Enzymes in Biomass Conversion - American Chemical Society

Chapter 22

Cellulase Insights through Recombinant DNA Approaches 1

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K. O. Elliston , M. D. Yablonsky , and D. E. Eveleigh Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch022

1

Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ 08903 Waksman Institute, Busch Campus, Rutgers University, Piscataway, NJ 08855 2

Cellulases are inherently interesting. Induction of such complex multiple synergistic components is efficiently accomplished though the substrate is extracellular and insoluble. Their study has been focused historically on applied aspects, initially on means of inhibiting their degradative action, with recent emphasis on obtaining enhanced yields/activities to promote efficient hydrolysis of cellulose to glucose syrups. Cloning of cellulases has opened new vistas for basic study. It facilitates purification of the individual components, a major advance in a notoriously difficult arena. It has aided development of the concept of the triple (binding, hinge and active site) domains of cellulase, besides allowing their further characterization via site specific mutagenesis. Evolutionary relationships have been clarified. The rDNA methodology also directly aids the application of cellulases through improvement and/or hyperproduction of specific components, thereby allowing them to be mixed optimally for routine and new applications.

Cellulases are central to the world's carbon cycle and energyflowas a result of cellulose being the world's most abundant natural polymer. Production guesstimates are in the range of 1 χ 10 tons per year. This abundant, but recalcitrant structural component is recycled through the action of cellulases. Cellulases also engender economic interest as they are both prime agents of decay and yet also a potential key to conversion of waste biomass (agricultural, food and forestry residues) into fermentation feedstocks (7). Furthermore, they are crucial in cell wall synthesis of plants and fungi, facilitating morphogenic development by permitting selective intercalation of cellulose building blocks within the wall. The study of cellulases initially focussed on means of inhibiting their activities in connection with man's desire to prevent decay of cellulosic materials - from timber to tents. More recently when energy oil supplies were restricted in the 1970's, interest in cellulase surged with a variety of proposals to reduce the energy deficit through use of cellulosic residues as an energy resource. In general, these scenarios envisaged conversion of biomass, especially waste biomass, to glucose and thence by fermentation to oxychemicals, e.g., ethanol as both a liquid transportation fuel and also an octane enhancer. 11

0097-6156/91/0460-0290$06.00/0 © 1991 American Chemical Society

In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch022

22. ELLISTON ET AL.

Cellulase: Recombinant DNA Approaches

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As a result of these studies, major advances in our understanding of cellulases occurred over the last decade. The previously somewhat restricted study of a few fungi (e.g., Trichoderma reesei, Phanerochaete chrysosporium) was greatly enlarged to include other fungi, besides bacterial and plant systems. Even so, the surface has just been scratched as there must be myriads of cryptic cellulolytic species in forest Utter and agricultural soils that have been ignored because they do not produce copious amounts of readily recoverable extracellular enzymes. One majorfindingis the confirmation that cell-bound cellulases (cellulosomes) are efficient and occur widely (2,5). General screening has also resulted in the discovery of cellulases with alkaline pH optima. These latter have been shown to act effectively as adjuncts in laundry detergents and are already on the market in Japan (see Horikoshi, this symposium). Molecular and biochemical studies have inspired novel ideas on the mechanisms of how a cellulase enzyme attacks a large insoluble substrate (Grohmann et al, this symposium). Such concepts in conjunction with molecular biological advances have advanced the idea of a cellulase being comprised of three facets; an active site domain, a hinge region and a cellulose binding zone. It has been proposed that 25 known cellulase gene sequences fit into six natural families based on comparison of DNA sequences and hydrophobic cluster analyses (2,4). Such analyses are significant in relation to practical application and evolutionary perspective. We believe that considerably greater insight can be gained from this type of analysis. With this in mind we present a comparison of cellulases based on domain analysis and multiple sequence alignments especially with regard to the cellulase of Microbispora bispora currently under study in our laboratory (5). Domain Analysis Analyses of the cellulases have concentrated mainly on comparison of groups of enzymes based upon both their overall structure, as well as their primary sequence. These analyses have shown these enzymes generally to be made up of three distinct regions; the catalytic domain, the cellulose binding domain, and a linker region also known as the hinge region. An example sequence, the Microbispora bispora endoglucanase is shown in Figure 1. Interestingly, some sequences contain two or more binding regions, each separated by a linker domain (6). We have recently undertaken a reconsideration of cellulase sequences by analyzing each domain of the sequence separately. Here we describe initial observations and methodologies we are developing for the analysis and comparison of cellulase domains. Novel methods of classification will give greater insight into theoretical, evolutionary and practical approaches to the analysis of cellulase. The analysis of cellulase sequences is facilitated by first dividing the sequences into each of the three predominant domains; the catalytic, binding and linking domains. The organization of some cellulase genes is such that the catalytic domain at the amino terminus is followed by the linking domain and the binding domain, e.g., Fig. 1. However, several cellulases are characterized by a carboxy terminal catalytic domain, with the binding and linking domains located at the amino terminal end. Cellulase sequences can readily be subdivided into the representative domains by sequence homology. We decided on initial groupings based upon a FastA analysis of each sequence, using 30 cellulase sequences as the database. The FastA analysis is based upon homology or similarity comparisons as developed by Lipman and Pearson (7,8), and implemented by the Genetics Computer Group sequence analysis package (9). Each of the catalytic and binding domains were then edited out of the primary sequence, using homology to several known catalytic and binding domains and a series of homology searches were run with each sequence. The linker or hinge region was used as the dividing region when deciding domain boundaries, as this sequence is readily observed by its repeating P/S/T motif. The P/S/T motif is characterized by a repeating pattern of proline-serine or proline-threonine residues which separate the binding and



V////////////////////SZ21

401

458

J 400

300

200

100

Figure 1. The domain representation of the Microbispora bispora endoglucanase. The signal peptide was calculated using the program Sigcleave (Rice, P., European Molecular Biology Laboratory, Heidelberg, FRG, personal communication, 1990), while the domains were identified by sequence similarity to known cellulolytic domains and cellulose binding domains. The border determination was made using the PT box as a boundary. The linker domain is simply identified as the region from the first observed proline in the obvious repeat, to the last observed member of the repeat.

GQSITQLWNGDLSTSGSNVTVRNVSWNGNVPAGGSTSFGFLGSGTGQLSSSITCSAS.

Binding Domain

DGCIATPGVFVPDRAYELAMNAAPPTYSPSPTPSTPSPSPSQSDPGSPSPSPSQPPAGRACEATYALVNQWPGGFQAEVTVKNTGSSPINGWTVQWTLPS

Hinge Region

ADEMASRLRGADIANSADGIALNVSNYRYTSGLISYAKSVLSAIGASHLRAVIDTSRNGNGPLGSEWCDPPGRATGTWSTTDTGDPAIDAFLWIKPPGEA

201

301

ΚΙ ΡIMWYAMPNRDCGGP SAGGAPNHTAYRAWI DE IAAGLRNRPAVIILEPDALPIMTNCMSPSEQAEVQASMAYAGKKFKAASSQAKVYFDAGHDAWVP

MSRIRRFIATALAAATAGVGAIVTAIASAGPAHAYDSP

Catalytic Domain

101

1

Signal Peptide

Microbispora bispora Endoglucanase


22.

ELUSTON ET A L


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catalytic domains of most cellulases. Grouping of domains into families was based solely on observed sequence similarity, and multiple sequence alignments (10) were performed on each family to confirm the family relatedness.


Catalytic Domains An analysis of catalytic domains results in some distinctive family groupings. These groupings are listed in Table 1. The striking characteristic of these groups is that they all share significant sequence similarity when they are analyzed using only their homologous domains. TTie inclusion of the often non-homologous hinge and binding domains tends to dilute the similarity observed. The catalytic domain sequences do not seem to be strongly, or at least solely influenced by phylogeny. For example, we see in Family 1 the presence of both bacterial and fungal cellulase sequences. This family has a different relationship between its members than do Families 4 and 5, which are comprised of members of a single species. However, we do notice that cellulase sequences from a particular organism can be distributed into several different families. Multiple sequence alignments of each family highlight specific, and distinct features in each group. These features may be sufficient to describe both the similarity between the members of a group, and to discriminate them from the members of other groups. We are currently developing methods that may be able to provide the functionality to make these determinations. A representative sequence alignment of the catalytic domain Family 1 is shown in Figure 2. Such alignments are very useful in the determination of key residues that may be involved in the conserved enzymatic activities of these subunits. Binding Domains The binding domains can also be classified into families on the basis of sequence homology (Table 2). However, this relationship is not the same as that of the catalytic domain (cf. Tables 1 and 2), suggesting that perhaps these two domains have evolved separately and have only recently been united. The relationships observed in the substrate binding domain seem to be most strongly influenced by the relationship present between the organisms themselves. This is in contrast to the catalytic domains where we noted that the relationships are not strongly influenced by phylogeny. Thisfindingsuggests the possibility that cellulase domains have evolved separately. One particular example that is notable, is the substrate binding domains of the P. fluorescens CMCase and the xylanase from the same organism. These two enzymes are active against quite distinct substrates, namely highly crystalline cellulose and amorphous xylan, yet their binding domains share a high level of similarity. This level of similarity is missing from the catalytic domains of these enzymes. These data suggest that substrate binding may be a more generalizable function, and that the evolution of these domains may be different than for the catalytic domains. Again, multiple sequence alignments can help us to elucidate the key residues involved in the cellulose substrate binding function of these domains. Figure 3 shows just such an alignment, using the sequences of the members of the binding domain Family 1. This multiple sequence alignment is particularly interesting as the Pseudomonas domains share very significant sequence homology with each other, as well as the other substrate binding domains, while binding different substrates. linker Domain The linker region, sometimes referred to as the PSbox or hinge region, has a very different type of conservation than do the other domains. In fact, these domains can


294

ENZYMES IN BIOMASS CONVERSION

Table 1. Cellulase catalytic domain families, organized by similarity scores as determined by successive FastA searches

1

FAMILY 1


CellulomonasflmiEndoglucanase A (11) Microbispora bispora Endoglucanase I (5) Streptomyces sp. Cellulase A (12) Trichoderma reesei Cellobiohydrolase II (13)

FAMILY 2 Bacillus sp. Cellulase (14) Bacillus sp. Cellulase A (14) Bacillus sp. Cellulase Β (15) Bacillus sp. Cellulase C (16) Bacillus subtilis Cellulase (17) Bacillus subtilis Cellulase Β (18) Clostridium acetobutylicum Cellulase (19) Erwinia chrysanthemi Cellulase Ζ (20)

FAMILY 3 Clostridium thermocellum Cellulase D (21) PseudomonasfluorescensCMCase (22)

FAMILY 4 Clostridium thermocellum Cellulase A (23) Clostridium thermocellum Cellulase Β (24) Clostridium thermocellum Cellulase D (21)

FAMILY 5 Trichoderma reesei Cellulase (25) Trichoderma reesei Cellulase A (26) Trichoderma reesei Exocellobiohydrolase I (27)

Each of the representative members of a family demonstrated a similarity score of at least 5 standard deviations from the mean score when the search was made using the PIR database, release 24 (28).



22. ELLISTON ET AL.

Microbispora C. fimi EndA Streptomyces Trichoderma CBHII

Microbispora C. fimi EndA Streptomyces Trichoderma CBHI


295

.

PQSNAAKWVAANPNDPRTPVIRDR IAAVPTGRW RAWQAASGTDK ALLEK IALTPQAYW AEAGVHAWLDANPGDHRAPLIVER IGSEPEAVW TRVPPVGSGTATYSGNPFVGVTPWANAYYASEVSSLAIPSLTGAMATAAAAVAKVPSFMW

FAN-YNPSTVRAEVDAYVGAAAAAGRKIPIMVVYAMPNRDCG GPSAGGAPNH VGNWADASHAQAEVADYTGRAVAAG-KTPMLVVYAIPGRDCG SHSGGGVSEFAGAYNPGTITQQVAEVTSRRQQPPGQLPVWPYMIPFRDCG NHSGGGAPSF LDTLDKTPLMEQTLADIRTANKNGGNYAGQFVVYDLPDRDCAALASNGEYSIADGGVAKY *

*

* *•*

**


TAYRAWIDEIAAGLRNRPAVIILEPDALPIMTNCMS Ρ SEQAEVQASMAYAGKKFKA SEYARWVDTVAQGIKGNP-IVILEPDALAQLGDC SGQGDRVGFLKYAAKSLTL AAYAEWSGLFAAGLGSEPWVVLSPMRFRWI-DCLE NQQRAERLAALQASPEAVTD KNYIDTIRQIVVEYSDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYAVTQLNL


ASSQAKVYFDAGHDAWV PADEMASRLRGADIANSAD GIALNVSNYR KG—ARVYIDAGHAKWL SVDTPVNRLNQVGF-EYAV GFALNTSNYQ ANPEARVYYDVGHSAWH APAAIAPTLVEAGILEHGA GIATNISNYR —PNVAMYLDAGHAGWLGWPANQDPAAQLFANVYKNASSPRALRGLATNVANYNGWNITS


YTSGLISYAKSVLSAIGASHLRA VIDTSRNGNGPLG SEWCDPPGR TTADSKAYGQQISQRLGGKKF VIDTSRNGNGSNG EWCNPRGR TTTDETAYASAVIAELGGG-LGA VVDTSRNGNGPTA ADLVNTR— PPSYTQGNAVYNEKLYIHAIGPLLANHGWSNAFFITDQGRSGKQPTGQQQWGDWCNVIGT


ATGTWSTTDTGDPAIDAFLWIKPP—GEADGCIATPGV FVPDRAYELAMN ALGERPVAVNDGSGLDALLWVKLPGESDG-ACNGGPAAGQWWQEIALEMARN ARW -TVTRC PGVDAFLWITCPVTDGGDGPVFSPPK LQLPRKPAAGRG GFGIRPSANTGDSLLDSFVWVKPGGECDGTSDSSAPR FDSHCALPDALQ

.*...*..

...

.*

Figure 2. Multiple sequence alignments of the Catalytic domain family 1. As with catalytic domain analysis, these alignments were calculated using the program Clustal (10). Identities in all sequences of the group are indicated by an "*", while conservative substitutions in all members of the alignment are indicated by a "Λ


296



Table 2. Cellulase binding domain families, organized by similarity scores (see Table 1)

FAMILY 1 CellulomonasfimiEndoglucanase A (11) CellulomonasfimiExoglucanase (29) Microbispora bispora Endoglucanase I (5) PseudomonasfluorescensCMCase (22) PseudomonasfluorescensXylanase A (30) FAMILY 2 Bacillus sp. Cellulase (14) Bacillus sp. Cellulase A (14) FAMILY 3 Clostridium thermocellum Clostridium thermocellum Clostridium thermocellum Clostridium thermocellum Clostridium thermocellum Clostridium thermocellum

Cellulase A Cellulase Β Cellulase D Cellulase Ε Cellulase X Xylanase Ζ

(23) (24) (21) (31) (31) (32)

FAMILY 4 Trichoderma reesei Cellulase A (26) Trichoderma reesei Cellulase (25) Trichoderma reesei Cellobiohydrolase Π (13) Trichoderma reesei Cellobiohydrolase I (33) Phanerochaete chrysosporium Exoceliobiohydrolase I (34)



Exo

C.

NVTVRNVSWNGNVPAGGSTSFGFLGSGTGQLSSS

IT

AVTVRNAPWNGSIΡAGGTAQFGFNGSHTGTNAAP

QVSVTSLPWNGSIPTGGTASFGFNGSWAGSNPTPASFSLN

*

*•

* e #

**•

PYSASNLSWNGNIQPGQSVSFGFQVNKNGGSAERPSVGGSICSGSVASSS

TAF

GTTC

CSAS

.

Figure 3. Multiple sequence alignments of the cellulose binding domain family 1 are presented. These alignments were calculated using the program Clustal (10). Identities in all sequences of the group are indicated by an "*", while conservative substitutions in all members of the alignment are indicated by a

Pseudomonas XynA

Pseudomonas CmcA PYAASALGWNANIQPGQTAEFGFQGTKGAGSRQVPAVTGSVCQ

fimi

EndA

C. fimi

Microbispora

.*.*.*.,..*

*

C. fimi E x o GPAGCQVLWGVNQWNTGFTANVTVKNTSSAPVDGWTLTFSFPSGQQVTQAWSSTVTQSGS Pseudomonas CmcA NCQYV-VTNQWNNGFTAVIRVRNNGSSAINRWSVNWSYSDGSRITNSWNANVTGN-N Pseudomonas XynA TCSYN-ITNEWNTGYTGDITITNRGSSAINGWSVNWQYAT-NRLSSSWNANVSGS-N

VTNQWPGGFGANVTITNLGD-PVSSWKLDWTYTAGQRIQQLWNGTASTNGG

EAT—YAL—VNQWPGGFQAEVTVKNTGSSPINGWTVQWTLPSGQSITQLWNGDLSTSGS

VDYA

EndA

C.

fimi

Microbispora


298


only be very loosely organized by homology as they only seem to preserve specific characteristics rather than distinct sequences. At this point, no real determination of the families can be made. Such a determination awaits the development of a method for comparing very diverse sequences.


Summary The classification of cellulase sequences into domains allows the further recognition of distinct sequences that characterize each family. To make these sequences evident, one must use multiple sequence alignments of the members of each individual family. We have concentrated on the Microbispora bispora endoglucanase, and present some initial multiple sequence alignments of the catalytic and binding domain families of which it is a member. A domain analysis of the cellulase sequences illuminates some striking relationships between these diverse enzymes. The domains of many of these genes have apparently either evolved separately, or have experienced some sort of directed evolution. The presence of strong relationships between binding domains within a species, with the classification of the catalytic domains into different families, suggests the former hypothesis. It does appear that cellulase gene evolution may be taking place piecemeal. That is, the binding domains may have evolved independently of the catalytic domains, with the distinct possibility that they were recruited subsequent to the development of the catalytic domains. The linker or hinge domain may simply be a region of protein sequence that efficiently links the divergent catalytic and binding domains. We have observed that in many multi domain proteins there are one or several proline residues, often associated with threonine or serine, in the region between the domains. This could be a common method of domain association that links distinctly separate domains and thus facilitates their coordinate evolution and development into a contiguous protein sequence. That would explain the observation of both amino terminal catalytic domains, as in the M. bispora endoglucanase, as well as carboxy terminal catalytic domains, as in the T. reesei CBH II, existing within the same catalytic domain family. The results of this analysis are obviously preliminary. We present them in the hope that we can stimulate both discussion and research in the area of cellulase domain analysis. It is our intention to fully develop the sequence analysis of these domains, and to develop logical criteria for family membership within each of the domain families. Acknowledgments This work was supported by the New Jersey Agriculture Experiment Station (K-01111-04-90) as well as the U.S. Department of Energy. Literature Cited 1.

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Yablonsky, M . D.; Elliston, K. O.; Eveleigh, D. E. In Enzyme systems for lignocellulose degradation; Coughlan, M. P., Ed.; Elsevier Applied Science: London, UK, 1989; pp 73-83.



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Wong, W. K. R.; Gerhard, B.; Guo, Z. M.; Kilburn, D. G.; Anthony, R.; Warren, J.; Miller, R. C. J. Gene 1986, 44, 315-324.

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Teeri, T. T.; Lehtovaara, P. K. S.; Salovuori, I.; Knowles, J. Gene 1987, 51, 43-52.

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