly higher than that of unfractionated cellulosomes andwas similarly affected by calcium, DTT, and cellobiose. Of thesix subunits in this subpopulation, three were known cellulo-somal proteins, but the other three were not. Another subcel-lulosomal preparation (minicellulosome) was constructed byusing CipA, exoglucanases S5 and S8 (CelS), and endoglu-canase S11. Adding CipA to the other three enzymes increasedAvicel hydrolysis sevenfold (39).

Dockerin-Containing Proteins

Proteins containing a dockerin domain are considered com-ponents of the cellulosome. To date, a large number of thegenes encoding dockerin-containing proteins have been clonedand characterized. However, the intriguing questions concern-ing how many exist in a clostridial genome and the nature ofthese gene products remain unresolved. These questions arenow partially answered, thanks to the availability of the C.thermocellum draft genome sequence. We have submitted theC. thermocellum ATCC 27405 genomic DNA to the U.S. De-partment of Energy Joint Genome Institute for sequencing,and a draft genome sequence is now available (http://genome.jgi-psf.org/draft_microbes/cloth/cloth.home.html). Searchingof the draft genome sequence for genes encoding dockerin-containing proteins yielded at least 72 entries, with only 28 ofthem having been previously identified through genetic cloning(368, 370). These dockerin-containing proteins are listed inTable 2. Besides CipA, there are enzymes of various glycosylhydrolase families, including families 5 (5 entries, including CelO,CelB, and CelG), 8 (1 entry [CelA]), 9 (14 entries, includingCbhA, CelK, CelD, CelN, CelR, CelQ, CelF, and CelT), and 48(1 entry [CelS]) and multifunctional proteins (8 entries, includingCelJ, CelH, XynZ, XynY, and CelE). In addition, there are atleast four xylanases (XynA, XynB, XynC, and XynD), one lichen-ase (LicB), one chitinase (ChiA), one mannanase (ManA), onexyloglucanase (XghA), four putative hemicellulases, five putativeglycosidases, four putative carbohydrate esterases, and five puta-tive pectinases. Also interesting is that the list contains 1 putativeprotease, 2 putative protease inhibitors, and 13 proteins withunknown functions (including CseP).

It is thus clear that in C. thermocellum alone, there are asurprisingly large number of proteins which can potentially be“inserted” into the cellulosome through their dockerin domains.The cellulosome may therefore be a versatile extracellular or-ganelle whose functions can be tailored by incorporating differentdockerin-containing subunits. The roles of the cellulosome inclostridial physiology must be complicated, and a clear pictureawaits further studies. Inevitably, these studies will have to revealhow the organism coordinates the expression of these dockerin-containing proteins in concert with the expression of noncellulo-somal proteins. As described below, we know only very littleabout the regulatory processes at this time.

CARBON SOURCE NUTRITION

C. thermocellum has been reported to utilize a number ofdifferent sugars, but quite a bit of disagreement is seen in

various reports. McBee (217) reported growth of strain 651with cellulose, hemicellulose, cellobiose, and xylose but notwith glucose, fructose, sorbitol, or mannitol. Growth on xyloseby strain ATCC 27405 (the designation given by the AmericanType Culture Collection to the 651 strain) was not confirmedby us (304). We also did not observe growth on xylan, a com-ponent of hemicellulose (93). Patni and Alexander (264, 265)found C. thermocellum 651 to grow on mannitol, glucose, xy-lose, and fructose. Ng et al. (248) obtained no growth of strainLQ8 on glucose and xylose but later obtained growth on glu-cose of strain LQ8Rl, derived from LQ8 (181). Strain YS hasbeen reported to grow only on cellulose or cellobiose (76). C.thermocellum I-1-B grows on esculin in addition to cellulose,cellobiose, glucose, and fructose (289). Recently, we found thatC. thermocellum ATCC 27405 grows well on laminaribiose, a�-1,3-linked glucose dimer, with a growth rate and extent com-parable to those obtained on cellobiose (M. Newcomb andJ. H. D. Wu, unpublished data). The growth of a number ofstrains on glucose in complex medium after a lag of 30 h hasbeen reported (125, 126, 212, 346). We later obtained growth ofstrain ATCC 27405 on glucose, fructose, and sorbitol (but not onmannitol) in chemically defined medium, but only after an excep-tionally long lag of over 100 h (138). With cellobiose as the solecarbon source, C. thermocellum achieved its maximum cell densitywithin 20 to 24 h. When a cellobiose-grown inoculum was trans-ferred to chemically defined medium with either glucose, fruc-tose, or sorbitol as the sole carbon source, there followed a longlag of over 100 h. At the end of this time period, growth occurredon these three carbon sources, and subsequent transfers intomedium with the same carbon source allowed growth to occurwithin 24 h. Freier et al. (85) reported growth of strain JW20 oncellulose, cellobiose, and xylooligomers and, after long-term ad-aptation, on glucose, fructose, and xylose. We thus felt that theuse of carbon sources other than cellulose and cellobiose oftendepends on the presence of high levels of yeast extract and/orrequires long periods of adaptation.

One possible explanation considered for the long lag periodwas physiological adaptation; i.e., the pathways for the utiliza-tion of glucose, fructose, and sorbitol could require inductionor deregulation, and thus the cells would not grow immediatelyon these carbon sources because they could not internalize andutilize these carbon sources immediately. To understand themode of sugar utilization in this organism, we felt it essential tostudy the differences observed when cellobiose-grown cellswere fed cellobiose, glucose, and fructose separately and tocompare these results with those for glucose-grown cells fedglucose and fructose-grown cells fed fructose (253). One of thekey factors related to growth is the energy status of the cell. Inanaerobic bacteria, the proton motive force is generatedthrough the activity of the proton-translocating ATPase, whichhydrolyzes ATP (generated via glycolysis) and causes the ex-trusion of protons. According to the chemiosmotic theory(229), the transmembrane electrical and chemical potentialsgenerated in this manner create a driving force to performwork, and a number of cellular processes can be driven in thismanner (119). The three major components related to theenergetics of a cell are (i) the chemical potential (�pH), (ii)the electrical potential (�Y) (which together constitute theproton motive force, �p), and (iii) ATP, which is the drivingforce, especially in anaerobic bacteria. Cell bioenergetics have



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TABLE 2. Dockerin-containing proteins found in the draft genome of C. thermocellum

Protein family orputative function group Gene producta Putative function and/or reading frameb Mol

mass (kDa) Reference(s) Module structurec

Structural component CipA (�) Scaffoldin, Chte02002467, ZP_00312244 197 88, 96, 374 2(Coh1)-CBM3a-7(Coh1)-UN-Doc2

Cellulase/�-glucanase CelO Cellobiohydrolase, Chte02003367,ZP_00311446

75 372 CBM3b-GH5-Doc1

— Chte02003044, ZP_00311743 103 GH5-CBM6-Fn3-Doc1CelB Endoglucanase, Chte02000960,

ZP_0031359364 105 GH5-Doc1

CelG (�) Endoglucanase, Chte02001813,ZP_00312831

63 188 GH5-Doc1

— Chte02001497, ZP_00313148 58 GH5-Doc1CelA (�) Endoglucanase, Chte02000171,

ZP_0031435953 30 GH8-Doc1

CbhA (�) Cellobiohydrolase, Chte02001488,ZP_00313140

138 375 CBM4-Ig-GH9-2(Fn3)-CBD3b-Doc1

CelK (�) Cellobiohydrolase, Chte02001490,ZP_00313141

101 152, 374 CBM4-Ig-GH9-Doc1

CelD Endoglucanase, Chte02001777,ZP_00312895

72 143 Ig-GH9-Doc1

— Chte02001890, ZP_00312800 110 GH9-CBM3c-CBM3b-Doc1— Chte02001086, ZP_00313565 105 GH9-CBM3c-CBM3b-Doc1CelN (�) Endoglucanase, Chte02000571,

ZP_0031403582 373 GH9-CBM3c-Doc1

CelR (�) Endoglucanase, Chte02001003,ZP_00313635

85 GH9-CBM3c-Doc1

CelQ (�) Endoglucanase, Chte02001251,ZP_00313301

80 9 GH9-CBM3c-Doc1

CelF endoglucanase, Chte02000967,ZP_00313600

82 245 GH9-CBM3c-Doc1

— Chte02001891, ZP_00312801 80 GH9-CBM3c-Doc1— Chte02001467, ZP_00313120 89 GH9-CBM3c-Doc1— Chte02002786, ZP_00311957 82 GH9-CBM3c-Doc1— Chte02000166, ZP_00314354 63 GH9-Doc1CelT (�) Endoglucanase, Chte02001327,

ZP_0031323569 172 GH9-Doc1

CelS (�) Exoglucanase, Chte02001227,ZP_00313420

83 341 GH48-Doc1

Xylanases XynD (�) Xylanase, Chte02000364, ZP_00314122 72 CBM22-GH10-Doc1XynC (�) Xylanase, Chte02001156, ZP_00313489 70 120 CBM22-GH10-Doc1XynA, XynU (�) Xylanase, Chte02001934, ZP_00312745 74 121 GH11-CBM4-Doc1-NodBXynB, XynV (�) Xylanase connecting error? 50 121 GH11-CBM4-Doc1

Other hemicellulases LicB (�) Lichenase, Chte02000203, ZP_00314391 38 367 GH16-Doc1ChiA (�) Chitinase, Chte02000170, ZP_00314358 55 366 GH18-Doc1ManA (�) Mannanase, Chte02001325,

ZP_0031323467 114 CBM-GH26-Doc1

— Chte02000583, ZP_00314046 67 GH26-Doc1— Chte02002707, ZP_00312042 71 GH30-CBM6-Doc1— Chte02002259, ZP_00312443 47 GH53-Doc1— Chte02002200, ZP_00312458 86 GH81-Doc1

Putative glycosidases — Chte02003048, ZP_00311747 104 GH2-CBM6-Doc1— Chte02003396, ZP_00311430 88 GH39-2(CBM6)-Doc1— Chte02003047, ZP_00311746 59 GH43-CBM6-Doc1— Chte02002202, ZP_00312459 64 GH43-CBM13-Doc1— Chte02000090, ZP_00314291 75 GH43-2(CBM6)-Doc1

Xyloglucan hydrolase XghA (�) Xyloglucanase, Chte02002261,ZP_00312445

92 GH74-CBM2-Doc1

Putativecarbohydrateesterases

— Chte02001642, ZP_00312970 91 Fn3-CE12-Doc1-CBM6-CE12

— Chte02001749, ZP_00312868 55 CE3-CE3-Doc1— Chte02001822, ZP_00312838 55 Doc1-CE6

Continued on following page



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been linked to different phenomena, including product forma-tion (43, 45, 241), chemotaxis (156, 316), and the growth statusof the cell (150). Although there were a few reports on themetabolism of sugars by C. thermocellum, aspects such as therate of utilization of sugars, formation of sugar phosphates,modes of sugar phosphorylation, and bioenergetics had notbeen examined in any detail.

We carried out a study of the bioenergetics of C. thermocellum;the mode by which cellobiose, glucose, and fructose were metab-olized; the mechanism of the lag observed when a cellobiose-grown inoculum was transferred to a medium with glucose orfructose as the sole carbon source; and correlations between en-

ergy-related parameters and cellulase production (253). Long lagsof 180 to 200 h were observed, followed by rapid growth onglucose or fructose. During the lag on fructose, true cellulaseproduction increased markedly, but during the lag on glucose,production was poor. These observations were similar to thosepreviously reported by Johnson et al. (138). When cells began togrow on fructose, cellulase levels were still elevated compared tothose in cellobiose-grown cultures. The final cell densities reachedwith glucose and fructose were similar to that achieved on cello-biose. Repeated transfers on glucose or fructose resulted ingrowth of the culture within 24 h, and repeated transfers onfructose (but not glucose) resulted in maintenance of high cellu-

TABLE 2—Continued

Protein family orputative function group Gene producta Putative function and/or reading frameb Mol

mass (kDa) Reference(s) Module structurec

— Chte02003045, ZP_00311744 54 CE1-CBM6-Doc1

Putative pectinases — Chte02001171, ZP_00313369 92 GH28-Doc1— Chte02002538, ZP_00312205 60 PL1-Doc1-CBM6— Chte02000923, ZP_00313704 98 PL1-Doc1-CBM6-PL9— Chte02002537, ZP_00312204 42 PL10-UN-Doc1— Chte02002767, ZP_00311987 89 Doc1-CBM6-PL11

Multifunctionalcomponents

CelJ (�) Cellulase, Chte02001252, ZP_00313302 178 1 CBM30-Ig-GH9-GH44-Doc1-UN

CelH Endoglucanase, Chte02003109,ZP_00311690

102 361 GH26-GH5-CBM9-Doc1

— Chte02003393, ZP_00311428 111 GH30-GH54-GH43-Doc1— Chte02001578, ZP_00313008;

Chte02001577, ZP_00313007, frameshift?

79 GH54-Doc1-GH43

— Chte02003394, ZP_00311429 66 GH54-GH43-Doc1ZynZ (�) Xylanase, Chte02002412, ZP_00312306 92 106 CE1-CBM6-Doc1-GH10XynY Xylanase, Chte02002344, ZP_00312390 120 83 CBM22-GH10-CBM22-Doc1-CE1CelE (�) Endoglucanase, Chte02001748,

ZP_0031286790 122 GH5-Doc1-CE2

Putative proteaseand proteaseinhibitors

— Chte02001648, ZP_00312975 40 Subtilisin-like serine protease-Doc1

— Chte02000228, ZP_00314411 64 Fn3-Doc1-serpin— Chte02000229, ZP_00314412 68 Doc1-serpin

Components withunknown function

— Chte02002760, ZP_00311980 117 2(UN)-UN-UN(CelP 550-870)-Doc1

— Chte02003046, ZP_00311745 105 UN-CBM6-Doc1CseP (�) Chte02000570, ZP_00314034 (structural

component)62 373 UN-Doc1

— Chte02002500, ZP_00312225 58 Doc1-UN— Chte02000182, ZP_00314370 51 Doc1-U— Chte02001566, ZP_00313102 76 Doc1-UN— Chte02002663, ZP_00312096 65 2(UN)-Doc1— Chte02003357, ZP_00311454 135 UN-UN-UN-Doc1

Chte02001465, ZP_00313118 40 Doc1-UNChte02001653, ZP_00312979 47 UN-Doc1Chte02000318, ZP_00314080 37 UN-Doc1Chte02001119, ZP_00313455 236 2(UN)-UN-Doc2Chte02002121, ZP_00312553 19 Doc1-Doc1

a The ORFs in the draft genome sequence, each containing at least a dockerin molecule, are listed. A gene designation indicates that the gene encoding thecomponent has been cloned and the encoded protein has been biochemically characterized; a dash indicates that this is not the case. �, the existence of the componentin the cellulosome has been experimentally verified.

b The enzymatic activity or function (putative or known) and the ORF number (Chte) from REFSEQ accession number AABG00000000 on the NCBI website(http://www.ncbi.nlm.nih.gov/RefSeq/; database as of 17 June 2004) are provided. The Chte number indicates the locus tag in the genome sequence (nucleotide searchRefSeq_DNA); the ZP number indicates the protein identification code (protein search RefSeq_Prot).

c Module classification according to P. M. Coutinho and B. Henrissat (Carbohydrate- Active Enzymes server at http://afmb.cnrs-mrs.fr/CAZY/index.html, 1999). Coh,cohesion; Doc, dockerin; CBM, carbohydrate-binding module; GH, glycosyl hydrolase family; Fn3, fibronectin III; Ig, Ig-like fold; CE, carbohydrate esterase; PL, pectinlyase; UN, module with unknown function; serpin, serine protease inhibitor homologous module. Adapted from reference 368 with permission of Wiley-VCH Verlag.



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lase titers. The growth rates of C. thermocellum growing on cel-lobiose, glucose, and fructose were comparable.

Data on the energetics of C. thermocellum, obtained with invivo 13C-NMR measurements (251, 328), revealed that fructosewas transported into cellobiose-grown cells at a higher rate thancellobiose, which in turn was transported faster than glucose.When these rates were compared to those of glucose transport inglucose-adapted cells and fructose transport in fructose-adaptedcells, it became clear that the rate of sugar transport was not thecause of the long lags on glucose and fructose. The rates of sugarphosphate formation were comparable in all cases, leading to theconclusion that the initial steps in glycolysis were not limitinggrowth. The mode of sugar phosphorylation was found to be ATPdependent for all three sugars (251). Thus, there was no apparentcorrelation between the growth lag and the mode of sugar phos-phorylation. ATP measurements on cellobiose-grown cells fedcellobiose, glucose, or fructose and on fructose-grown and glu-cose-grown cells fed fructose and glucose, respectively, revealedthat in all cases there was no lack of ATP for carrying out cellularfunctions. In fact, the ATP levels were higher in cellobiose-growncells fed glucose and fructose than in such cells fed cellobiose. Thevalues of �Y, the transmembrane electrical potential, were com-parable irrespective of the carbon source fed to cellobiose-growncells. Thus, the �Y values did not offer an explanation for theinitial lags on glucose and fructose from a cellobiose inoculum. Itwas obvious that the hypothesis described above, i.e., that growthlags were due to a lack of ability to take up or metabolize fructoseand glucose, was incorrect. Two other hypotheses were consid-ered next: (i) that the lag represents the time taken for a mutantcell (capable of utilizing glucose or fructose), which was present inthe cellobiose-grown inoculum, to grow, and (ii) that eventualgrowth is due to a mutation(s) during the extended lag phase.Plating experiments with cellobiose-grown cells showed that col-onies appeared suddenly at 180 to 200 h and not before. Thus, thelag was not due to mutant cells carried over from the inoculum(253). Instead, the occurrence of mutations at a high frequency (2� 104) was found to be the mechanism involved. The mutationswere stable in that after mutant cells were grown on fructose orglucose and put through a transfer on cellobiose, they still grewrapidly on fructose or glucose. These findings (138, 253) consti-tuted one of the early reports of adaptive mutation (48, 112, 350).

Another interesting finding was that during the lag in glucoseor fructose, the cells have a much lower �pH (or internal pH)than do cells actively growing in cellobiose, glucose, or fructose(250). This low pH gradient and the lack of growth is terminatedby the mutation which consistently occurs. We postulated that�pH (or internal pH) might be the critical factor for the lag ingrowth. Two alternative hypotheses might explain the relationshipbetween �pH and the initial lack of growth on glucose and fruc-tose. Either (i) the breakdown and internalization of the disac-charide cellobiose contribute to the formation of the essential pHgradient or (ii) the transport of glucose and fructose via protonsymport dissipates the pH gradient and effectively preventsgrowth until the mutation(s) somehow corrects the problem.

REGULATION OF CELLULASE PRODUCTION

Carbon Source Regulation

Growth conditions markedly affect the cellulolytic activity ofC. thermocellum (85, 138, 177, 253) as well as the profile of

cellulosomal components (21). The overall cellulase activity ishigher when cells are grown on cellulose than when they aregrown on cellobiose (21, 177, 340, 352). Although total endo-glucanase activity in C. thermocellum is constitutive and notsubject to carbon source repression (93, 115, 304), transcrip-tion of endoglucanase genes celA, celD, and celF appears to becontrolled temporally (227). Transcripts were not detected un-til the late exponential growth stage, probably at the time thatthe cellobiose concentration had markedly decreased. We ob-served that C. thermocellum carefully regulates its productionof true cellulase activity, with enzyme production being depen-dent upon the source and availability of carbon and energy(138). True cellulase activity was formed when the bacteriumwas grown with cellulose, cellobiose, glucose, fructose, or sor-bitol, but specific titers varied more than 200-fold between thedifferent substrates. Unexpectedly, marked derepression re-sulting in very high transient rates of synthesis was observedwhen C. thermocellum was adapting, during long-term (over100-h) lag phases, to growth on fructose or sorbitol but notwhen it was adapting long term to growth on glucose. High-level enzyme production was also observed when carbon andenergy availability was limited by growth on crystalline cellu-lose. High cellulase production on fructose was repressed rap-idly by addition of cellobiose (138). Superior enzyme produc-tion on fructose and sorbitol is thought to reflect theirinactivity as carbon source repressors, in contrast to the re-pressive activity of cellobiose and glucose (340; J. H. Wang andJ. H. D. Wu, Abstr. Annu. Meet. Soc. Ind. Microbiol., abstr. p.25, 1989). There is no evidence that a specific inducer is in-volved in regulation of cellulase synthesis (138), despite a claimthat cellobiose is an inducer (40). At present, it appears thatcarbon source repression and/or growth rate (see below) is theprincipal mechanism of regulation of cellulase synthesis in C.thermocellum. It is worth noting, however, that laminaribiosewas recently shown to serve as an inducer for genes encodingcellulases which are also active on �-1,3-glucan (246) (for de-tails, see “Negative regulation” below).

As mentioned above, when C. thermocellum was grown onthe different carbon sources, the amount of true cellulase ac-tivity varied. The true cellulase titer was highest in cells grownon fructose, intermediate in cells grown on glucose, and lowestin cells grown on cellobiose. Due to the fact that the mode ofsugar phosphorylation is ATP dependent for all three sugarsstudied (251), no correlation could be drawn between sugartransport and the (de)regulation of cellulase. Since the intra-cellular ATP level was highest on fructose, lower on glucose,and lowest on cellobiose, there was a direct (but not linear)relationship between cellular ATP and true cellulase produc-tion. There was no obvious relationship between �pH, rates ofuptake of inorganic phosphate and of sugars, and the truecellulase titer. However, �p, the proton motive force, and,more directly, its electrical potential component, �Y, showed anonlinear inverse relationship with true cellulase titer (252).

Recent studies by Dror et al. (76), using batch cultures aswell as continuous culture, have shown that CelS transcriptionis controlled by growth rate under conditions of limitation ofthe carbon or nitrogen source. Expression under carbon re-striction was higher than that under nitrogen limitation, sug-gesting some effect of carbon source on control of transcrip-tion. Transcriptional activity was found to be inversely



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proportional to growth rate. Expression of celS was threefoldhigher in the mid-exponential growth phase on cellulose(growth rate of 0.23 h1) than on cellobiose (growth rate of0.35 h1). These data confirmed those of Johnson et al. (138),who observed higher levels of cellulase with cells growingslowly on crystalline cellulose or during adaptation to growthon fructose or sorbitol. Dror et al. (76) also observed twomajor transcriptional start sites at positions 140 and 145 bpupstream of the translational start site of celS. Potential pro-moters showed homology to A ( 70) and D in B. subtilis.Earlier, the Beguin group in Paris, France, had proposed thatregulation of cellulase synthesis may be accomplished, at leastin part, by sigma factors directing RNA polymerase to certainpromoters. They found similarities between the promoter se-quences of celA and celD and promoters preferred by A and D in B. subtilis (35, 227).

The control of CipA and cell surface anchoring proteinsOlpB and Orf2p was similarly found to be a function of growthrate (77). Thirty-eight to 66 transcripts of these genes per cellwere observed under cellobiose limitation at the low growthrate of 0.04 h1 in continuous culture, and 2 to 13 transcriptswere observed at an exponential growth rate of 0.35 h1 inbatch culture on cellobiose. Under nitrogen limitation in thechemostat, transcript levels were lower, reaching 25 to 31 percell, again indicating a contribution to regulation by carbonsource repression. Transcription of the cell surface gene sdbAwas not influenced by growth rate. Two transcriptional startsites were located at 81 and 50 bp upstream of cipA’stranslational start site. The potential promoters showed ho-mology to A and L ( 54) of B. subtilis. Although transcriptionfrom the A-like promoter was observed only under carbonlimitation, transcription from the L-like promoter occurredunder all growth conditions examined.

In the mesophile C. cellulovorans, crystalline cellulose pro-motes assembly of cellulosomes (213). When grown with cel-lobiose, the cellulosomal proteins were not organized but werepresent free in the medium. Upon addition of crystalline cel-lulose to the extracellular broth, cellulosomes formed. Growthon cellulose also induces production of cellular protuberances(42), but these are not observed during growth on glucose,fructose, cellobiose, or CMC. Addition of soluble carbohydrateto cellulose-grown cells led to a rapid disappearance of theprotuberances within minutes. The cellulosome of C. cellulo-vorans contains enzymes not only for the degradation of cel-lulose but also for the breakdown of xylan, lichenan, pectin,and mannan (318), like the cellulosome of C. thermocellum.The cellulosomal enzyme mannanase, encoded by manA, isrepressed by cellobiose compared to acid-swollen cellulose asrevealed by Western blot analysis. Studies at the protein levelby zymograms and sodium dodecyl sulfate-polyacrylamide gelelectrophoresis revealed that the composition of xylanases andtheir quantities depend on the carbon source used (165). Arecent study using Northern dot blot technique to semiquantifyexpression of various cellulase, hemicellulase, and pectin lyasegenes revealed that cellulose, xylan, or pectin, when used as thecarbon source, gave rise to high-level expression of most genes(117). Moderate expression was observed with cellobiose orfructose. Low-level expression was obtained with lactose, man-nose, and locust bean gum, and the lowest or no expression wasobserved with glucose, galactose, maltose, and sucrose. These

results provide evidence of coordinate expression of cellulaseand hemicellulase genes, the existence of catabolite repression,and influence of hemicellulose on cellulose degradation. Onthe other hand, an enzyme preparation from pectin-grown cellswas found to be more effective in generating protoplasts fromplant cells than preparations from glucose-, cellobiose-, xylan-,or locust bean gum-grown cells (322). Cellulosomal proteomeprofiles were found to be more affected by the carbon sourcethan noncellulosomal enzymes (116). As in C. thermocellum,the promoter regions (of cbpA, engE, manA, and hbpA) harbora conserved sequence exhibiting strong similarity to the A

consensus promoter sequences of gram-positive bacteria (118).

Cellulase Gene Clusters

Despite the fact that cellulase and xylanase genes of C.thermocellum are mostly scattered over the chromosome (110),

FIG. 4. Cellulase gene clusters found in C. thermocellum. The ar-row indicates the transcription direction and the approximate size ofthe gene. GH, glycosyl hydrolase family.



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several gene clusters have been found, suggesting the existenceof operons as units of gene regulation (Fig. 4). The first twooperons found are in the cipA cluster as two tandem operons,including cipA plus olpB and orfp2 plus olpA (“Attachment ofcellulosomes to the cell surface” above). More recently, a non-cellulosomal endoglucanase, CelI, was shown to be clusteredwith cellulosomal cellulase CelN and a possible cellulosomalstructural, component CseP (for cellulosomal element protein)(373). cseP codes for a type I dockerin-containing protein andis in the opposite strand and likely transcribed independentlyfrom celI and celN. The recombinant CseP displayed no de-tectable enzymatic activities. Although the exact function ofCseP is unknown, Western blot analysis revealed that the cel-lulosome contains a substantial amount of this protein (373).Another gene cluster consists of the genes encoding the non-cellulosomal endoglucanase CelC, a LacI-like protein, the li-chenan-degrading enzyme LicA, and a potential membraneprotein with eight putative transmembrane regions (86). We(246) reported that this gene cluster is immediately upstreamof the genes encoding ManB (171) and CelT (172). The clustertherefore has a total of six genes (246). The gene cluster ispreceded by a putative transposase gene and orf2. All of thesegenes are on the same strand. As described below, our dataindicate that the LacI-like protein regulates the expression ofthe gene cluster by binding to the promoter region. The fourthgene cluster consists of celA, chiA, and orfZ (366). CelA is anoncellulosomal endoglucanase. ChiA (family 18) is the onlychitinase in the cellulosome. OrfZ has a domain of unknownfunction and a dockerin. Finally, a five-gene cluster consistingof genes encoding five dockerin-containing proteins was foundin analyzing the C. thermocellum genome (M. Newcomb, T.Dabral, and J. H. D. Wu, unpublished data). Interestingly,none of these five genes have been previously cloned. The firstgene product is a putative family 2 glycosyl hydrolase. Thesecond encoded protein is a putative family 43 glycosyl hydro-lase, which contains a family 4 CBD. The third encoded pro-tein contains a domain of unknown function and a family 6CBD. The fourth encoded protein is a putative esterase with afamily 6 CBD. The last gene product is a putative family 5cellulase with a family 4 CBD and a fibronectin-like domain.Gene clusters that presumably resulted from gene duplicationinclude the gene pairs of celK-cbhA (374) and xynB-xynA (121).As the genome sequencing of C. thermocellum is being com-pleted, more cellulase and hemicellulase gene clusters may bedetected.

In mesophilic clostridia, large cellulase gene clusters havebeen found (Fig. 5). Interestingly, these gene clusters all startwith a scaffoldin gene, followed by a gene encoding a family 48enzyme (exoglucanase working from the reducing end), one totwo endoglucanase genes, and another gene encoding an exo-glucanase working from the nonreducing end (293). Similari-ties among these gene clusters indicate that these clostridia areclosely related. Alternatively, the similarities may be the resultsof lateral gene transfer. In C. cellulovorans, the nine-gene clus-ter contains the genes encoding the scaffolding protein CbpA,the exoglucanase ExgS, several family 9 endoglucanases, themannanase ManA, and the hydrophobic protein HbpA con-taining a surface layer homology domain and a hydrophobic(or cohesin) domain (318, 321). The sequence of the nine-genecluster is cbpA-exgS-engH-engK-hbpA-engL-manA-engM-engN

and spans about 22 kb in length. Several possible transcriptionterminators have been found between some of the genes.Northern hybridization revealed that the cellulosomal cbpAgene cluster is transcribed as polycistronic mRNAs of 8 and 12kb. The 8-kb mRNA codes for CbpA and ExgS, and the 12-kbmRNA codes for CbpA, ExgS, EngH, and EngK (118). On theother hand, manA is transcribed as a monocistronic messenger.

In C. cellulolyticum, a similar gene cluster has been reported.The sequence of the 10-gene, 20-kb cluster is cipC-celF-celC-celG-celE-orfXp-celH-celJ-manK-celM. Results of Northernblot analysis revealed that celC and celG are expressed as apolycistronic transcriptional unit which possibly includes athird gene (12).

The genome sequence of C. acetobutylicum revealed at least11 genes encoding dockerin- or cohesin-containing proteins(254). Ten of these genes form a large cluster, similar to the C.cellulovorans and C. cellulolyticum gene clusters, with the se-quence cipA-celA-celB-celC-orfXp-celD-celE-celF-manG-celH(279). The existence of these sequences suggests that the bac-terium is capable of producing a cellulosome-like structure,and such a structure has been identified (279), as mentionedabove. However, the bacterial strain is incapable of hydrolyzingamorphous or crystalline cellulose, although it hydrolyzesCMC. In addition, most of the xylanase genes in this bacteriumare located in a predicted operon (254). In C. josui, a genecluster containing at least four genes has been reported (149).The scaffoldin gene cipA is the first member of this cluster,followed by celD, celB, and celE.

Negative Regulation

The task of elucidating the mechanisms controlling the bio-synthesis of biomass-degrading enzymes in clostridia is obvi-ously complicated by the large number of genes and proteinsinvolved. In C. thermocellum, the long list of the cellulosomalgenes is further complicated by many noncellulosomal enzymecomponents. The large number of genes involved necessitatesthe use of a genomics approach. By searching the genome, wehave identified three C. thermocellum proteins, GlyR1, GlyR2,and GlyR3 (formerly CelR1, CelR2, and CelR3, respectively),that are homologous to E. coli LacI (246). Each of theseputative regulatory proteins contains two major domains. Ahelix-turn-helix DNA-binding domain is N terminal to a sugar-binding domain. The domain structure of the GlyR proteins isthus similar to the LacI structure and suggests that they belongto the Lac I family of negative regulators. Among them, GlyR3is encoded by a member of the celC gene cluster mentionedabove, including the genes encoding CelC, GlyR3, LicA, aputative membrane protein, ManB, and CelT, respectively.The DNA-binding domain of GlyR3 shows a helix-turn-helixstructure very similar to that of LacI. On the other hand, thefolding of the sugar-binding domain of GlyR3, as predictedby homology modeling, is substantially different from that ofits equivalent in LacI (246). The results are consistent withour expectation that GlyR3 is a DNA-binding protein reg-ulated by a sugar other than lactose. Our experimental ev-idence indicates that GlyR3 binds to the celC promoterregion and that such binding is specifically inhibited by lami-naribiose (246; M. Newcomb and J. H. D. Wu, unpublisheddata). Thus, laminaribiose appears to serve as an inducer of



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the gene cluster by inactivating binding of GlyR3 to thepromoter region. This is the first demonstration, after a longsearch for transcription regulators of the C. thermocellumcellulase system, that the cellulase genes can be regulated bynegative control. The demonstration will undoubtedlyprompt efforts to find additional transcription factors thatregulate cellulase formation in clostridia. Thorough under-standing of the cellulase and hemicellulase regulatory mech-anisms will be crucial for deregulating their productionthrough rational genetic manipulations.

CLOSTRIDIAL COCULTURES

In addition to our basic interest in the cellulolytic complex ofC. thermocellum, we are intrigued by the possibility that theanaerobic and thermophilic clostridia will someday represent apractical means of directly converting cellulose and hemicel-lulose to fuel ethanol. In principle, the concept of a thermo-philic ethanol fermentation is very simple, i.e., a fermentationat high temperature (60 to 70°C), eliminating or reducing the

need for power-consuming cooling and aeration of large reac-tor vessels and facilitating the constant removal of the volatileethanol by evaporation and distillation, thus maintaining a lowethanol concentration in the fermentor and circumventing theneed for an organism that is resistant to very high ethanolconcentrations; only moderately resistant cultures would benecessary. Research on the optimization of ethanol productionby clostridia has focused on substrate utilization (61, 283;D. I. C. Wang and G. C. Avgerinos, Abstr. Am. Inst. Chem.Eng. Annu. Meet., 1983), substrate pretreatment (79, 205),manipulation of fermentation conditions (140, 335), mutationand screening or selection for better cellulase producers (93),ethanol-tolerant strains (10, 127, 317), and strains which do notproduce acids (Wang and Avgerinos, Abstr. AIChE Annu.Mtg., 1983).

No microbial system that simultaneously synthesizes a cel-lulase system at the high activities required and produces eth-anol at the required high selectivity (i.e., without acetate andlactate as side products) is presently known. One could con-ceivably convert an excellent ethanol producer, such as S. cer-

FIG. 5. Cellulase gene clusters found in mesophilic clostridia. The arrow indicates the transcription direction and the approximate size of thegene. The sequence of the C. josui cluster is incomplete, and additional genes of the cluster may exist.



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evisiae, into a cellulase producer, but this would be extremelydifficult because of the complexity of cellulase systems; indeed,such efforts have failed in the past to yield commercially inter-esting cultures. On the other hand, it would be much simpler togenetically modify an excellent cellulase producer that pro-duces ethanol, acetate, and lactate so that mainly ethanol isproduced. This approach has the added benefit of addressingthe more serious economic problem, that of product selectivity,rather than the less important problem (i.e., for a thermophile)of ethanol sensitivity. C. thermocellum is the organism ofchoice for such an effort, since it is the most thoroughly de-scribed cellulolytic ethanol-producing thermophile. Studies(198) revealed that continuous cultures of C. thermocellumgrown on pretreated hardwood can achieve essentially com-plete hydrolysis in a 12-h residence time. Pretreatment oflignocellulose has been discussed by Grethlein (109), Grethleinand Converse (108), and Khan et al. (155).

Because C. thermocellum is capable of utilizing only thehexoses and not the pentose sugars generated from celluloseand hemicellulose, the use of mixed-culture (i.e., dual-culture)systems is of great interest. C. thermocellum has been cultivatedwith thermophilic, anaerobic bacteria that are capable of uti-lizing pentose as well as hexose sugars, i.e., Clostridium ther-mosaccharolyticum (282, 335, 338), Clostridium thermohydro-sulfuricum (95, 247, 282), Thermoanaerobacter ethanolicus(347) and Thermoanaerobium brockii (181). One such directprocess was studied at M.I.T. (11, 335, 338), and involved amixture of C. thermocellum and C. thermosaccharolyticum (78).This combination forms closely associated, syntrophic, andvery stable dual cultures (345). Cellulose is broken down by thecellulase complex of C. thermocellum to cellobiose and cello-dextrins, which are then utilized by the organisms to produceethanol; unfortunately, acetate and lactate are also formed.

C. thermocellum is unable to utilize the pentose sugars,mainly xylose and xylobiose, formed by its breakdown of hemi-cellulose. The other anaerobic thermophile in the dual culture,C. thermosaccharolyticum, uses the pentoses to form ethanol,acetate, and lactate. The latter organism also uses the cellobi-ose resulting from cellulase action faster than C. thermocellum,thus preventing buildup of this inhibitory and repressive dis-accharide. Residual lignin formed from the breakdown oflignocellulosic materials could be sold for adhesive formula-tions, burned as fuel, or converted into steam and electricity(47).

The dual-culture process has great potential, but the prob-lem of side products such as acetate and lactate that decreasethe yield of ethanol and can act as weak uncouplers and slowcell growth (129) must also be addressed. The method of acidproduction is clear: acetate production involves the activity ofphosphotransacetylase and acetate kinase, whereas lactate for-mation involves lactate dehydrogenase (Fig. 6). These enzymesshould be genetically removable without disrupting growth ofthe organism. Physiological manipulations can also be impor-tant; growth of C. thermosaccharolyticum during transientstates in a continuous-culture reactor resulted in an ethanolselectivity (moles of ethanol per mole of other products) inexcess of 11 (199). The purification and characterization ofacetate kinase from C. thermocellum have been described (192,193).

The other problem with the thermoanaerobes is the buildup

of ethanol which can inhibit cell growth (128). The basis andextent of ethanol sensitivity in thermoethanologenic bacteriaare still unclear. Proposed mechanisms include ethanol-in-duced changes in the cell membrane, overreduction of thepyridine nucleotide pool, and inhibition of alcohol dehydroge-nase. Although clostridia have lower ethanol tolerance thanyeast, certain developments indicate the difference not to be asgreat as generally thought. In batch culture, it had been shownthat growth of C. thermocellum ATCC 27405 was 50% inhib-ited at 4 to 16 g of ethanol per liter (127, 289). However,ethanol-resistant strains have which are 50% inhibited only athigher levels, e.g., at 25 and 48 g per liter, have been obtained(see the table in reference 14). Another isolated strain, C.thermocellum strain I-1-B is 50% inhibited by 27g of ethanolper liter, compared to 16g per liter for ATCC 27405 (289). Itproduced 23.6g of ethanol per liter from 80g of cellulose perliter; other products were 8.5g of lactate per liter, 2.9g ofacetate per liter, and 0.9g of formate per liter. Such an ethanoltiter is high for a monoculture.

Growth of C. thermosaccharolyticum strain HG-8 in contin-uous culture was not inhibited by 21g of ethanol (endogenousplus exogenous) per liter and was 50% inhibited by 28 to 29g/liter at its optimum temperature of 60°C (159). At 55°C, 36 to40 g of ethanol per liter was required to inhibit growth by 50%

FIG. 6. Ethanol, lactate, and acetate fermentation by C. thermocel-lum. 1, enzymes of Embden-Meyerhof pathway; 2, lactate dehydroge-nase; 3, pyruvate-ferredoxin oxidoreductase; 4, acetaldehyde dehydro-genase; 5, alcohol dehydrogenase; 6, phosphotransacetylase; 7, acetatekinase.



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(14, 159). These data indicate a much greater ethanol toler-ance than previously thought (130, 199). At 30°C, 7°C belowthe optimum temperature for S. cerevisiae growth, the ethanolconcentration causing 50% inhibition in continuous culture ofyeast was about 50 g/liter (24, 100). Thus, at temperatures 5 to7°C below those for optimum growth (lowering the tempera-ture decreases the toxicity of ethanol), the difference in levelscausing 50% growth inhibition in continuous culture betweenS. cerevisiae and C. thermosaccharolyticum is rather small, i.e.,ca. 50 versus 40 g/liter, respectively. Of course, ethanol sensi-tivity becomes even less important in a thermophilic processwhere ethanol can continuously be removed by evaporationand distillation. True cellulase activity of C. thermocellum israther resistant to ethanol, with 50% inhibition requiring 8%ethanol (by weight) (36).

Although strains which show decreased acid production orincreased ethanol tolerance have been obtained by mutagen-esis followed by screening or selection (60, 127, 173, 317, 338),genetic stability has been a problem (60, 78, 228) and furthergenetic work is needed. It is encouraging that a hypercellulo-lytic strain isolated from C. thermocellum Ym4 showed stabilitythrough five subcultures (236). Since the number of variablesand mutant strains which can be examined at one time infermentors is limited, we scaled down the M.I.T. cocultureprocess from fermentors into flasks by using our simple chem-ically defined medium with Solka Floc as a carbon source(335). Prior to these studies, flask experiments worked effec-tively only at low substrate concentrations, e.g., 5 g of celluloseper liter; higher cellulose concentrations yielded no additionalethanol. We were able to raise the substrate concentration to40g/liter and obtain increased ethanol production by raisingthe iron concentration in the medium. Ethanol titers of 12 to16 g/liter were attained with carbon balances of 91 to 97%. Theflask system can be used to attack the problems mentionedabove in order to remove the bottlenecks to economicprogress.

APPLICATION OF RECOMBINANT DNA TECHNOLOGYTO THE SELECTIVITY PROBLEM

A major obstacle to using a clostridial coculture for ethanolproduction is the branched fermentation pathway shared bymembers of this genus, which results in a reduced ethanol yielddue to formation of other fermentation products, notably ac-etate and lactate (Fig. 6). One potential solution is the elimi-nation of metabolic branches, which would result in ethanolformation being the sole means for the cell to rid itself ofexcess reducing equivalents. In doing so, factors which arelikely to affect gene transfer in the two thermophilic organisms,C. thermocellum and C. thermosaccharolyticum, have to be in-vestigated. Genetic manipulation with the goal of increasingethanol yield has been hampered by the paucity of informationon the molecular genetics of C. thermocellum and C. thermo-saccharolyticum. One obvious approach is to knock out thegenes (encoding acetate kinase and/or phosphotransacetylaseand lactate dehydrogenase) that are responsible for thebranched metabolic pathways. The DNA sequences of thesetarget genes can be obtained by cloning the respective genesfrom C. thermocellum and C. thermosaccharolyticum into E.coli or by searching the genomic sequences, if available, for

these genes. Thereafter, a knockout vector can be constructedby using a nonreplicative, temperature-sensitive or otherwiseunstable plasmid containing a fragment(s) of the target gene,to be introduced into the respective organism by a methodsuch as electroporation. Insertional inactivation (single cross-over) or deletion (double crossover) of the endogenous, func-tional genes in the chromosome via homologous recombina-tion would yield stable knockout strains directing the substratemainly to ethanol.

A prerequisite for any such manipulation would be the abil-ity to introduce foreign DNA into these bacteria. Althoughmany genes from thermophilic clostridia have been cloned,reports on the introduction of DNA into these organisms arealmost nonexistent, except for a recent breakthrough (334)(see below). In addition to one initial report of polyethyleneglycol-mediated transformation of the thermophile C. thermo-hydrosulfuricum (synonymous with Thermoanaerobacter ther-mohydrosulfuricus [185]) with plasmid pUB110 (311), someprogress was made in a joint Dartmouth College-M.I.T. inves-tigation of C. thermocellum and C. thermosaccharolyticum deal-ing with restriction endonuclease systems (157). Restriction-modification systems are usually composed of an endonucleasethat digests DNA at a specific base sequence and a methylasethat protects the sequence from digestion by its action at aspecific position on one or more bases in that same sequence.Restriction-modification systems can also be obstacles to trans-formation, as they digest incoming DNA that is not properlymethylated. The action of endonucleases had been shown to bethe primary barrier to achieving efficient transformation of C.acetobutylicum with DNA that is not protected by appropriatemethylation (223). Accordingly, Klapatch et al. (157) first in-vestigated endonuclease activity, the use of interspecific meth-ylase expression to protect DNA in situations where endonu-clease attack would otherwise occur, and the identity of therestriction site.

Cell extracts were used to determine whether a restrictionsystem was operative in C. thermocellum. It was found thatextracts prepared by high pressure disruption or protoplastextraction exhibited sequence-specific restriction endonucleaseactivity with little nonspecific exonuclease activity. Next to beexamined was the question of whether an interspecific meth-ylase could protect DNA from endonuclease cleavage. TheDam methylation system of E. coli completely protected allDNAs tested (totaling 100 kb, ensuring that most base paircombinations were exposed). Based on both the Dam recog-nition sequence and the similarity in action of cell extracts andMboI DNA digests, the C. thermocellum restriction enzymerecognition sequence appeared to be 5�-GATC-3�. Since plas-mids prepared from Dam E. coli strains or from B. subtilis,which does not exhibit Dam methylation, were digested by thecell extract, it was concluded that the genome of C. thermocel-lum exhibits a Dam� phenotype. These results suggested thatE. coli shuttle plasmids used to transform C. thermocellumshould be prepared from a Dam� E. coli host to escape diges-tion at this commonly occurring (on average, once every 256bp) site. In C. thermocellum ATCC 27405, isoschizomers ofBclI (TGATCA) and EcoRII (CC[A/T]GG) had been re-ported (363), but digestion by extracts at these sequences wasnot observed, despite the fact that their respective restrictionsites were present in the DNA studied. The finding that C.



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thermocellum DNA prepared in a dam� host is resistant toendonuclease attack has been confirmed (256).

The action of C. thermosaccharolyticum extracts againstmethylated and unmethylated DNA was also investigated(157). Two independently prepared cell extracts that were ex-posed to 100 kb of DNA did not exhibit restriction endonu-clease activity at 55°C. In contrast to that of C. thermocellum,the genome of C. thermosaccharolyticum was digested by MboI,indicating that it has a Dam phenotype. It is noteworthy thata third thermophile, C. thermohydrosulfuricum, exhibits restric-tion activity at GATC which is prevented by Dam methylationfound in that organism (272). Nonspecific degradation of bothlinear and circular DNA was observed by Klapatch et al. (157)upon incubation with cell extracts from either C. thermocellumor C. thermosaccharolyticum at 63°C but not at 55 or 60°C.DNase activity had been reported previously for both organ-isms (184), but the effect of temperature on this activity hadnot been addressed.

Further work was aimed at creating a stable strain by usinghomologous recombination-mediated targeted mutagenesis(65). As mentioned above, one key prerequisite for carryingout this approach is the development of a transformation sys-tem to allow introduction of modified target genes into the cellfor homologous recombination with the endogenous genes. Apreliminary report on successful transformation of C. thermo-cellum had appeared (331), but the efficiency of gene transferwas not disclosed. Recently, electrotransformation of C. ther-mocellum was achieved by using plasmid pIKm1 with selectionbased on resistance to erythromycin and lincomycin (334).Transformation efficiency was optimized. Dam methylation in-creased the transformation efficiency by 7- or 40-fold, depend-ing on the host strain.

C. thermosaccharolyticum was successfully transformed byelectrotransformation, and the foreign erythromycin resistancecharacter was expressed (65, 158). Cells were prepared in 20%glycerol and electroporated using a high field strength (10kV/cm) with a time constant of approximately 16 ms. Theglycerol electroporation buffer, which those authors success-fully used for C. thermosaccharolyticum, had been used withClostridium perfringens (7) to increase transformation efficiencyover that obtained with phosphate-buffered sucrose. Evidencesupporting the reproducible transformation with pCTC1 in-cluded a positive hybridization signal on a Southern blot oftotal genomic DNA from transformed C. thermosaccharolyti-cum when probed with pCTC1, visualization on agarose gels ofpCTC1 in 100% of the tested transformed C. thermosaccharo-lyticum clones, correlation of resistance to erythromycin withthe presence of the plasmid, transformation of E. coli withpCTC1 isolated from C. thermosaccharolyticum, reisolation ofthe plasmid from E. coli, and a dose response of the number ofC. thermosaccharolyticum transformants with increasingamounts of DNA. A 95% survival frequency was observedduring electroporation with C. thermosaccharolyticum. The av-erage transformation frequency was low but reproducible andwas similar to the initial frequencies seen with other pioneer-ing transformation studies using various organisms. The trans-formation efficiency was low when the plasmid was prepared inE. coli, but it was two orders of magnitude higher when plasmidproduced in C. thermosaccharolyticum was used, i.e., 52 trans-formants per �g of DNA. The preferred growth temperature

of C. thermosaccharolyticum is 60°C, but in the experiments ofKlapatch et al. (158), cells were grown at 45°C. A lower growthtemperature was used because increased plasmid segregationalinstability of pAMb1 replicons with increasing temperaturehad been reported (311). Klapatch et al. (158) also obtainedindirect evidence of this in the form of lowered plasmid yieldsof alkaline lysis maxipreparations of transformed C. thermo-saccharolyticum when cells were grown at 60°C. Isolation ofpCTC1 from E. coli following passage through C. thermosac-charolyticum and examination of the restriction pattern did notproduce any changes from the expected pattern. Since C. ther-mosaccharolyticum HG8 does not exhibit restriction endonu-clease activity and lacks DNA methylation activity, propermethylation of the incoming DNA does not appear to be thereason why C. thermosaccharolyticum-prepared DNA trans-formed 100-fold better that E. coli-prepared DNA. One pos-sible explanation is that the topology of DNA affects geneexpression (75, 339) and may affect replicon recognition. Ther-mophilic bacteria have been reported to contain unique topo-isomerase activity, and this proper “twisting” of the DNA mol-ecule may be reflected in increased recognition of the repliconby host cell replication factors and increased transformationefficiency (54).

The Dartmouth-M.I.T. group also attempted the isolation ofthe clostridial acetate kinase and phosphotransacetylase genes,ack and pta, respectively, in E. coli (65). We used strains of E.coli that lack ack, pta, or both. Such mutants grow more slowlyas colonies on acetate minimal plates than does the wild-typeparent (190). This constituted a convenient screening system,since the recipient mutants should grow slowly unless the cor-rect gene has been inserted and expressed. To test the system,we transformed overexpression plasmids (pT7-7) containingack (designated pML703) and pta (designated pML702) fromthe thermophile Methanosarcina thermophila (182) into the E.coli mutants and examined growth on minimal acetate plates.Transformants TA3514(pML702) and TA3515(pML703) pro-duced colonies at 24 h. The mutants without inserts, the mu-tants with the “wrong” insert, and the double mutant witheither insert failed to produce colonies until 72 h. Activity testsshowed TA3515(pML703) to contain 7.7 U of acetate kinaseper mg, Ack TA3515 had 0.1 U/mg, and Pta TA3514 con-tained 6.0 U/mg. Thus, the screening procedure was effective.In addition, we showed for the first time that an acetate kinasegene from a thermophile could complement an acetate kinasedeficiency in the mesophile E. coli K-12. The deduced aminoacid sequences of the ack genes from M. thermophila and E.coli show 60% similarity and 40% identity (182). This work wasimportant since the M. thermophila genes provide an alterna-tive path in case of failure to isolate the clostridial genes; i.e.,one could inactivate the M. thermophila genes in E. coli andincorporate these deletion-bearing constructs into the clostrid-ial chromosomes via homologous recombination by using theelectrotransformation methodology described above.

Ozcengiz et al. (255) investigated three different strategiesfor cloning ack from C. thermocellum into E. coli: (i) heterol-ogous complementation, (ii) heterologous probing, and (iii)PCR-based amplification of a C. thermocellum ack fragmentand homologous probing. The use of a PCR-based approachwas found to be best.

Recently, success has been achieved by the Lynd laboratory



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at Dartmouth College, where an efficient gene transfer systemwas developed for C. thermocellum (334). Genes ldh and ackhave been individually eliminated from Thermoanaerbacteriumsaccharolyticum, resulting in markedly decreased production oflactate and acetate, respectively. Plans are in place for elimi-nating both genes in a single strain and repeating this excitingwork in C. thermocellum.

The DNA-shuffling technique for evolution of improved en-zymes has been applied to genes engB and engD of C. cellulo-vorans to increase thermostability (244).

Another exciting development is the cloning and expressionof the genes encoding the EngB endoglucanase and the mini-CbpA1 scaffoldin of C. cellulovorans in B. subtilis and theisolation of minicellulosomes from the gram-positive aerobe(57). This could potentially lead to the production of “designercellulosomes” in B. subtilis.

CLOSING COMMENTS

Today the world is being inundated with urban, agricultural,and industrial waste. Landfill space to handle this waste isbecoming unavailable, and its cost is rising rapidly. A potentialsolution to this problem is the conversion of lignocellulosicbiomass into motor fuel, i.e., ethanol, by a coculture of ther-mophilic, anaerobic microorganisms (i.e., a cellulolytic strainsuch as C. thermocellum and a saccharolytic strain such as C.thermosaccharolyticum). Together, these strains attack cellu-lose and hemicellulose and convert the sugars produced toethanol.

Attention has focused on anaerobic thermophiles as “eth-anologens” for the following reasons: (i) thermophiles arethought to be robust and contain stable enzymes; (ii) anaer-obes generally have a low cellular growth yield, and hencemore of the substrate is converted to ethanol; (iii) thermophilicfermentations are less prone to detrimental effects of contam-ination; and (iv) growth at higher temperatures may facilitatethe removal and recovery of volatile products such as ethanol.Our experience suggests that even more important are theadvantages of cellulase production in situ and the high rates ofmetabolism of cellulose and hemicellulose.

In addition to addressing a pollution problem, the clos-tridial coculture system is potentially capable of dramati-cally increasing the use of ethanol as a major liquid fuel withrenewable photosynthetic biomass as feedstock. The majorobstacle to an economic process is the production of the sideproducts, acetate and lactate, which limits conversion yield.In principle, the concept of a thermophilic ethanol fermen-tation is a very simple one involving a high-temperaturefermentation with a reduced need for power-consumingcooling and agitation or aeration of reactor vessels and withthe four biologically mediated events involved in ethanolproduction (cellulase and hemicellulase formation, celluloseand hemicellulose hydrolysis, hexose fermentation, and pen-tose fermentation) consolidated in a single process step. Acombination of recombinant DNA technology and meta-bolic engineering research is sure to overcome the bottle-neck described above. The selectivity problem is being ad-dressed by using a molecular genetics approach involvinginactivation of the genes encoding acetate- and lactate-pro-ducing enzymes of both members of the coculture system.

ACKNOWLEDGMENTS

We thank Lee R. Lynd and Mark Laser of Dartmouth College fortheir advice on ethanol as a fuel and Sara Ladd for proofreading themanuscript.

The cellulase work by J.H.D.W.’s group was supported by grantsfrom DOE (DE-FG02-94ER20155) and USDA. We also thank LinkFoundation for providing a graduate fellowship.

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