ANNUAL REVIEWS Annu. Rev. Microbiol. 1990. 44:219-48
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MOLECULAR BIOLOGY OF
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CELLULOSE DEGRADATION
Unite de Physiologie Cellulaire, Departement des Biotechnologies, Institut Pasteur,
75724 Paris Cedex 15, France KEY WORDS:
ccllulases, cellulase genes
CONTENTS INTRODUCTION . . ... . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . ..... . . ... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .
219
MULTIPLICITY AND ORGANIZATION OF CELLULASE GENES.....................
221
EXPRESSION OF CELLULASE GENES .......................................................
222 222 224
Expression in the Native Host.. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . Expression in Foreign Hosts . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. . . . STRUCTURE AND FUNCTION OF CELLULASES .. . . .. . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . Common Elements in Cellulase Sequences . . . ......... . . . . . . . . . . . . . . . ... ... . . . . . . . . . . . . . . . .. Modification of Specific Residues.. . . . . . . . . . . . . ..... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... GENETIC MANIPULATION OF CELLULOLYTIC ORGANISMS . . . . .. . . . . . . . . . . . . . . . . . Trichoderma reesei .. " ....... . .. ....... ;.... "".... .. . . . ......... ........ . . . . . . ............... . . Erwinia chrysanthemi . ... . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . ... . . . .... . . . . . . . . . Rumen Bacteria """" . . """"""""""""""""". """""""""". """""""". Actinomycetes . . . .. . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . " . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .
Clostridia .... . . . . . .. . . . . . . . . . .. . . . . . . . ... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . ..
CONCLUDING REMARKS
"""
" "' . ...... . . . . "... . . . . . . . ........
225 225 236 237 237 238 238 239 239 239
INTRODUCTION Cellulose is by far the most abundant carbohydrate available from plant biomass, with an estimated synthesis rate of 4 x 1010 tons/year (27). As such,
it has attracted the interest of many microorganisms, which use it as a carbon source, and, more recently, of biotechnologists, who wish to use it as a renewable source of fuels and chemicals. 219
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220
BEGUIN
The enzymatic conversion of cellulose into glucose is far from straightfor ward, due to the physical nature of the substrate. In its native form, cellulose is composed of largely crystalline fibers in which the hydrogen bonds hold together the individual molecules. Furthermore, the fibers are embedded in a matrix of hemicellulose and lignin, which further reduces their accessibility to cellulolytic enzymes. All organisms that can degrade crystalline cellulose secrete more or less complex cellulase systems. Such systems are composed of a variety of enzymes with different specificities and modes of action, which act in syner gism to hydrolyze cellulose . The enzymology of fungal and bacterial cellulase systems has been re viewed elsewhere (27, 29, 165), and is briefly recalled in this article for the sake of clarity. Fungal (e.g. Trichodermal reesei) cellulases have been ordered into three major categories: (a) cellobiohydrolases (CBH) , which attack cellulose molecules stepwise from the nonreducing end, liberating cellobiose subunits; (b) endoglucanases (EG) , which cleave ,B-glucosidic bonds at random in the middle of cellulose molecules; (c) ,B-glucosidases, which hydrolyze cellobiose and low melecular weight cellodextrins into glucose. The synergism between the three types of enzyme was explained by proposing that EGs attack the amorphous regions of cellulose fibers, thereby creating sites for CBHs which would then proceed into the crystalline regions of the fiber. ,B-glucosidases would perform the last step of hydrolysis and prevent the build-up of cellobiose, which inhibits CBHs. The model is, however, most likely an oversimplification because , for example, it does not explain the synergism between CBHs (35), or, conversely, the absence of synergism between some CBHs and some EGs (166). The interpretation of these observations is still debated. The enzymology of bacterial cellulases is even less well defined. All cellulolytic bacteria secrete a variety of EGs, most of which show little activity toward crystalline cellulose. Few exoglucanases have been character ized (7, 30, 44, 48), only two have been definitely identified as CBHs (30, 44), and only one has been shown to act synergistically with EGs to degrade crystalline cellulose (30). "Fungal-like" prokaryotes such as Actinomycetes and the related Corynebacteria (Cellulomonas) might degrade cellulose, according to a mechanism similar to that of fungi, with cellulolytic enzymes that can be found nonassociated in the culture medium (29). Conversely, in many anaerobic bacteria (rumen bacteria, Clostridium thermocellum), the various cellulase components are found in tightly associated multimolecular complexes, whose quaternary structure seems to be a key feature responsible for the efficient degradation of crystalline cellulose (89) . The complexes are originally associated with the surface of the cells, where they mediate binding to the substrate, and they are subsequently released in the growth medium
MOLECULAR BIOLOGY OF CELLULASES
221
(to, 38, 167). The best known example is the cellulase complex of Clostridi um thermocellum. C. thermocellum cellulase contains at least 14-18 different polypeptides forming a very stable extracellular structure termed cellulosome (28,90,91). The cellulosome has a high affinity for cellulose, which appears to be mediated, at least in part, by a noncatalytic binding factor; Mr 250,000, termed SL (168). Although C. thermocellum cellulase degrades crystalline cellulose very efficiently with cellobiose as the main product, no exo-acting CBH has been characterized so far. Mayer et al (t03) have proposed a model in which cellulose would be attacked simultaneously by regularly spaced catalytic subunits lining up along the cellulose molecules. In recent years, studies of the molecular biology of cellulase genes and their products has developed very fast, opening new fields of investigation such as the organization of cellulase genes, their regulation at the molecular level, and the study of structural features required for enzyme activity. This article reviews advances in these areas that were made possible by rDNA technology and protein chemistry. For reviews of the techniques used in cloning and screening for cellulase genes, the reader is referred elsewhere (13, 14).
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=
MULTIPLICITY AND ORGANIZATION OF CELLULASE GENES
Cellulolytic microorganisms have long been known to secrete a variety of cellulolytic enzymes, but to what extent such multiple cellulases arose from the expression of different genes or from post-translational modifications of the same gene product was not clear. Post-translational modification, such as glycosylation or proteolysis occurring in aging cultures (93, t07) certainly should not be neglected as a source of cellulase diversity. All truly cellulolytic organisms that have been studied so far, however, contain a set of cellulase genes. The majority of cloned genes encode EGs, which are easiest to screen for. The most cellulase- and hemicellulase-encoding genes were found in C. thermocellum, which had at least 15 EG genes, two xylanase genes, and two f3-glucosidase genes (51, 68). The actual number may be even greater: a collection isolated from a thermophilic Clostridium sp. closely resembling C. thermoceUum contained 4 EG genes, 1 ,B-glucosidase gene, and 2 xylanase exoglucanase genes whose restriction maps did not match those of the C. thermocellum genes cloned previously (132). Another example is Ruminococ cus albus. Up to to different EG genes were cloned fromR. albus 8 (73), two fromR. albus SY3 (13 1), three fromR. albus AR67 (158), one fromR. albus AR68 (158), and one from R. albus F-40 (79, 115). The reason for such a diversity of genes encoding enzymes that have similar activities is not known. Furthermore, the interpretation of data from laboratories working with differ ent clone collections originatigg from the same organism becomes quite
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BEGUIN
confusing, particularly if the clones were not obtained from the same type strain. Indeed, cross-hybridization studies by Ware et al (158) demonstrated that strains AR67 and AR68 of R. albus do not belong to the same species. Furthermore, if matching of restriction maps is good evidence for the pres ence of a common DNA fragment, the converse is not necessarily true: DNA rearrangements occurring either because of artifacts of gene bank construction or the in vivo insertion of transposable elements may introduce misleading modifications of the restriction pattern. Thus, the safest way to assess the nonidentity of two clones is their lack of cross-hybridization (68, 131). In many cases, cloned cellulase genes are not clustered, and transcription studies have so far failed to detect polycistronic mRNAs (see below). A few examples of genes located close to other carbohydrase genes have been reported, however. The two homologous EG genes of Bacillus sp. N-4 are separated by 551 bp. The genes are framed by conserved determinants and the tandem arrangement may have originated from homologous recombination involving these determinants (41) . A xylanase gene was found to start 158 bp downstream from the Pseudomonas fluorescens var. cellulosa EGA (en donuclease A) gene (63) . A lichenase gene was found 3-4 kb upstream from the C. thermocellum gene celC encoding EGC (endonuclease C) (139). Finally, at least one EG gene and one ,B-glucosidase gene of Cellvibrio mutus appear to be clustered in a 9.8 kb region that also encodes chitinase and amylase activity (169). T. reesei cellulase genes each contain 2-3 introns, ranging between 57 and 174 bp, with splice junctions similar to those found in other genes of filamentous fungi (123, 133, 1 42, 150) . The position of the introns does not match between homologous genes, nor does it correspond to the border between different structural domains of the proteins. Therefore, introns do not seem to be involved in the shuffling of T. reesei cellulase domains, and they may have been introduced recently in the evolution of the genes. EXPRESSION OF CELLULASE GENES Expression in the Native Host
Two types of mechanisms control the synthesis and secretion of cellulase. In most organisms, cellulase production is repressed in the presence of high concentrations of readily metabolized carbon sources (74, 106, 146, 172). Furthermore, in several systems, the synthesis of cellulase is induced by cellobiose or sophorose, which are generated from cellulose in the presence of low, constitutive cellulase and ,B-glucosidase-associated transglucosidase ac tivities (87, 146). Cellulase synthesis appears to be regulated at the level of mRNA transcrip tion. For example, the levels of T. reesei cbhl mRNA increased in the
MOLECULAR BIOLOGY OF CELLULASES
223
presence of Avicel or sophorose, and were repressed upon addition of glucose
(34). The induction of cbhl transcription in the presence of Avicel could be prevented by adding antibodies to the major cellulase components, but induc tion by sophorose was insensitive to the presence of antibodies. Therefore, induction of
cbhl transcription in the presence of Avicel appears to require
constitutive levels of cellulase and transglucosidase that are needed to gener ate low molecular weight inducers such as sophorose from cellulose. For all cellulase genes studied so far, transcripts are monocistronic, usually
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with 3' ends located in the vicinity of palindromic structures presumably acting as terminators. Identified determinants preceding transcriptional start sites tend to resemble promoters described in related species. The T.
reesei cbh2 mRNA has multiple start sites located 35 to 48 bp downstream from a typical TATA box, as commonly found in eukaryotes (150); the 5' end of
Erwinia chrysanthemi celZ mRNA is preceded by a promoter similar to those recognized by Escherichia coli (7'70 (58); and the two transcripts of C. thermocellum ceLA are initiated downstream from determinants resembling B. subtilis (7'43 and a28 promoters (16). Transcription o f the B. subtilis DLG EG gene starts downstream from a typical B. subtilis (7'43 promoter (130). Transcription of Cellulomonas fimi genes cenA, cenB, cenC, and cex, encoding EGA, EGB, EGC and exoglucanase (EXG), was studied under inducing [in the presence of carboxymethylcellulose (CMC)] , noninducing (in the presence of glycerol), and repressing conditions (in the presence of glucose) (52, 53, 110). Transcripts for
cex and cenC were only detectable in
cells grown in the presence of CMC. Cells grown in the presence of CMC and, to a lesser extent, cells grown in the presence of glycerol contained cenA
mRNA, but the transcript was not present with glucose. Transcription of cenB was greatest in the presence of CMC, but was also found in the presence of
cenB was initiated from two promoters: cenBpl and cenBp2. The high transcription rate found in induced cells resulted from cenBpl, whereas cenBp2 was responsible for the low-rate
glycerol and glucose. Transcription of
transcription found in noninduced and repressed cells. The cenBpl promoter exhibited significant homology with the -10 and 35 regions of the ermp2 promoter of Streptomyces erythraeus and the -10 region of the cex promoter was similar to the tsrpl promoter of Streptomyces azureus. The -10 regions of the cen A and cenC promoters shared the consensus TXPyCCT, whereas the -35 regions of the cenA, cenC and cex -
promoters shared the consensus CTPyXCGC. Cellulase synthesis in
Thermomonospora fusca is subject to a dual control
by induction and growth rate-dependent repression by readily metabolized carbon sources. The latter could be alleviated, at least in part, by cAMP (96). At the protein level, the expression of
T. fusca EGs EJ, E2, and E5 is
coordinately regulated (96), and for cultures grown on different carbon
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sources, the concentration of Es present in the culture supernatant is corre lated with the concentration of the corresponding celE mRNA present in the cells (97). Mapping of celE transcripts (presumably isolated from cellulose grown cells) yielded three closely spaced start sites and a single termination site (97). Using partially purified cell extracts of T. fusca, Lin & Wilson (98) identified a protein that binds specifically to a 21 bp region starting 50 bp downstream from the start site of celE mRNA. The binding region comprised a nearly perfect 18 bp palindrome. The celE-binding protein was present in cellobiose-grown, but not in glucose-grown wild type cells. Thus, the celE binding protein is likely involved in the regulation of EG Es synthesis. Expression in Foreign Hosts
Most cloned genes expressed detectable endoglucanase or cellobiosidase activity in E. coli, and in many cases, expression was independent from vector promoters. However, fusion of the genes with promoters and ribosom al initiation sites from highly expressed genes of E. coli usually resulted in considerably higher yields (76, 117, 137). The vast majority of cloned gene products bear a signal peptide and are partially or entirely transported to the periplasm of E. coli. However, the presence of an N-terminal, hydrophobic signal peptide is not always a prereq uisite for transfer to the cytoplasm. The cellodextrinase encoded by the celA gene from Ruminococcusflavefaciens FD-I was found to be transported to the periplasm in E. coli, in spite of the fact that it lacks an expected signal peptide (1. Thomson, personal communication). Likewise, truncation of 263 codons from the 5' end of the P. fluorescens var. cellulosa xynA gene did not prevent secretion of xylanase XYNA into the periplasm of E. coli (63). Both proteins carry hydrophobic domains close to the COOH end, and it would be interest ing to check if the latter play a role in transmembrane transfer. A few cloned cellulases are secreted by E. coli into the culture medium (26, 85, 1 0 1 ) . The factors allowing extracellular secretion in E. coli are unknown. For example, the EG expressed from the genes cloned from B . subtilis strains PAP115 (70% extracellular) (101), IF01034 (24% extracellular) (85), and DLG « 1 % extracellular) ( 1 29) differ widely in their ability to be secreted extracellularly from E. coli. However, the sequences of the three enzymes, including the signal peptides, are extremely similar; most of the divergences occur in the COOH region, which is not required for secretion (99). Besides E. coli, cellulase genes have also been introduced into a variety of other hosts, such as B . suMlis (45, 77, 94, 1 45), Bacillus megaterium (94), Bacillus stearothermophilus (145), Brevibacterium lactoJermentum (120), Streptomyces lividans (45, 46), Lactobacillus plantarum (8, 135), Zymomo nas mobilis (20, 95), and Saccharomyces cerevisiae (31, 121, 122, 141, 144,
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MOLECULAR BIOLOGY OF CELLULASES
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163). The choice of an alternate host was motivated by (a) ability to secrete proteins into the extracellular medium (most cases); (b) closer evolutionary kinship, allowing more efficient expression (45, 46); and (c) usefulness of S. cerevisiae or Z. mobilis for ethanol production and of L. plantarum for silage pretreatment. In most cases, heterologous gene cloning does not yet provide a practical alternative: to native cellulolytic organisms, if only for the lack of a complete set of cellulolytic enzymes. However, clones often represent a useful source of cellulase components devoid of contamination by other gene products (15, 46, 122, 137, 138) in spite of spurious proteolytic processing (12, 21) or glycosylation (31, 122, 141), whose effects on activity and specificity are still ill defined. Synthesis of individual components may have defined applications beyond n�search purposes. Conceivably, cellulase components present in limiting amounts may be supplied from appropriate clones. Addition of cloned {3-g1ucosidase to the extracellular C. thermocellum cellulase complex resulted in enhanced production of glucose and relief of cellobiose-induced product inhibition (78). Furthermore, some applications should not require the whole set of enzymes required for native cellulose hydrolysis. Barley {3-glucan, which impedes filterability in the beer brewing process, can be hydrolyzed with no adverse effect on taste by brewer's yeast carrying and expressing the T. reesei egl gene (122). In the silage process, the production of endoglucanase by lactic acid bacteria may assist the softening of cell walls, making substrate more rapidly available for lactic acid fermentation, but actual solubilization of cellulose would be unnecessary and even undesirable. Finally, individual cellulase genes may be useful in the development of new host-vector systems for secretion (31 , 66, 77). STRUCTURE AND FUNCTION OF CELLULASES Common Elements in Cellulase Sequences
The sequence of close to 50 fungal and bacterial cellulases and {3-g1ucosidases has been determined. As shown in Figure 1, the structure of many of the enzymes is composed of domains that are more or less conserved and are integrated in different orders in various proteins. The longest domains (showing similarity between more than 250-300 residues) correspond to catalytic "cores." Gene deletion, proteolytk truncation or even recombination of two different cores in a single protein (21, 49, 57, 62, 92, 134, 153, 156, 160) indicate that the cores behave as independent entities endowed with catalytic activity and defined specifici ties toward soluble model substrates. Although the cores exhibit considerable diversity, Henrissat et al (70) suggested that they could be ordered into six CATALYTIC DOMAINS
226
B EGUIN EGB.•.
100 aa
I---i
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Figure 1
EGAM.bi.
�
Domains of extended similarity between some cellulases or xylanases (based upon
information in Ref. 14). (B.s.) B. subtilis; (C.sa) C. saccharolyticum; (C.t.) C. thermocellum; (C.jl.) C.flavigena; (Cfi.) C.fimi; (M.bi.) M. bispora. Domains showing significant similarity are tilled with the same pattern.
different families based on hydrophobic cluster analysis (HCA) (43). The validity of the HCA-derived classification has since been borne out by the comparison of further sequences, most of which fit in one of the previously established categories (Table 1) (71). By far the largest family is Family A. Cellulases of type A have been found in quite different organisms, including gram-positive and gram-negative bac teria, aerobic and anaerobic bacteria, as well as the fungus T. reesei. Type A cellulases can be divided into five subtypes. Within each subtype, similarities can usually be detected by classical comparison algorithms (>25% identi ties). However, members of different subtypes show considerable divergence, and pairwise comparison usually fails to reveal obvious resemblance except when using more sophisticated techniques such as HCA. Nevertheless, align ment of all A-type enzymes reveals that they share small conserved regions distributed along the sequence. Besides typical patterns of hydrophobic amino acids, some of the conserved regions contain residues that are identical in most or all sequences. Taking the sequence (including signal peptide) of C. thermocellum EGB, as a reference, Arg9g, His155, AsnZ03, Gluz04, and Glu363 are conserved in all enzymes. ProZ05 is conserved in all proteins except C. thermocellum EGC; His311 is conserved in all enzymes except Schizophyllum commune EGI; and ProtoO is conserved in all sequences except those of the Al subfamily. All members described to date are EGs, although they may differ in enzyme specificities. For example, the ratio of xylanase to CMCase activity is 1: 10 for C. thermocellum EGE (62), 1: 100 for C. thermocellum EGH (171a), and nondetectable for Butyrivibrio fibrisolvens EG (17). C. thermocellum EGC and Fibrobacter succinogenes (formerly Bacteriodes suc cinogenes) EG3 are distinguished by their ability to hydrolyze cellotriose (l05, 125). Furthermore, C. thermocellum EGC was shown to cleave some of the 13-1,3 linkages found in barley ,B-glucan (138). Family B comprises three clearly related EGs from C. fimi, the Actinomy cetes Streptomyces sp. KSM-9, and Microbispora bispora, as well as from T. reesei CBHII, which shares a more distant, but significant similarity with the
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Table 1 Family
Families of homologous catalytic domains in cellulases and xylanases Subfamily
Enzyme
Organism
Mode of action
Reference
Family A Subfamily AI EGB
Clostridium thermocellum
Endoglucanase
55
ORFA" b EGB
Cellulomonas flavigena
Endoglucanase
3
Caldocellum saccharolyticum
Endoglucanase
134
EG
Bacillus subtilis
Endoglucanase
101
EGA
Bacillus sp. strain N-4
Endoglucanase
42 42
Subfamily A2
�
EG3A
Bacillus circulans
Endoglucanase
65
EG
Clostridium acetobutylicum
Endoglucanase
174
0 r tIl (j e r ;1> ::tl ttl
EGZ
Erwinia chrysanthemi
Endoglucanase
59
0
EGC
Clostridium thermocellum
Endoglucanasellichenase
136
EG3
Fibrobacter succinogenes S85
Endoglucanasellichenase
lOS
0 -
Further
Quick links to online content
Copyright © 1990 by Annual Reviews Inc. All rights reserved
MOLECULAR BIOLOGY OF
Annu. Rev. Microbiol. 1990.44:219-248. Downloaded from www.annualreviews.org by University of Sussex on 09/24/12. For personal use only.
CELLULOSE DEGRADATION
Unite de Physiologie Cellulaire, Departement des Biotechnologies, Institut Pasteur,
75724 Paris Cedex 15, France KEY WORDS:
ccllulases, cellulase genes
CONTENTS INTRODUCTION . . ... . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . ..... . . ... . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .
219
MULTIPLICITY AND ORGANIZATION OF CELLULASE GENES.....................
221
EXPRESSION OF CELLULASE GENES .......................................................
222 222 224
Expression in the Native Host.. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . Expression in Foreign Hosts . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. . . . STRUCTURE AND FUNCTION OF CELLULASES .. . . .. . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . Common Elements in Cellulase Sequences . . . ......... . . . . . . . . . . . . . . . ... ... . . . . . . . . . . . . . . . .. Modification of Specific Residues.. . . . . . . . . . . . . ..... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... GENETIC MANIPULATION OF CELLULOLYTIC ORGANISMS . . . . .. . . . . . . . . . . . . . . . . . Trichoderma reesei .. " ....... . .. ....... ;.... "".... .. . . . ......... ........ . . . . . . ............... . . Erwinia chrysanthemi . ... . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . ... . . . .... . . . . . . . . . Rumen Bacteria """" . . """"""""""""""""". """""""""". """""""". Actinomycetes . . . .. . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . " . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .
Clostridia .... . . . . . .. . . . . . . . . . .. . . . . . . . ... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . ..
CONCLUDING REMARKS
"""
" "' . ...... . . . . "... . . . . . . . ........
225 225 236 237 237 238 238 239 239 239
INTRODUCTION Cellulose is by far the most abundant carbohydrate available from plant biomass, with an estimated synthesis rate of 4 x 1010 tons/year (27). As such,
it has attracted the interest of many microorganisms, which use it as a carbon source, and, more recently, of biotechnologists, who wish to use it as a renewable source of fuels and chemicals. 219
0066-4227/90/1001-0219$02.00
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The enzymatic conversion of cellulose into glucose is far from straightfor ward, due to the physical nature of the substrate. In its native form, cellulose is composed of largely crystalline fibers in which the hydrogen bonds hold together the individual molecules. Furthermore, the fibers are embedded in a matrix of hemicellulose and lignin, which further reduces their accessibility to cellulolytic enzymes. All organisms that can degrade crystalline cellulose secrete more or less complex cellulase systems. Such systems are composed of a variety of enzymes with different specificities and modes of action, which act in syner gism to hydrolyze cellulose . The enzymology of fungal and bacterial cellulase systems has been re viewed elsewhere (27, 29, 165), and is briefly recalled in this article for the sake of clarity. Fungal (e.g. Trichodermal reesei) cellulases have been ordered into three major categories: (a) cellobiohydrolases (CBH) , which attack cellulose molecules stepwise from the nonreducing end, liberating cellobiose subunits; (b) endoglucanases (EG) , which cleave ,B-glucosidic bonds at random in the middle of cellulose molecules; (c) ,B-glucosidases, which hydrolyze cellobiose and low melecular weight cellodextrins into glucose. The synergism between the three types of enzyme was explained by proposing that EGs attack the amorphous regions of cellulose fibers, thereby creating sites for CBHs which would then proceed into the crystalline regions of the fiber. ,B-glucosidases would perform the last step of hydrolysis and prevent the build-up of cellobiose, which inhibits CBHs. The model is, however, most likely an oversimplification because , for example, it does not explain the synergism between CBHs (35), or, conversely, the absence of synergism between some CBHs and some EGs (166). The interpretation of these observations is still debated. The enzymology of bacterial cellulases is even less well defined. All cellulolytic bacteria secrete a variety of EGs, most of which show little activity toward crystalline cellulose. Few exoglucanases have been character ized (7, 30, 44, 48), only two have been definitely identified as CBHs (30, 44), and only one has been shown to act synergistically with EGs to degrade crystalline cellulose (30). "Fungal-like" prokaryotes such as Actinomycetes and the related Corynebacteria (Cellulomonas) might degrade cellulose, according to a mechanism similar to that of fungi, with cellulolytic enzymes that can be found nonassociated in the culture medium (29). Conversely, in many anaerobic bacteria (rumen bacteria, Clostridium thermocellum), the various cellulase components are found in tightly associated multimolecular complexes, whose quaternary structure seems to be a key feature responsible for the efficient degradation of crystalline cellulose (89) . The complexes are originally associated with the surface of the cells, where they mediate binding to the substrate, and they are subsequently released in the growth medium
MOLECULAR BIOLOGY OF CELLULASES
221
(to, 38, 167). The best known example is the cellulase complex of Clostridi um thermocellum. C. thermocellum cellulase contains at least 14-18 different polypeptides forming a very stable extracellular structure termed cellulosome (28,90,91). The cellulosome has a high affinity for cellulose, which appears to be mediated, at least in part, by a noncatalytic binding factor; Mr 250,000, termed SL (168). Although C. thermocellum cellulase degrades crystalline cellulose very efficiently with cellobiose as the main product, no exo-acting CBH has been characterized so far. Mayer et al (t03) have proposed a model in which cellulose would be attacked simultaneously by regularly spaced catalytic subunits lining up along the cellulose molecules. In recent years, studies of the molecular biology of cellulase genes and their products has developed very fast, opening new fields of investigation such as the organization of cellulase genes, their regulation at the molecular level, and the study of structural features required for enzyme activity. This article reviews advances in these areas that were made possible by rDNA technology and protein chemistry. For reviews of the techniques used in cloning and screening for cellulase genes, the reader is referred elsewhere (13, 14).
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=
MULTIPLICITY AND ORGANIZATION OF CELLULASE GENES
Cellulolytic microorganisms have long been known to secrete a variety of cellulolytic enzymes, but to what extent such multiple cellulases arose from the expression of different genes or from post-translational modifications of the same gene product was not clear. Post-translational modification, such as glycosylation or proteolysis occurring in aging cultures (93, t07) certainly should not be neglected as a source of cellulase diversity. All truly cellulolytic organisms that have been studied so far, however, contain a set of cellulase genes. The majority of cloned genes encode EGs, which are easiest to screen for. The most cellulase- and hemicellulase-encoding genes were found in C. thermocellum, which had at least 15 EG genes, two xylanase genes, and two f3-glucosidase genes (51, 68). The actual number may be even greater: a collection isolated from a thermophilic Clostridium sp. closely resembling C. thermoceUum contained 4 EG genes, 1 ,B-glucosidase gene, and 2 xylanase exoglucanase genes whose restriction maps did not match those of the C. thermocellum genes cloned previously (132). Another example is Ruminococ cus albus. Up to to different EG genes were cloned fromR. albus 8 (73), two fromR. albus SY3 (13 1), three fromR. albus AR67 (158), one fromR. albus AR68 (158), and one from R. albus F-40 (79, 115). The reason for such a diversity of genes encoding enzymes that have similar activities is not known. Furthermore, the interpretation of data from laboratories working with differ ent clone collections originatigg from the same organism becomes quite
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confusing, particularly if the clones were not obtained from the same type strain. Indeed, cross-hybridization studies by Ware et al (158) demonstrated that strains AR67 and AR68 of R. albus do not belong to the same species. Furthermore, if matching of restriction maps is good evidence for the pres ence of a common DNA fragment, the converse is not necessarily true: DNA rearrangements occurring either because of artifacts of gene bank construction or the in vivo insertion of transposable elements may introduce misleading modifications of the restriction pattern. Thus, the safest way to assess the nonidentity of two clones is their lack of cross-hybridization (68, 131). In many cases, cloned cellulase genes are not clustered, and transcription studies have so far failed to detect polycistronic mRNAs (see below). A few examples of genes located close to other carbohydrase genes have been reported, however. The two homologous EG genes of Bacillus sp. N-4 are separated by 551 bp. The genes are framed by conserved determinants and the tandem arrangement may have originated from homologous recombination involving these determinants (41) . A xylanase gene was found to start 158 bp downstream from the Pseudomonas fluorescens var. cellulosa EGA (en donuclease A) gene (63) . A lichenase gene was found 3-4 kb upstream from the C. thermocellum gene celC encoding EGC (endonuclease C) (139). Finally, at least one EG gene and one ,B-glucosidase gene of Cellvibrio mutus appear to be clustered in a 9.8 kb region that also encodes chitinase and amylase activity (169). T. reesei cellulase genes each contain 2-3 introns, ranging between 57 and 174 bp, with splice junctions similar to those found in other genes of filamentous fungi (123, 133, 1 42, 150) . The position of the introns does not match between homologous genes, nor does it correspond to the border between different structural domains of the proteins. Therefore, introns do not seem to be involved in the shuffling of T. reesei cellulase domains, and they may have been introduced recently in the evolution of the genes. EXPRESSION OF CELLULASE GENES Expression in the Native Host
Two types of mechanisms control the synthesis and secretion of cellulase. In most organisms, cellulase production is repressed in the presence of high concentrations of readily metabolized carbon sources (74, 106, 146, 172). Furthermore, in several systems, the synthesis of cellulase is induced by cellobiose or sophorose, which are generated from cellulose in the presence of low, constitutive cellulase and ,B-glucosidase-associated transglucosidase ac tivities (87, 146). Cellulase synthesis appears to be regulated at the level of mRNA transcrip tion. For example, the levels of T. reesei cbhl mRNA increased in the
MOLECULAR BIOLOGY OF CELLULASES
223
presence of Avicel or sophorose, and were repressed upon addition of glucose
(34). The induction of cbhl transcription in the presence of Avicel could be prevented by adding antibodies to the major cellulase components, but induc tion by sophorose was insensitive to the presence of antibodies. Therefore, induction of
cbhl transcription in the presence of Avicel appears to require
constitutive levels of cellulase and transglucosidase that are needed to gener ate low molecular weight inducers such as sophorose from cellulose. For all cellulase genes studied so far, transcripts are monocistronic, usually
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with 3' ends located in the vicinity of palindromic structures presumably acting as terminators. Identified determinants preceding transcriptional start sites tend to resemble promoters described in related species. The T.
reesei cbh2 mRNA has multiple start sites located 35 to 48 bp downstream from a typical TATA box, as commonly found in eukaryotes (150); the 5' end of
Erwinia chrysanthemi celZ mRNA is preceded by a promoter similar to those recognized by Escherichia coli (7'70 (58); and the two transcripts of C. thermocellum ceLA are initiated downstream from determinants resembling B. subtilis (7'43 and a28 promoters (16). Transcription o f the B. subtilis DLG EG gene starts downstream from a typical B. subtilis (7'43 promoter (130). Transcription of Cellulomonas fimi genes cenA, cenB, cenC, and cex, encoding EGA, EGB, EGC and exoglucanase (EXG), was studied under inducing [in the presence of carboxymethylcellulose (CMC)] , noninducing (in the presence of glycerol), and repressing conditions (in the presence of glucose) (52, 53, 110). Transcripts for
cex and cenC were only detectable in
cells grown in the presence of CMC. Cells grown in the presence of CMC and, to a lesser extent, cells grown in the presence of glycerol contained cenA
mRNA, but the transcript was not present with glucose. Transcription of cenB was greatest in the presence of CMC, but was also found in the presence of
cenB was initiated from two promoters: cenBpl and cenBp2. The high transcription rate found in induced cells resulted from cenBpl, whereas cenBp2 was responsible for the low-rate
glycerol and glucose. Transcription of
transcription found in noninduced and repressed cells. The cenBpl promoter exhibited significant homology with the -10 and 35 regions of the ermp2 promoter of Streptomyces erythraeus and the -10 region of the cex promoter was similar to the tsrpl promoter of Streptomyces azureus. The -10 regions of the cen A and cenC promoters shared the consensus TXPyCCT, whereas the -35 regions of the cenA, cenC and cex -
promoters shared the consensus CTPyXCGC. Cellulase synthesis in
Thermomonospora fusca is subject to a dual control
by induction and growth rate-dependent repression by readily metabolized carbon sources. The latter could be alleviated, at least in part, by cAMP (96). At the protein level, the expression of
T. fusca EGs EJ, E2, and E5 is
coordinately regulated (96), and for cultures grown on different carbon
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sources, the concentration of Es present in the culture supernatant is corre lated with the concentration of the corresponding celE mRNA present in the cells (97). Mapping of celE transcripts (presumably isolated from cellulose grown cells) yielded three closely spaced start sites and a single termination site (97). Using partially purified cell extracts of T. fusca, Lin & Wilson (98) identified a protein that binds specifically to a 21 bp region starting 50 bp downstream from the start site of celE mRNA. The binding region comprised a nearly perfect 18 bp palindrome. The celE-binding protein was present in cellobiose-grown, but not in glucose-grown wild type cells. Thus, the celE binding protein is likely involved in the regulation of EG Es synthesis. Expression in Foreign Hosts
Most cloned genes expressed detectable endoglucanase or cellobiosidase activity in E. coli, and in many cases, expression was independent from vector promoters. However, fusion of the genes with promoters and ribosom al initiation sites from highly expressed genes of E. coli usually resulted in considerably higher yields (76, 117, 137). The vast majority of cloned gene products bear a signal peptide and are partially or entirely transported to the periplasm of E. coli. However, the presence of an N-terminal, hydrophobic signal peptide is not always a prereq uisite for transfer to the cytoplasm. The cellodextrinase encoded by the celA gene from Ruminococcusflavefaciens FD-I was found to be transported to the periplasm in E. coli, in spite of the fact that it lacks an expected signal peptide (1. Thomson, personal communication). Likewise, truncation of 263 codons from the 5' end of the P. fluorescens var. cellulosa xynA gene did not prevent secretion of xylanase XYNA into the periplasm of E. coli (63). Both proteins carry hydrophobic domains close to the COOH end, and it would be interest ing to check if the latter play a role in transmembrane transfer. A few cloned cellulases are secreted by E. coli into the culture medium (26, 85, 1 0 1 ) . The factors allowing extracellular secretion in E. coli are unknown. For example, the EG expressed from the genes cloned from B . subtilis strains PAP115 (70% extracellular) (101), IF01034 (24% extracellular) (85), and DLG « 1 % extracellular) ( 1 29) differ widely in their ability to be secreted extracellularly from E. coli. However, the sequences of the three enzymes, including the signal peptides, are extremely similar; most of the divergences occur in the COOH region, which is not required for secretion (99). Besides E. coli, cellulase genes have also been introduced into a variety of other hosts, such as B . suMlis (45, 77, 94, 1 45), Bacillus megaterium (94), Bacillus stearothermophilus (145), Brevibacterium lactoJermentum (120), Streptomyces lividans (45, 46), Lactobacillus plantarum (8, 135), Zymomo nas mobilis (20, 95), and Saccharomyces cerevisiae (31, 121, 122, 141, 144,
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163). The choice of an alternate host was motivated by (a) ability to secrete proteins into the extracellular medium (most cases); (b) closer evolutionary kinship, allowing more efficient expression (45, 46); and (c) usefulness of S. cerevisiae or Z. mobilis for ethanol production and of L. plantarum for silage pretreatment. In most cases, heterologous gene cloning does not yet provide a practical alternative: to native cellulolytic organisms, if only for the lack of a complete set of cellulolytic enzymes. However, clones often represent a useful source of cellulase components devoid of contamination by other gene products (15, 46, 122, 137, 138) in spite of spurious proteolytic processing (12, 21) or glycosylation (31, 122, 141), whose effects on activity and specificity are still ill defined. Synthesis of individual components may have defined applications beyond n�search purposes. Conceivably, cellulase components present in limiting amounts may be supplied from appropriate clones. Addition of cloned {3-g1ucosidase to the extracellular C. thermocellum cellulase complex resulted in enhanced production of glucose and relief of cellobiose-induced product inhibition (78). Furthermore, some applications should not require the whole set of enzymes required for native cellulose hydrolysis. Barley {3-glucan, which impedes filterability in the beer brewing process, can be hydrolyzed with no adverse effect on taste by brewer's yeast carrying and expressing the T. reesei egl gene (122). In the silage process, the production of endoglucanase by lactic acid bacteria may assist the softening of cell walls, making substrate more rapidly available for lactic acid fermentation, but actual solubilization of cellulose would be unnecessary and even undesirable. Finally, individual cellulase genes may be useful in the development of new host-vector systems for secretion (31 , 66, 77). STRUCTURE AND FUNCTION OF CELLULASES Common Elements in Cellulase Sequences
The sequence of close to 50 fungal and bacterial cellulases and {3-g1ucosidases has been determined. As shown in Figure 1, the structure of many of the enzymes is composed of domains that are more or less conserved and are integrated in different orders in various proteins. The longest domains (showing similarity between more than 250-300 residues) correspond to catalytic "cores." Gene deletion, proteolytk truncation or even recombination of two different cores in a single protein (21, 49, 57, 62, 92, 134, 153, 156, 160) indicate that the cores behave as independent entities endowed with catalytic activity and defined specifici ties toward soluble model substrates. Although the cores exhibit considerable diversity, Henrissat et al (70) suggested that they could be ordered into six CATALYTIC DOMAINS
226
B EGUIN EGB.•.
100 aa
I---i
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Figure 1
EGAM.bi.
�
Domains of extended similarity between some cellulases or xylanases (based upon
information in Ref. 14). (B.s.) B. subtilis; (C.sa) C. saccharolyticum; (C.t.) C. thermocellum; (C.jl.) C.flavigena; (Cfi.) C.fimi; (M.bi.) M. bispora. Domains showing significant similarity are tilled with the same pattern.
different families based on hydrophobic cluster analysis (HCA) (43). The validity of the HCA-derived classification has since been borne out by the comparison of further sequences, most of which fit in one of the previously established categories (Table 1) (71). By far the largest family is Family A. Cellulases of type A have been found in quite different organisms, including gram-positive and gram-negative bac teria, aerobic and anaerobic bacteria, as well as the fungus T. reesei. Type A cellulases can be divided into five subtypes. Within each subtype, similarities can usually be detected by classical comparison algorithms (>25% identi ties). However, members of different subtypes show considerable divergence, and pairwise comparison usually fails to reveal obvious resemblance except when using more sophisticated techniques such as HCA. Nevertheless, align ment of all A-type enzymes reveals that they share small conserved regions distributed along the sequence. Besides typical patterns of hydrophobic amino acids, some of the conserved regions contain residues that are identical in most or all sequences. Taking the sequence (including signal peptide) of C. thermocellum EGB, as a reference, Arg9g, His155, AsnZ03, Gluz04, and Glu363 are conserved in all enzymes. ProZ05 is conserved in all proteins except C. thermocellum EGC; His311 is conserved in all enzymes except Schizophyllum commune EGI; and ProtoO is conserved in all sequences except those of the Al subfamily. All members described to date are EGs, although they may differ in enzyme specificities. For example, the ratio of xylanase to CMCase activity is 1: 10 for C. thermocellum EGE (62), 1: 100 for C. thermocellum EGH (171a), and nondetectable for Butyrivibrio fibrisolvens EG (17). C. thermocellum EGC and Fibrobacter succinogenes (formerly Bacteriodes suc cinogenes) EG3 are distinguished by their ability to hydrolyze cellotriose (l05, 125). Furthermore, C. thermocellum EGC was shown to cleave some of the 13-1,3 linkages found in barley ,B-glucan (138). Family B comprises three clearly related EGs from C. fimi, the Actinomy cetes Streptomyces sp. KSM-9, and Microbispora bispora, as well as from T. reesei CBHII, which shares a more distant, but significant similarity with the
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Table 1 Family
Families of homologous catalytic domains in cellulases and xylanases Subfamily
Enzyme
Organism
Mode of action
Reference
Family A Subfamily AI EGB
Clostridium thermocellum
Endoglucanase
55
ORFA" b EGB
Cellulomonas flavigena
Endoglucanase
3
Caldocellum saccharolyticum
Endoglucanase
134
EG
Bacillus subtilis
Endoglucanase
101
EGA
Bacillus sp. strain N-4
Endoglucanase
42 42
Subfamily A2
�
EG3A
Bacillus circulans
Endoglucanase
65
EG
Clostridium acetobutylicum
Endoglucanase
174
0 r tIl (j e r ;1> ::tl ttl
EGZ
Erwinia chrysanthemi
Endoglucanase
59
0
EGC
Clostridium thermocellum
Endoglucanasellichenase
136
EG3
Fibrobacter succinogenes S85
Endoglucanasellichenase
lOS
0 -
