FEMS MicrobiologyLetters78 (1991) 201-206 © 1991 Federationof EuropeanMicrobiologicalSocieties0378-1097/91/$03.50 ADONIS 037810979100128T

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FEMSLE04325

Cloning of xylanase gene of Streptomyces flavogriseus in Escherichia coli and bacteriophage X-induced lysis for the release of cloned enzyme Ranjana Srivastava, Simi S. Ali and Brahm S. Srivastava Division of MicrobialGenetics, CentralDrug Research Institute, Lucknow, lndia

Received12 June 1990 Revisionreceived29 October 1990 Accepted 30 October 1990 Key words: Streptomyces flavogriseus; Escherichia coli; Bacteriophage h; Xylanase gene 1. SUMMARY The xylanase gene of Streptomyces flavogriseus was cloned in pUC8 plasmid and expressed in Escherichia coli lysogenic for hci857, h-Induced lysis of E. coli at 42 °C allowed efficient release of cloned enzyme activity in extracellular environment. The xylanase gene was located in the 0.8-kb HindlII fragment and coded for 18000 M r xylanase.

2. INTRODUCTION Streptomycetes are known to utilize xylan in its native hemicellulose form. These organisms produce extraceUular xylanases which are the primary hydrolytic agents for this commonly occurring

Correspondence to: R. Srivastava,Divisionof MicrobialGenetics, Central Drug Research Institute, Lucknow226001, India.

hemicellulose [1]. Xylanases have been purified and characterized from several streptomycetes [14] and the structural genes encoding xylanases have been cloned [5,6]. S. flavogriseus is an interesting strain which possesses hemicellulose debranching activities mandatory for extensive degradation of lignocellulosic biomass [1]. The xylanase complex of this strain has been studied extensively by Ishaque and Kluepfel [7] and in our laboratory (unpublished observations). We describe in this paper cloning of xylanase gene from S. flavogriseus in Escherichia coli and efficient release of recombinant enzyme by bacteriophage h-induced lysis of bacteria.

3. MATERIALS AND METHODS 3.1. Bacterial strains and plasmid S. flavogriseus 45 CD, obtained from Dr. D. Kluepfel, Institut Armand Frappier, Quebec, Canada, was used as source of xylanase gene. E. coli JM83 lysogenic for bacteriophage hcI857 was

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used as host strain and plasmid pUC8 as vector for cloning [8].

3.2. Media S. flavogriseus was maintained on YEME agar slants [9] at 4 ° C. The basal medium for xylanase production has been described earlier [7]. Tryptone Soya broth (TSB) was used for growth of Streptomyces for isolation of total DNA [9]. E. coli was grown in LB broth [8]. The media was solidified by the addition of 1.2% (w/v) Bacto-agar (Difco). Ampicillin (50 /~g/ml) and 5-bromo-4chloro-3-indolyl-/3-D-galactoside (Xgal) (40 ~tl/ml) were used in media for selection and growth of transformants. Larchwood xylan (Sigma, U.S.A.) was used throughout.

3.3. Isolation of DNA, restriction analysis and cloning Total DNA was isolated from S. flavogriseus [9]. Large-scale purification of plasmid pUC8 DNA by alkaline lysis followed by CsC1-EtdBr gradient, small-scale plasmid isolation, digestion with restriction endonuclease, dephosphorylation, ligation, transformation, gel electrophoresis and other techniques were used following Maniatis et al. [8]. S. flaoogriseus DNA (fragments larger than 50 kb) was digested with HindlII enzyme into fragments of 0.5-10 kb in length. Fragments in the size range 0.5-5 kb (1 /~g sample) isolated from agarose gel were ligated to 0.25 /~g HindlII digested and dephosphorylated pUC8 DNA. The recombinant plasmid molecules were used to transform competent E. coli JM83 ()~cI857) cells. The transformants were scored on LB plates containing ampicillin and XGal [8]. Transformants containing recombinant plasmids were grown overnight on LB agar containing xylan (0.2%) and ampicillin at 32°C. The plates were shifted at 42°C for 30 min to induce the lysogen followed by 2 h incubation at 40°C. Enzyme activity was detected using the Congo red staining method [10]. A clear halo was observed around clones which had digested xylan in the surrounding medium.

3.4. Enzyme studies Spore suspension prepared from 7-day-old agar slant culture of S. flauogriseus was used as inoculum for submerged culture in basal xylan medium. Incubation was carried out on a rotary shaker at 240 rpm at 30°C. Extracellular and intracellular xylanase activities were analysed after 24 to 72 h as described earlier (ref. 7; see also below). E. coli [pSX4] was grown in LB in presence and absence of xylan at 32°C. After 24 h, cytoplasmic, membrane and periplasmic fractions of E. coli were prepared as described by Minton et al. [11] for localization studies. For enzyme preparation, E. coli [pSX4] was grown in LB broth with vigorous shaking at 32°C for about 3 h until the cells reached a concentration of 2.5 x 108 cells/ml (ODss 0 0.25). The lysogen was induced at 42°C followed by subsequent incubation at 37°C for 2-3 h till lysis appeared. DNase was added to a final concentration of 25 # g / m l and lysate was incubated at 37°C for 30 min followed by centrifugation at 50000 x g for 30 min at 4°C. Supernatant was collected and concentrated. fl-Xylanase was assayed at 40 °C by incubating 1 ml of enzyme solution with 1 ml 200 mM-sodium acetate buffer, pH 6.5 containing 1% (w/v) larchwood xylan.The amount of reducing sugars released was determined by the dinitrosalicylic acid (DNS) method [7] with D-xylose as standard. One unit of xylanase was defined as the release of 1 #mol of reducing sugars as xylose in 1 min by 1 ml of enzyme which corresponds to one international unit (IU). Proteins were determined by the Lowry method [12].

3.5. SDS-PAGE and zymogram Culture supernatants of S. flavogriseus and E. eoli [pSX4] lysate containing xylanase activity were concentrated in microconcentrater (Amicon, Denver, MA) and were electrophoresed on a 0.1% SDS-polyacrylamide (7.5%) slab gel. fl-Xylanase activity was detected by the replica gel procedure [13]. Replica gels contained 0.2% (w/v) xylan in 200 mM-sodium acetate buffer, pH 6.5. Congo red staining was used to reveal the zone of activity which appeared as yellow halo in red background.

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Protein bands were revealed by staining with Coomassie brilliant blue.

4. RESULTS AND DISCUSSION

4.1. Growth and enzyme production by S. flavogriseus S. flavogriseus IAF 45-CD is a well characterized xylanolytic strain [7,14]. Growth of S. flavogriseus in basal xylan medium reached maximum level after 48 h. Average value of 50 I U / m l of xylanase was consistently obtained in the supernatant which is comparable to that obtained earlier [7,14]. No intracellular xylanase was detected. Xylanase activity was optimal at 50 °C and pH 6.5. Xylanase synthesis occurred only in medium containing xylan. The enzyme synthesis was repressed by xylose or glucose when present alone or along with the inducer xylan (Table 1). 4.2. Cloning of the xylanase gene from S. flaoogriseus in E. coil A gene bank of S. flavogriseus was constructed by shotgun cloning in E. coli JM83 (Aci857) using the HindlII site of the vector pUC8. Approximately 20 000 a m p r transformants were obtained, out of which 90% formed white colonies on XGal plate indicating the presence of inserts [8]. All the Table 1 Production of xylanase activity by S. flaoogriseus and E. coli [pSX4] when grown on different carbon sources The Streptomyces and E. coli cells were grown in the presence of different carbon sources (10 mg/ml), Xylanase activity in cell free extracts and culture supernatant was determined and expressed as total xylanase activity (IU) as described in METHODS. ND, not determined Addition to growth medium

Total xylanase activity (IU) S. flavogriseus

E. coil [pSX4]

None Xylan Xylose Glucose Xylan + xylose Xylan + glucose

3.5 50.0 3.5 3.9 7.5 4.0

7.85 8.50 7.50 7.56 ND ND

Fig. 1. Screening of E. coli xylanase positive clones (1'). Transformants were plated on LB xylan agar. The enzyme activity was detected by Congo red staining.

white transformants were plated on LB xylan plates supplemented with ampicillin. The enzyme was released by X-induced lysis at 42 ° C. Positive clones were detected by appearance of zone clearing around the colony (Fig. 1). Five positive clones were obtained. On further analysis, three were shown to contain a common HindIII fragment of approximately 0.8 kb while the other two clones had an insert of 1.3 kb. All five clones had the insert in the same orientation as determined by the presence of Pst I restriction site (Fig. 2). Clones with a 0.8 kb insert yielded higher enzyme activity and one of them was chosen for further studies, the plasmid it contained being designated pSX4.

pSX4 3.SKb Avail

pUC8 2.TKb

stmptomxc*m DN&

0JKb

Fig. 2. Restriction map of the recombinant plasmid pSX4. The thick line indicates DNA from S. flavogriseus, cloned into the HindIII site of pUC8 (thin line).

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Restriction fragment analysis performed on pSX4 enabled the construction of a physical map shown in Fig. 2. When E. coli JM83 (hc/857) was transformed with recombinant plasmid pSX4, all transformants were positive for xylanase activity, indicating the presence of xylanase gene on the plasmid.

4.3. SDS-PAGE and zymogram analysis of the xylanase produced by S. flaoogriseus and E. coli [pSX4] Zymogram analysis of S. flavogriseus extracellular fraction after SDS-PAGE revealed xylanase activity in three bands corresponding to molecular mass 42 , 30 and 18 kDa. The 18 kDa subunit consistently displayed significant xylanase activity. These three forms were observed throughout the growth period and again the 18 kDa protein displayed maximum xylanase activity. When S. flavogriseus extracellular xylanase was fractionated by HPLC, Johnson et al. [1] obtained three distinct endo fl-(1,4)-endoxylanases, one neutral and two anionic activities eluting at 0.23 and 0.34 M NaC1. Whether these multiple xylanases are consequences of post-translational modification of a single gene product, or there exist multiple xylanase genes, is still not clear. Zymogram analysis of E. coli [pSX4] intracellular fraction after SDS-PAGE revealed xylanase activity in only one band corresponding to protein of 18 kDa (Fig. 3). A similar observation was made with the other four xylanase positive clones. This seems to indicate that all carry the same gene coding the 18 kDa protein. 4.4. Expression and release of cloned xylanase E. coli JM83 (hd857), whether it carried the plasmid pUC8 or not, produced no xylanase activity when grown in LB. Clone E. coli [pSX4] showed significant xylanase activity (7.85 IU/ml) in LB devoid of any inducer. Clone grown in presence of xylan, xylose or glucose produced similar level of xylanase (Table 1). This is in contrast to glucose and xylose repression observed in S. flavogriseus. Absence of induction and repression could be due to absence of regulatory elements in the insert. In E. coli [pSX4] approximately 90% of the total xylanase activity was localized in the in-

A

B

kOQ

/-5.0

2Z,.O I/~.3

Fig. 3. Zymograms of xylanases produced by S. flavogriseus (lane A) and E. coli [pSX4] (lane B). Prior to activity staining, enzyme preparations were electrophoresed on SDS-polyacrylamide (7.5%) gel.

tracellular fraction and the rest was present in culture supernatant. To ensure that efficient fractionation had occurred, the activities of marker enzymes B-lactamase (periplasmic), glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic) and N A D H : O 2 oxidoreductase (membrane) were also determined (data not shown). X-Induced lysis resulted in release of total xylanase activity in the extracellular medium. On LB xylan plates, lysis of cells obtained by induction of lysogen allowed release and detection of enzyme activity in less than 3 h. One of the major reasons for previous cloning of Streptomyces xylanase genes in the Streptomyces host was to allow excellent secretion of cloned enzyme [5,6]. To overcome this limitation in E. coli, a h lysogen was used for release of total enzyme activity. This system has been used for efficient release of cloned Vibrio cholerae antigens [15]. The temperature (42°C) to induce h ideally coincided with the optimum temperature for enzyme activity.

ACKNOWLEDGEMENTS We are grateful to Prof. B.N. Dhawan, Director of this institute for facilities and support and Prof.

205 D a v i d A. H o p w o o d , J o h n I n n e s I n s t i t u t e , for critical r e a d i n g of m a n u s c r i p t . T h i s is c o m m t m i c a t i o n n u m b e r 4673 o f this institute.

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[6] Mondou, F., Shareck, F., Morosoli, R. and Kluepfel, D. (1986) Gene 49, 323-329. [7] Ishaque, M. and Kluepfel, D. (1981) Biotechnol. Lett. 3, 481-486. [8] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [9] Hopwood, D.A., Bib, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schrempf, H (1985) in Genetic Manipulation of Streptomyces, pp. 72-78, John Innes Foundation, Norwich. [10] Teather, R.M. and Wood, P.J. (1982) Appl. Environ. Microbiol. 43, 777-780. [11] Minton, N.P., Atkinson, T. and Sherwood, R.F. (1986) J. Bacteriol. 156, 1222-1227. [12] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [13] Beguin, P. (1983) Anal. Biochem. 131, 333-336. [14] Kluepfel, D. and Ishaque, M. (1982) Dev. Indust. Microbiol. 23, 389-396. [15] Srivastava, R., Khan, A.A. and Srivastava, B.S. (1985) Gene 40, 267-272.