Journal of Biotechnology, 17 (1991) 177-188 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0168-1656/91/$03.50 ADONIS 0168165691000583

177

BIOTEC 00582

Cloning of three endoglucanase genes from Therrnomonospora curvata into Escherichia coli David G. Presutti, Thomas A. Hughes and Fred J. Stutzenberger Department of Microbiology, Clemson Unioersity, Clemson, South Carolina, U.S.A. (Received 17 October 1989; revision accepted 20 July 1990)

Summary A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated.

Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels. Cellulase; Cloning; Endoglucanase; Thermomonospora curvata

Introduction The enzymatic hydrolysis of cellulose to metabolizable sugars is important in terms of carbon recycling and as a basis for eventual development of biomass conversion systems for alternate fuels production. The microbial cellulolytic enzyme systems are interesting and complex, involving synergistic action of multiple components which vary in their ratios according to cultural conditions and species of microbe. Endo-l,4-fl-glucanases (EC 3.2.1.4) cleave the cellulose polymer at random internal sites to yield free chain ends. Exo-l,4-fl-glucanases (EC 3.2.1.91) cleave cellobiose units from the non-reducing ends of these chains, fl-glucosidases (EC 3.2.1.21) catalyze the hydrolysis of cellobiose to glucose (Eriksson, 1979). Correspondence to: F.J. Stutzenberger, Dept. of Microbiology, Clemson University, Clemson, SC 296341909, U.S.A.

178

In recent years the cellulase genes from a wide variety of microorganisms have been cloned (Teeri et al., 1983; Cornet et al., 1983; Robson and Chambliss, 1986; Collmer and Wilson, 1983; Johnson et al., 1986; Taylor et al., 1987). Genes encoding thermostable cellulases are particularly attractive candidates for manipulation because thermostability provides a variety of advantages in industrial applications (reviewed by Margaritis and Merchant, 1986). In this paper we report the cloning of three endoglucanase genes from the thermophilic actinomycete, T. curvata. This organism, isolated from municipal waste compost, secretes a highly active, thermostable multicomponent cellulase complex that readily degrades crystalline cellulose (Fennington et al., 1982; Stutzenberger, 1972).

Materials and Methods

Materials Restriction endonucleases and T4 DNA ligase were from BRL, Sigma or US Biochemical. Nick translation kits and Biotin-ll-dUTP were from BRL. RNase A, lysozyme, calf alkaline phosphatase, 4-methylumbelliferyl-/3-D-cellobioside (MUC) and 4-methylumbelliferyl-fl-D-glucoside (MUG) were from Sigma. Carboxymethylcellulose (CMC; types 7L and 7H) was from Hercules, Inc. Nitrocellulose membranes (0.45/~m) were from Schleicher and Schuell.

Bacterial strains and plasmids Thermomonospora curvata was isolated from municipal solid waste compost at the Public Health Service and Tennessee Valley Authority composting plant at Johnson City, TN (Stutzenberger, 1971). The cosmid recipient E. coli strain used was X2602 (F-, lacY1, glyU44, galK2, galT2, tyrT58, met81, hsdS3) (obtained from Roy Curtiss III, Washington Univ., St. Louis, MO). E. coli DH1 (Hanahan, 1983) was used for subcloning. The plasmid used for cosmid cloning was pHC79 (Hohn and Collins, 1980). Endoglucanase genes were subcloned using E. coli-B. subtilis shuttle vector pLP1202 (Ostroff and Pene, 1984). A T. fusca endoglucanase plasmid, pD318 (obtained from David Wilson, Cornell University), was also used in hybridization experiments. Culture media Thermomonospora curvata was grown in mineral salts medium consisting of: (NH4)zSO4, 1 g; MgC1z • 6HzO, 0.1 g; CaC12 • 2H20, 11 mg; thiamine, 1 mg; biotin, 1 mg; 1.0 M potassium phosphate buffer (pH 8.2), 100 ml; and distilled water to 1 1. For isolation of chromosomal DNA the medium was supplemented with cellobiose (5 g 1-1). Escherichia coli strains were grown in LB medium (Tryptone, Difco, 10 g 1-1; yeast extract, Difco, 5 g 1-l; NaCI, 10 g 1-1; pH 7.5) or M9 salts (Maniatis et al., 1982) containing 0.2% glycerol as a carbon source. The

179 above media were supplemented with ampicillin (50 gg ml-a), tetracycline (25 #g ml-1), 0.1% carboxymethylcellulose (CMC, Hercules 7L), and 1.5% agar where necessary. Isolation of DNA T. curvata cultures (500 ml) were grown for 18 h and harvested by centrifugation. Chromosomal DNA was extracted from the cell pellet using the method described by Collmer and Wilson (1983). Plasmids were isolated by the method of Birnboim and Doly (1983) or Holmes and Quigley (1981). Large recombinant cosmids were extracted by the method of Godson and Vapnek (1973) and purified on cesium chloride-ethidium bromide density gradients. Cloning procedures Three T. curvata DNA samples containing 1.7/~g each were digested with 0.7 U BamHI (Sigma) for 5, 10 and 20 rain. The DNA samples were pooled and purified by extractions with phenol and subsequently dissolved in 10 #1 of water. Cosmid vector pHC79 (20 #g) was digested completely with BamHI and alkaline phosphatase-treated. The partially digested T. curvata DNA sample (5 gg) was ligated with 8 #g of BamHI-cut, alkaline phosphatase-treated pHC79 in a total volume of 30 gl containing 2 U T4 DNA ligase (BRL). The ligation mixture was maintained at 4°C for approximately 16 h. Bacteriophage X packaging extracts were prepared by "Protocol II" and samples of the ligation mixture were packaged as described by Maniatis et al. (1982). Phage samples were plated on exponential phase E. coli X2602 grown in the presence of 10 mM MgC1z (as described by Maniatis et al., 1982). Transformants were selected on LB (ampicillin, 50 gg ml-1) agar plates and scored for tetracycline sensitivity on LB agar (tetracycline, 25 #g ml-1) plates. Competent cells for subcloning were prepared by the procedure for preparation of frozen competent cells described by Hanahan (1983) except that hexamine cobalt (III) chloride and dithiothreitol were omitted. Screening of clones for CMCase activity Transformants were screened for CMCase activity by a modification of the technique of Teather and Wood (1982). E. coli clones were scored on LB agar (ampicillin, 50 gg ml-1; 0.1% CMC 7L). After incubation at 37°C overnight, the plates were incubated at 60°C from 3-16 h. The colonies were washed off the plates and the plates stained with a 0.5% aqueous solution of Congo Red for 20 min. The plates were then washed with 1 M NaC1. CMCase positive clones were also screened for activity against the fluorescent substrates MUC and MUG. Colonies from overnight cultures were scraped off plates with toothpicks and suspended in 200 #1 1 mM MUC (in 0.1 M MES [2-(N-morpholino)-ethanesulphonic acid], pH 6.0) or 1 mM MUG (in 0.1 M KHPO4, pH 7.0) in the wells of 96-well microtiter plates. The

180 plates were sealed and incubated at 60°C (MUC) or 52°C ( M U G ) for 16 h. Fluorescence was detected by placing the plates on a short-wave UV transilluminator.

Labelling of probes and hybridization of DNA digests DNA probes were labelled with Biotin-ll-dUTP using a BRL nick translation kit. DNA restriction digests were electrophoresed on 0.7% agarose gels. Transfer of D N A to nitrocellulose sheets was carried out by the Southern method (1975). Hybridizations were performed as described by the Blu-Gene Non-Radioactive Nucleic Acid Detection System (BRL). All hybridizations were carried out at 60°C in the absence of formarnide. Post-hybridization washes were also performed at 60°C.

Preparation of enzyme extracts E. coli strains were grown at 37°C in M9 medium supplemented with 50/~g ml -a ampicillin and appropriate amino acids and vitamins, using 0.2% glycerol as a carbon source. E. eoli cultures were grown to late exponential phase using 1% inocula from overnight M9 cultures. Extracts were prepared from cell pellets by resuspending in 10 mM MES, pH 6.1, and sonicating on ice for 3 1-min bursts at 60% of maximum with a Fisher Sonic Dismembrator 300. Cell debris was removed by centrifugation for 5 min in an Eppendorf centrifuge 5414. Enzyme assays Endoglucanase assays were performed using reaction mixtures containing 2% CMC (Hercules type 7L) buffered to pH 6.1 with 0.1 M MES buffer and 0.1 ml enzyme sample. After incubation at 65°C for 2.5 h, reducing sugars (as glucose equivalents) were determined by the Bernfeld method (1955). Viscosimetric assays were performed using Cannon-Fenske type 200 viscometers. Assay mixtures contained 4.75 ml of 1% CMC (Hercules type 7H in 0.1 M MES; pH 6.1) and 0.25 ml of the enzyme sample (preheated to 65°C before mixing). Efflux times were taken at time = 0 min (initial reading) and at time = 10 rain. The specific fluidity (~sp) of each sample was calculated for each incubation time by the following formula: ~sp= T-To~To; T = efflux time sample, To= efflux time of completely degraded 1% CMC (1% CMC digested to completion with crude T. curvata endoglucanase). The specific fluidity values for the time = 0 min and time = 10 min readings were calculated and expressed as 100% (where ~sp for time = 0 rain is 100%). The subsequent values were graphed (~sp% vs incubation time) and the slope of the line calculated. The slope x ( - 100) was set as the number of units ml-~ enzyme sample.

181 Results Cloning of ~-endoglucanase genes

A total of 419 amp r, tet s clones were obtained. E. coli X2602 and X2602 (pHC79) showed no detectable endoglucanase activity. Four clones able to hydrolyze CMC were detected using the Congo red plate assay (Fig. 1). Cosmids isolated from these clones were designated; pTC146, pTC520, pTC717 and pTC749. None of the clones showed any detectable activity against MUC or MUG. B a m H I digestions of these cosmids, electrophoresed on a 0.7% agarose gel, are shown in Fig. 2. Probing of restriction digests

To verify the origin of the inserted DNA in these cosmids and to determine the number of distinct genes which had been cloned, hybridizations were performed using different DNA probes. A preliminary hybridization was performed using biotin-labelled, EcoRI-digested pD318 as a probe (not shown). The pD318 probe hybridized to a 13 kb fragment of pTC146 but did not hybridize to fragments contained in pTC520, pTC717 or pTC749. The 13 kb B a m H I fragment was eluted from a 0.7% agarose gel, purified by extraction with phenol and ligated to B a m H I digested pLP1202. The resulting plasmid (pTCA1) expressed endoglucanase activity on Congo red-stained LB-CMC plates. The cellulase gene contained in pTC520 was

Fig. 1. Zonesof hydrolysisformedon Congored-stained LB-CMCplates. (A) E. coli X2602 (pTC520); (B) E. coli X2602(pTC717); (C) E. coli X2602(pHC79); (D) E. coli X2602(pTC146); (E) E. coli X2602 (pTC749).

182

23130

9416 6557 4361

2322 2027

Fig. 2. Restriction analysis of cosmids encoding endoglucanase activity. Lane 1 = H i n d l I l digested DNA. Lane 2 = B a m H l cut pTC749. Lane 3 = B a m H I cut pTC717. Lane 4 = B a m H I cut pTC520. Lane 5 = B a m H I cut pTC146.

also subcloned to a single BamHI fragment (10 kb) which encoded an active endoglucanase. This fragment was also ligated to BarnHI-digested pLP1202. The resulting plasmid was called pTC12. The 13 kb B a m H I fragment contained in pTC146 and the 10 kb fragment contained in pTC520 (isolated from E. coli DH1 subclones containing pTCA1 and pTC12 respectively) were then used to probe BamHI digestions of the endoglucanase cosmids. The biotin-labelled 13 kb B a m H I fragment of pTC146 was used to probe BamHI digests of pTC146, pTC520, pTC717, pTC749, T. curoata chromosomal DNA and a BglI digest of pD318. The probe hybridized with the 13 kb fragment of pTC146 as well as T. curvata chromosomal DNA and one of the BglI fragments of pD318 (Fig. 3). No hybridization was observed with any of the inserts in pTC520, pTC717 or pTC749. DNA samples were again separated by electrophoresis and transferred to a nitrocellulose sheet. These were probed with the biotin-labelled 10 kb B a m H I

183

3

4

5

6

!i!i~i!i!i~i~ii!,i~ i~!ii_i!~i~!~ii!~

23131 941~ 65S

4a6

232 202

i i~i!! ~

Fig. 3. Probing of the endoglucanase cosmids, T. cumata DNA and pD318 with pTC146 (from pTCA1) probe. Lane 1 = pTC146 probe hybridizing with 13 kb fragment of BamHI cut pTC146. Lanes 2, 3 and 4 = absence of hybridization of pTC146 probe with BamHI cut pTC520 (2), pTC717 (3) and pTC749 (4). Lane 5 = pTC146 probe hybridizing with a 13 kb BamHI fragment of T. curvata chromosomal DNA. Lane 6 = pTC146 probe hybridizing with BglI fragment of pD318.

f r a g m e n t of pTC520. T h e p T C 5 2 0 p r o b e h y b r i d i z e d to the 10 k b insert of pTC520, a f r a g m e n t of T curvata c h r o m o s o m a l D N A a n d a 10 k b f r a g m e n t of p T C 7 1 7 (Fig. 4). N o h y b r i d i z a t i o n was o b s e r v e d with inserts c o n t a i n e d in p T C 1 4 6 o r pTC749. B a m H I - d i g e s t e d p T C 7 4 9 was b i o t i n - l a b e l l e d a n d used to p r o b e B a m H I - d i g e s t e d T. curvata c h r o m o s o m a l D N A . H y b r i d i z a t i o n o f the l a b e l l e d D N A with c o r r e s p o n d ing D N A f r a g m e n t s of T. curvata D N A was o b s e r v e d ( n o t shown). Expression o f endoglucanase genes in E. coli Results o f e n d o g l u c a n a s e assays of extracts o b t a i n e d f r o m E. coli t r a n s f o r m a n t s are shown in T a b l e 1. These assays were p e r f o r m e d b o t h v i s c o m e t r i c a l l y a n d b y

184 (

1

2

3

4

5 ~i! ~i ~

23130-9416-6557--, 4361---

2322-2027,--.

Fig. 4. Probing of the endoglucanase cosmids and T. curvata DNA with pTC520 (from pTC12) probe. Lane 1 = absence of hybridization of pTC520 probe with B a m H I cut pTC749. Lane 2 = pTC520 probe hybridizing with 10 kb B a m H I fragment of pTC717. Lane 3 = pTC520 probe hybridizing with 10 kb B a m H I fragment of pTC520. Lane 4 = absence of hybridization of pTC520 probe with B a m H I cut pTC146. Lane 5 = pTC520 probe hybridizing with 10 kb B a m H I fragment of T. curoata DNA.

release of reducing sugars. The original transformant X2602 (pTC146) was lost and we have been unable to retransform E. coli X2602 with the cosmid pTC146. We therefore attempted to transform competent E. coli DH1 with cosmids pTC146, pTC520, pTC717 and pTC749. However, transformants were obtained only with pTC146 and pTC717. Because strain DH1 (pTC146) showed much higher activity than the other three cosmids harbored in X2602 (and because X2602, pTC146, did not initially appear to have a great deal more activity than the other three from

185 TABLE 1 Expression of Thermomonospora curvata endoglucanase genes in E. coli Strain

Dry cell wt (viscosimetric) Units mg- 1

Dry cell wt (reducing sugar) Units a mg- 1

X2602 (pTC520) X2602 (pTC717) X2602 (pTC749) X2602 (pTCA1) DH1 (pTC146) DH1 (pTC717) DH1 (pTCA1) DH1 (pTC12)

0.549 0.350 0.282 0.752 6.755 0.750 7.545 0.869

4.47 × 10 - 4 ND b ND 4.14×10 4 2.44× 10 -3 5.02 X 1 0 - 4 3.21 × 10 -3 6.26 × 10-4

a Units expressed as micromoles reducing sugar per min b Not detectable under the conditions employed. observation of Congo red plate assays), E. coli X2602 was transformed with pTCA1 (subclone of cellulase gene contained on pTC146) in order to compare expression of this gene in the two E. coli strains. Results from these additional transformants are included in Table 1.

Discussion A total of 419 cosmid transductants were obtained. Assuming a genome size for T. curvata similar to that of E. coli, and an average insert size of approximately 40 kb, the chance of isolating a particular gene intact from this library is approximately 98%. By hybridization analysis it was shown that three distinct endoglucanase genes have been cloned from T. curvata and expressed in E. coll. Multiplicity of endoglucanase forms in cellulolytic organisms is c o m m o n (Hazlewood et al., 1988; Hu and Wilson, 1988; Knowles et al., 1987; Lupo and Stutzenberger, 1988; Robson and Chambliss, 1989). Multiple endoglucanase genes are also commonplace in many cellulolytic organisms. Hazlewood et al. (1988), have shown that Clostridium thermocellum contains at least 15 different endoglucanase genes. At least 3 endoglucanases have been cloned from Cellulomonas fimi (Moser et al., 1989). Trichoderma reesei has at least 2 endoglucanase genes (Knowles et al., 1988; Penttil~i et al., 1986). From the closely related organism T. fusca, at least three endoglucanase have now been cloned (Collmer and Wilson, 1983; Hu and Wilson, 1988). However it appears that at least two additional endoglucanase genes m a y be present (Hu and Wilson, 1988). One of the genes isolated from T. curoata (contained on pTC15) showed homology to an endoglucanase gene from T. fusca. The T. fusca gene codes for endoglucanase E 5 (Collmer and Wilson, 1983; Hu and Wilson, 1988; Wilson, 1988). The plasmid containing this gene (pD318) was originally obtained to screen the T. curvata genomic library. However, this proved to be unnecessary. We did not have

186 available the other isolated T. fusca endoglucanase genes or T. fusca chromosomal DNA. Thus, at this time it is not known if the other T. curvata genes isolated in this study are homologous with other T. fusca endoglucanase genes. It is not unreasonable to expect that other cellulases remain to be cloned from T. curoata as well as T. fusca. Recent electron microscopic evidence (Bonner and Stutzenberger, 1988) suggests that T. curvata forms an extracellular cellulase complex similar to the "cellulosome" of C. thermocellum (Lamed and Bayer, 1988). The cellulosomes of C. thermocellum are very complex and consist of m a n y cellulases. If the cellulosome of T. curvata is as complex, more ceUulase genes may be isolated. Also, while 7". curoata culture fluid shows activity against M U C (data not shown), no M U C - h y d r o lyzing activity has been detected in any of the clones, indicating that genes encoding MUC-hydrolyzing enzymes are yet to be isolated. We are currently exploring the feasibility of the expression of these genes in other organisms. The expression of T. curvata cellulase genes in E. coli was relatively low, with the best subclone (X2602, pTCA1) producing only about 1/2000 of the activity produced by induced T. curvata cultures. This level of expression is comparable to that reported by CoUmer and Wilson (1983) for expression of T. fusca cellulase genes in E. coli. However, these investigators did not report whether these values were compared to induced or uninduced enzyme levels obtained from T. fusca. It is perhaps more appropriate to compare cellulase levels in E. coli transformants to uninduced levels in T. curvata. In this case, X2602 (pTCA1) produces about 1 / 1 0 of the cellulase activity produced by uninduced T. curvata cultures. However, it should be pointed out that the activity of these clones is being compared to the cumulative enzyme activity resulting from multiple endoglucanases in T. curvata cultures. We are currently characterizing the enzymes produced by E. coli transformants and exploring the feasibility of expression of T. curoata cellulase genes in other hosts.

Acknowledgements This study was funded by grants from the South Carolina Energy Research and Development Center and the US A r m y Research Office.

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Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli.

A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isol...
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