A Major Exoglucanase Component of - American Chemical Society

Chapter 5

CelS: A Major Exoglucanase Component of Clostridium thermocellum Cellulosome Kristiina

Kruus,

William K . Wang, Pei-Ching C h i u , Joting Ching, T z u u - Y i W a n g , a n d J. H. D a v i d W u

Downloaded by UNIV LAVAL on October 27, 2015 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch005

D e p a r t m e n t o f C h e m i c a l E n g i n e e r i n g , U n i v e r s i t y o f Rochester, Rochester, NY 14627-0166

The CelS protein (M = 82,000) is the most abundant protein species secreted by Clostridium thermocellum. It has been identified as a key catalytic component of the C. thermocellum cellulosome, an extremely complicated and large protein aggregate responsible for the degradation of crystalline cellulose. We have proposed that CelS acts synergistically with CelL ( M = 250,000), another major cellulosome component, to degrade crystalline cellulose through a novel enzyme-anchor mechanism distinguishable from that of the fungal cellulase system. However, the function and the properties of this key catalytic component remain to be elucidated. We have recently cloned the celS gene. Its D N A sequence cannot be classified into any of the known cellulase families. In fact, the celS sequence has led to the discovery of a new cellulase family. Two previously un-characterized gene fragments from other bacteria have been found to contain sequences highly homologous to the celS sequence. Furthermore, the N-terminal amino acid sequence of a novel exo-β-glucanase (Avicelase II) purified from C. stercorarium matches that of CelS. The members of this new cellulase family are so far found only in strict anaerobic bacteria. CelS may therefore be one of the key components distinguishing the anaerobic cellulase system from its fungal counterpart. Expression of the celS gene in Escherichia coli resulted in the formation of inclusion bodies. The solubilized and refolded gene product displayed activity typical of an exo-β-glucanase. Thermostability was enhanced by Ca , as for the crude enzyme. The celS gene therefore encodes a novel exoglucanase. r

r

++

Clostridium thermocellum, an anaerobic, thermophilic, cellulolytic, and ethanogenic bacterium, produces an extremely complicated extracellular cellulase complex called a cellulosome ( i ) . The cellulosome is formed by more than fourteen cellulase

0097-6156/94/0566-0084$08.00/0 © 1994 American Chemical Society

In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


5.

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CelS: A Major Exoglucanase

85

Component

components with a total molecular weight of millions (2-4), and accounts for most of the Avicelase activity (activity against A v i c e l , a microcrystalline cellulose preparation) in the culture filtrate of this bacterium (5). M a n y enzymatic properties displayed by the cellulosome are significantly different from those of the better-studied fungal cellulase system; In particular, the clostridial cellulase appears to have a higher specific activity against crystalline cellulose (6). Aggregation of cellulase components has been found in other cellulolytic bacteria (7,8). W i t h their unique multi-component structure and properties, these bacterial cellulase systems likely belong to a different and less well-studied class of cellulase systems. Difficulty in purifying individual cellulosome components has posed serious technical challenges and impeded progress in elucidating the mechanism and structure of the cellulosome. Molecular cloning approaches have led to the discovery of many endoglucanase genes, but have given no clue to the mechanism of the cellulosome. Initial insight into this complicated enzyme complex has been obtained by the identification of the two major cellulosome components, C e l L ( S or C i p A ) and CelS (or S ) , which degrade crystalline cellulose synergistically (5). A n "anchor-enzyme model" has been proposed to explain the synergism between these two subunits (9), in which C e l L functions as an anchor on the cellulose surface for C e l S , the catalytic subunit This mechanism is clearly different from the well-accepted, exo-endo synergism model based on the fungal cellulase system. Recent progress i n cloning and analyzing the D N A sequences of both celL (10) and celS (11,12) genes has confirmed the enzyme-anchor model and expanded it into a more sophisticated model L

s

(13) . This expanded enzyme-anchor model involves specific receptor-ligand interactions as the mechanism of the complex formation between the catalytic and the anchorage subunits. The enzyme-anchor model therefore provides a structural basis for the unique properties of the enzyme. Further characterization of CelS and C e l L , which could now be made available i n the recombinant form, w i l l eventually unveil the detailed and sophisticated mechanism by which the cellulosome degrades cellulose. In this article, recent progress i n cloning and expressing the celS gene i n E. coli and the initial characterization of the gene product w i l l be briefly presented. C e l S Belongs to a N e w Cellulase F a m i l y T h e Uniqueness o f the celS D N A Sequence. Due to the complexity and the stability of the C. thermocellum cellulosome, purification of individual components has proven to be extremely difficult. Molecular cloning and expression of individual genes i n a heterologous host appears to be the only feasible approach for elucidating the functions of cellulosome components and the possible synergism between them. U s i n g the carboxymethylcellulose ( C M C ) and Congo-Red plate screening method (14) , many cellulase genes have been cloned. It is not surprising that all the genes obtained are endo-enzymes since the screening method is specific for endoglucanase activity. However, it is somewhat puzzling that the bacterium harbors so many cel genes with seemingly similar function. M a n y of the D N A sequences of the cloned C. thermocellum cel genes have been determined. These genes fall into known cellulase families on the basis of primary structure homology (Table 1; 75). Most of the genes (celB, celC, celE, celG, and



86

ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION

celH) belong to Family A . There are two genes (celD and celF) in Family E and one each i n Family D (celA) and Family F (xynZ), respectively. They therefore share homology with themselves and with cellulases of other microorganisms including Trichoderma reesei, Bacillus sp., Schizophyllum commune, and Erwinia chrysanthemi, (Family A ) ; Cellulomonas uda (Family D ) ; Pseudomonas fluorescens (Family E ) ; and Cellulomonas fimi and Cryptococcus (Family F ) . In contrast to the endoglucanase genes described above, the celS gene was cloned by hybridization to a degenerate oligonucleotide probe derived from its N-terminal amino acid sequence. The D N A sequence of celS (11) does not have significant homology with other cel genes cloned from C. thermocellum or other microorganisms except the conserved, duplicated sequence at its C-terminus (discussed later). It therefore does not belong to any known cellulase family (Families A - F ; 75) reported so far. It is clear that the probe hybridization method has yielded a novel cellulase gene (Table 1). Table 1. Cloned C. thermocellum Gene Product

Size

CelA CelB CelC CelD CelE CelF CelG CelH XynZ

1,344 1,689 1,032 1,947 2,442 2,219 1,698 2,702 2,511

CelS

2,241

(b.p.)

Endoglucanase and Xylanase Genes 1

Reference

Observed M . W . (Da)

Cellulase Family

52,503 63,857 40,439 72,344 90,211 87,409 63,128 102,301 92,159

56,000 66,000 38,000 65,000

(16,17) (18,19)

90,000

D A A E A E A A F

80,670

82,000

NEW

(11,12)

Predicted M . W . (Da)

66,000

-

(2021) (2223) (24) (25) (26) (27) (28)

^ e n r i s s a t et al., (75).

A N e w Cellulase F a m i l y . Since CelS has been shown to play a critical role i n the degradation of crystalline cellulose, it would be surprising i f the celS gene is only found i n C. thermocellum and not i n other cellulolytic microorganisms. The celS and its related genes may have escaped cloning because the C M C a s e activity screening method is commonly used in cloning cellulase genes. Indeed, sequences highly homologous to the celS sequence have been identified from at least two other bacterial genes (Figure 1), the O R F 1 of C. cellulolyticum (29) and the celA of Caldocellum saccharolyticum (30). These two gene fragments are each located next to an endoglucanase gene and were therefore cloned during the cloning of the endoglucanase gene. The complete open reading frames of both homologous


5.

CelS

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KRUUS ET AL.

CelS A B C


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F i g u r e 1. A m i n o acid sequence comparison between CelS, the partial O R F (ORF1) preceding the celCCC gene of C cellulolyticum ( A ) , the partial O R F (also O R F 1 or celA) preceding the manA gene of Caldocellum saccharolyticum (B), and the 28 amino acid cartridge in an endoglucanase of Bacteroides ruminicola (C). B o x e d amino acids are identical or have similar chemical properties. Numbers indicate the position, within the sequence of each protein, of the first or last amino acid shown on a line. For (A) and (B), the numbers start with the first amino acid residues reported. For (C), the numbering refers to the second encoding O R F . Similar residues were: V , L , I , M , F ; R , K ; D , E ; N , Q ; Y , F , W ; S,T; G , A . (Taken from reference 77)


229

732 334


88


sequences have not been published. There were no clues to the functions of these two homologous open reading frames when they were originally cloned and sequenced. However, based on the degree of homology with celS, these two open-reading-frames most likely code for cellulolytic enzymes. The structure of the celS gene has thus led to the discovery of a new cellulase family. It is interesting to note that, although C. thermocellum is a thermophilic bacterium with the optimum growth temperature of 60°C, C. cellulolyticwn is mesophilic. O n the other hand, C. saccharolyticum has an optimum growth temperature of 68 - 70°C. The celS gene family therefore appears to have evolved to function in at least three different temperature ranges. Furthermore, the celA gene of C. saccharolyticum appears to codes for a "hybrid protein" with its C-terminal half resembling that of CelS and its N-terminal half homologous to an endoglucanase gene (cenB of C.fimi; 31). The celA is therefore likely a product of "domain shuffling" involving part of the CelS and an endoglucanase. Additionally, unlike CelS and O R F 1 of C. cellulolyticwn, the celA gene of C. saccharolyticum does not encode the C-terminal conserved, duplicated sequence. A s w i l l be discussed below, this conserved, duplicated sequence is the binding ligand to C e l L . This indicates that CelS family may function either alone (as in the case of C e l A ) or by forming a multi-subunit complex with other cellulase components (as i n the cases of CelS and O R F 1 ) . Finally, a l l three bacteria are strict anaerobes, suggesting that CelS may be unique to the anaerobic cellulase system. The Conserved Binding Ligand Sequence. The genetic evidence that CelS forms a protein complex with C e l L , which is essential i f the enzyme-anchor model is to be considered valid, is that CelS contains a conserved, duplicated sequence at its C-terminus. It has been shown (32) that this conserved, duplicated sequence serves as a binding ligand to the receptor sites of the C e l L protein as depicted i n the modified enzyme-anchor model (13). The list of the genes containing this ligand sequence continues to expand. A total of eighteen genes are compiled i n Figure 2. Twelve of them are from C. thermocellum, five from C. cellulolyticwn, and one from C. cellulovorans. This suggests that the cellulolytic enzymes of these Clostridia are organized as described by the modified enzyme-anchor model. These cellulase systems therefore likely represent a special class of cellulase adopting the enzyme-anchor mechanism which clearly distinguishes them from the fungal system. A C e l L - l i k e protein (CbpA) has been discovered i n C. cellulovorans (33). A CelS-like protein ( O R F 1) has been found in C. cellulolyticwn (29). It w i l l not be surprising i f both the C e l L and CelS equivalents are discovered later i n both bacteria. Cloning and Expression of celS in E. coli P C R Cloning and Expression of celS. T o obtain the intact celS gene, as opposed to the four overlapping clones used earlier to determine the sequence (11), we used the Polymerase Chain Reaction ( P C R ) technique i n which two sequences flanking the celS gene were used as the P C R primers and the C. thermocellum genomic D N A as the P C R template. The PCR-amplified gene product was cloned into the E. coli expression vector p R S E T - B for expression (Figure 3). The resulting D N A construct was sequenced to ensure that fusion of the celS structural gene to the expression


5. K R U U S E T A L .

CelS: A Major Exoglucanase Component

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F i g u r e 2. The alignment of the conserved duplicated sequence between C e l S , C i p A , C e l A , C e l B , C e l D , C e l E , C e l F , C e l G , C e l X , C e l H , X y n Z , and L i c B (38) of C . thermocellum, C e l C C A , C e l C C C , C e l C C D , C e l C C G , and O R F 1 of C . cellulolyticwn (29, 39, 40), and E n g B of C . cellulovorans (41). Boxed amino acids are identical or have similar chemical properties. Numbers indicate the position, within the sequence of each protein, of the first or the last amino acid shown on a line. Similar residues are: V , L , I, M , F ; R , K ; D , E ; N , Q; Y , F , W ; S, T . C i p A is also named as C e l L or S . L


441

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F i g u r e 3. M a p of the ce/S-pRSET B clone. The 2.1 kb Pstl-HindlW celS P C R fragment was cloned into the 2.9 kb p R S E T B expression vector. The cloned gene was inserted in-frame with the initiation A T G codon. Expression was controlled with the promotor recognized by T 7 R N A polymerase. The resulting gene product contained a 4 k D a N-terminal fusion encoded by the leader sequence. The plasmid was selected by growth on media containing the antibiotic ampicillin. The figure is not drawn to scale.


5.

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CelS: A Major Exoglucanase Component

91

vector is i n frame (data not shown). A n additional protein species (M = 86,000) was produced i n the E. coli strain harboring the insert gene (Lanes 3 and 4, Figure 4). The expression product was slightly larger than native CelS (Lane 1, Figure 4) due to the fused sequence from the expression vector. The identity of the gene product was verified by Western blotting, using a polyclonal antibody preparation specific to CelS. The antibody specifically recognized the rCelS (data not shown), indicating its authenticity. Phase-contrast microscopy revealed that the rCelS protein was produced in the form of inclusion bodies i n the host cells. Under an electron microscope, the inclusion body appeared as an amorphous structure inside the E. coli cell (Figure 5). The inclusion bodies existed even when the conserved, duplicated sequence at the C-terminus was deleted (data not shown). Downloaded by UNIV LAVAL on October 27, 2015 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch005

r

T h e Cellulase A c t i v i t y of r C e l S . T o obtain an active protein, the inclusion bodies were isolated by centrifugation and solubilized i n 5 M urea, which was later removed by dialysis. This resulted in a protein preparation containing predominantly rCelS (Lane 2, Figure 4). The preparation was subsequently used for characterizing the enzyme activity of CelS. A c t i v i t y on V a r i o u s Cellulose Substrates. The rCelS produced little reducing sugar when incubated with carboxymethylcellulose ( C M C ; Table 2). However, when A v i c e l was used as the substrate, almost twice as much reducing sugar was released (Table 2). A more dramatic increase in reducing sugar production was observed on amorphous cellulose (phosphoric acid-swollen Avicel). These results are typical of an exoglucanase, indicating that CelS is not an endoglucanase (commonly referred to as C M C a s e ) as are many other cel gene products identified from C. thermocellum (Table 1). Its lack of a significant activity on C M C explains why the celS gene could not have been cloned by an C M C - C o n g o red screening method.

T a b l e 2. A c t i v i t y of r C e l S on V a r i o u s Cellulosic Substrates 1

Reducing Sugar Released Glucose Equivalent (pg/ml) Incubation T i m e (hours)

0

15

24

CMC Avicel Amorphous Cellulose

0 0 0

6.2 9.5 39

6.8 13 48

incubation was carried out at 60°C i n a succinate buffer (pH 5.7) which contained 12 m M Ca+* and 0.5% (w/v) substrate.



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F i g u r e 4. S D S - P A G E analysis of the celS expression in E. coli. Lane 1, the crude C. thermocellum culture filtrate; Lane 2, the partially purified rCelS; Lane 3, the lysate of E. coli strain harboring the recombinant plasmid containing the celS gene; Lane 4, the lysate of E. coli strain harboring the control plasmid without the celS gene. The proteins were electroblotted to a P V D F membrane before staining with Coomassie blue.


5. K R U U S E T A L .


93

Component

+ +

Effect o f C a . Ca** has been found to stimulate the Avicelase activity of the crude C . thermocellum cellulase (6). The effect of Ca** on the rCelS activity was therefore examined using amorphous cellulose as the substrate. A s shown i n Figure 6, Ca** (12 m M ) d i d not have a significant effect at 60°C under the assay conditions. However, at higher temperature settings, the rCelS activity was stimulated by the Ca** (Figure 6), indicating that C a * enhanced the thermostability of the enzyme. The optimum temperature was 70°C in the presence of Ca** and was lower in the absence of Ca**. This observation is consistent with the effect of Ca** on the crude enzyme (6) and the S8-tr protein (34) discussed below. 4

A N e w F a m i l y o f Exo-P-glucanase. While CelS is produced by the C. thermocellum A T C C 27405, the S8 protein is the major component of the cellulosome of the Y S strain. A truncated form of S8 (S8-tr, M = 68,000) has been purified by partial proteolysis of the cellulosome and the subsequent purification of the dissociated proteolysis product (34). The purified S8-tr displays typical cellobiohydrolase activities. Its thermostability is enhanced by Ca**. Although the reported apparent molecular weight of S8 appears to be different from that of CelS (75,000 vs. 82,000), both protein species migrate at the same rate when subjected to the SDS-gel electrophoresis on the same gel (Figure 7). Furthermore, S8 has an N-terminal amino acid sequence identical to that of CelS (Figure 8; 35). Although the cloning and D N A sequence of the S 8 gene have not been reported, these two proteins appear to be identical. A s indicated i n Figure 7, S8 and CelS are both the most abundant protein species of the Y S and A T C C strains, respectively. The next major protein species are C e l L (of the A T C C 27405 strain) or S I (of the Y S strain). It is interesting to note that the S I protein is slightly but noticeably smaller than C e l L (Figure 7). The high level production of both protein species ( C e l S / C e l L or S8/S1) in two different strains suggest that they play important roles in the cellulolytic activity as described in the enzyme-anchor model.


r

Besides the S8 protein, the C. stercorarium Avicelase II (M = 87,000; 36) also has an N-terminal amino acid sequence highly homologous to that of CelS (Figure 8). The purified Avicelase II shows exoglucanase activities. However, unlike the conventional cellobiohydrolase which preferentially releases cellobiose from the non-reducing ends of cellulose chains, this enzyme releases cellobiose, cellotriose and cellotetraose from the non-reducing ends depending on the type of substrate. Avicelase II therefore appears to represent a novel type of exoglucanase. A name of "cellodextrinohydrolase" has been proposed to describe this new exoglucanase. The novel action of Avicelase II is consistent with the novelty of the CelS sequence. In addition, as in the case of CelS, the thermostability of Avicelase II is enhanced by Ca**. However, the gene sequence of this enzyme has not been reported, preventing sequence comparison beyond the N-terminal peptide. Furthermore, Avicelase II is slightly larger than CelS (87,000 vs. 82,000). Finally, there is a significant difference between the two proteins: CelS is part of a cellulase aggregate and Avicelase II appears to be a free enzyme. In fact, no protein aggregation has been reported in C. stercorarium. r

It appears that at least four microorganisms produce CelS-like protein (C. thermocellum [CelS or S8], C. cellulolyticum [ORF-1], C. stercorarium [Avicelase II], and C. saccharolyticum [CelA]), although CelS so far is the only member with



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F i g u r e 5. The transmission electron micrograph showing the inclusion body formation in a E. coli cell expressing the celS gene.

o *-

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Temperature (*C)

F i g u r e 6. The effects of temperature and Ca** on the activity of rCelS on the amorphous cellulose. The assay tubes contained 0.5% (w/v) phosphoric acid-swollen A v i c e l . The incubation time was 15 hours. The Ca** concentration was 12 m M .


KRUUSETAL.


95

Component


5.

F i g u r e 7. S D S - P A G E pattern of the total extracellular proteins of C . thermocellum A T C C 27405 and Y S strains, indicating that CelS (S ) and S8 have the same apparent molecular weight and that C e l L ( S or C i p A ) is noticeably larger than S I . s

L



1

Avicelase II

28

1

CelS

S8

GK 1

K D P K N G Y F S P D E G 1 P Y H S

1

PlY K[Q R[F L E L W E E L H D P S N G Y F S - X H G 1 P Y H A V

K D L F[X|E

F i g u r e 8. The alignment of the N-termini of CelS and S8 from the A T C C 27405 and Y S strains of C. thermocellum, respectively, and Avicelase II from C. stercorarium. B o x e d amino acids are identical or have similar chemical properties. Numbers indicate the position, within the sequence of each protein, of the first or last amino acid shown on a line. Similar residues were: V , L , I , M , F ; R , K ; D , E ; N , Q ; Y , F , W ; S,T; G , A . The first 27 amino acid residues of CelS presumably form the signal peptide for secretion. The N-terminus of the native CelS starts at the residue 28.

X S D D

G P T K A P T K P G T S Y

G P T K A P T K D G T S Y K D L F L E L Y


35

19

70

5.

KRUUSETAL.


Component

97

complete D N A and amino acid sequences reported. These proteins form a new family of exo-P-glucanases that may have a novel mode of action different from that of conventional cellobiohydrolases. A l l four bacteria are anaerobic, indicating that CelS may be a major component contributing to the unique properties of the anaerobic bacterial cellulase system.


Conclusion Compared to the well-studied T. reesei cellulase system, the C. thermocellum cellulase exhibits a higher specific activity toward crystalline cellulose. It also displays other unique properties such as requiring Ca** and reducing agent for maximum activity, and inhibition by non-polar chelating agents (6,37). A s we have previously proposed, those unusual properties are rooted in the unique structure of the cellulosome, best described by the enzyme-anchor model involving predominantly two proteins, CelS and C e l L . It is clear now that C e l L , a major component of the cellulosome, serves as a scaffolding protein of the cellulosome and an anchor for the catalytic subunits on the cellulose surface. Inclusion of this scaffolding/anchorage subunit i n the C. thermocellum cellulase system clearly distinguishes it from the fungal cellulases. CelS, the most abundant protein species secreted into the culture supernatant, has been identified to be the essential catalytic subunit for crystalline cellulose degradation. Cloning of the celS gene has led to the discovery of a new cellulase family. The members of this family so far have only been found in anaerobic bacteria. It codes for an exoglucanase clearly distinguishable from its fungal counterpart. The properties of the C. thermocellum cellulase system may therefore be further defined by the presence of CelS in the complex. It appears that an exciting, novel mechanism of enzymatic cellulose degradation is in the process of being unveiled. The mechanism involves an anchor/scaffolding protein (CelL) and a novel exoglucanase (CelS). The availability of the celS gene and the rCelS protein w i l l facilitate further characterization of its function and interactions with C e l L , and provide further insights into this intriguing, sophisticated, and non-conventional cellulolytic process which has only begun to be explored recently. Acknowledgments The research project on cellulase in our laboratory is financially supported by N R E L (XAC-3-13419-01) and U S D A (Alcohol Fuels Program; 93-37308-8988). W . K . W . appreciates the support of the L i n k Foundation Energy Fellowship. K . K . appreciates the scholarship from the Finnish Academy. Literature Cited 1. 2. 3.

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A Major Exoglucanase Component of - American Chemical Society

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