Cloning of the xylanase gene of Streptomyces lividans

Gene, 49 (1986) 323-329 Elsevier

323

GEN 01864

Cloning of the xylanase gene of Streptomyces lividuns * (Actinomy~etes; recomb~~t

DNA; expression; xfn; secretion; antixylanase antibodies; promoter)

Francine Mondou, Frangois Shareck, Rolf Motosoli and Dieter Kluepfel ** Centre de Recherche en Bact&iologie, InstitutArmand-Frappier, Universitkdu Qu&ec, Ville de Laval, QuLI,H7N423 T&l.(514)687-5010

(Canada)

(Received July 23rd, 1986) (Accepted September 22nd, 1986)

SUMMARY

The xylanase (xln) gene of ~tre~t~~~~es lividans 1326 was cloned by functional complementation of the xylanase-negative and /I- 1,4-glucan-glucanohydrolase-negative double mutant of S. lividans using the multicopy plasmid pIJ702. Three clones had a common 2-kb DNA fragment as determined by restriction mapping and Southern hybridization. These clones secreted a xylanase of M, 43 000 which reacted with specific antixylanase antibodies and corresponded exactly to the enzyme previously isolated from the wild-type strain. The DNA fragment likely carried the full structural gene, the xln promoter and also the regulatory sequence, since the xylanase activity was educible by xylan. Enzyme levels of up to 380 IU/ml of culture filtrate were obtained.

INTRODUCTION

Hemicellulose is one of the major components of li~ocellulosi~ biomass and consists largely of xylan, a /?-i,4-linked polymer of D-xylose. The enzymatic degradation of xylan plays an important role in the microbial decomposition of lignocellulose. Xylan-

* Since the submission of this manuscript, the molecular cloning of a xylauase gene from Streptomycessp. has also been reported elsewhere (Iwasaki et al., 1986). ** To whom correspondence and reprint requests should be addressed. Abbreviations: bp, base pair(s); CMC, carboxymethylcellulose; DMSO, dimethylsulfoxide; kb, 1000 bp; IU, international unit(s); p, plasmid; PAGE, polyacrylamide gel electrophoresis; R, resistance; RBB, Remazol brilliant blue; SDS, sodium dodecyt sulfate; Th, thiostrepton; TSB, Trypticase Soy Broth; .&I gene, xylanase gene.

ases have been purified and characterized from various microorganisms (Woodward, 1984) including several streptomycetes (Nakajima et al., 1984; Morosoli et al., 1986). There also have been two reports on the cloning and the expression of xylanase genes in Escherichia coii from two BaciIIus strains (Bemier et al., 1983; Panbangred et al., 1983). This approach of gene cloning in a heterologous host has shown serious limitations such as the secretion of the enzyme into the extracellular environment which represents a major difficulty for the development of a large scale production of xylanase by a genetically engineered Gram-negative microorganism. Recently, we have reported the production of cellulases and of a xylanase by S. lividans (Kluepfel et al., 1986). This gives us the possibility to attempt the homologous cloning of the cellulase and xylanase genes in this microorganism. In view of the rapid development of molecular biology techniques applic-

0378-l119/86/$03.500 1986Elsevier Science Publishers B.V. (BmmedtcalDivismn)

324

able to streptomycetes, in particular to S. lividuns (Hopwood et al., 1985) and, in an effort to enhance the biosynthesis of xylanase production, we constructed a strain of S. lividans which produces high levels of extracellular xylanase by cloning the gene encoding the biosynthesis of the xylanase in the multicopy plasmid pIJ702.

MATERIALS AND METHODS

(a) Strains S. lividuns66 (strain 1326) was obtained from Dr. D.A. Hopwood (John Innes Institute, Norwich, U.K.). From this strain we derived by mutagenesis S. lividuns lo- 164, a fi- 1,4glucanglucanohydrolase (endocellulase)- and xylanase-negative double mutant. S. lividans 313 1 harbouring the plasmid pIJ702, which carries the genes for Th resistance (fsr) and melanin synthesis (me/) (Katz et al., 1983), was kindly provided by Dr. E. Katz (Dept. of Microbiology, Georgetown University School of Medicine and Dentistry, Washington DC).

(b) Mutation and selection of mutants The mutation of S. lividuns 1326 was carried out with N-methyl-N’-nitro-N-nitrosoguanidine (Delic et al., 1970). The endocellulase-negative mutants were selected by replicating the surviving colonies on a solid minimal medium containing 1% CMC (Sigma Chem. Co., St. Louis, MO; medium viscosity, DS 0.65-0.90) as main carbon source. Visualization of endocellulase activity was achieved by Congo Red staining according to Teather and Wood (1982). The detection of xylanase-negative mutants was carried out in the same manner substituting the CMC by 1% oat spelts xylan (Sigma Chem. Co.). The Congo Red coloration was found to be applicable also for the detection of xylanase activity. In both cases, the absence of decoloration zones was taken as an indication for the absence of enzyme production. The mutants were purified twice on the same medium, further tested for enzyme activity (Kluepfel et al., 1986) and for spontaneous reversions. These mutants lacked any other nutritional

requirements. The mutant lo-164 was very stable, appeared to give the highest transformation efficiency and was used throughout this work. (c) Culture conditions The culture media for Streptomyces were TSB (IAF Productions LtCe, Laval, Que., Canada), Bennett medium (Jones, 1949), R5 (Hopwood et al., 1985) containing 400 pg tyrosine/ml, 100 pg methionine/ml and 5 pg CuSO, . SH,O/ml (Kendall and Cullum, 1984) for scoring the melanin phenotype, S medium (Okanishi et al., 1974), xylan medium (Kluepfel et al., 1986) or yeast extract-malt extract (YEME; Pridham et al., 1956; 1957) supplemented with 34% sucrose and 5 mM MgCl, * 2H,O (Bibb et al., 1977). The minimal medium had the following composition: xylan (or CMC), 10 g; KH,PO,, 1.5 g; K,HPO,, 5 g; (NH&SO,, 1.0 g; KCl, 0.5 g; Bacto yeast extract (D&o), 0.5 g; MgSO, * 7H,O, 0.5 g; agar, 17 g; and 1 ml of a trace metal solution containing CoCl, * 7H,O, 200 mg; FeSO, .7H,O, 500 mg; MnSO, * H,O, 160 mg; ZnSO, * 7H,O, 140 mg, all dissolved in 100 ml of water acidified to pH 3.0 and autoclaved. All ingredients except the MgSO, were dissolved in 1000 ml of distilled water and autoclaved. The magnesium salt was added aseptically after sterilisation to prevent the formation of precipitates. For the screening of xylanase-positive clones, the xylan was replaced by RBB-xylan which was prepared according to Biely et al. (1985) by binding xylan covalently to RBB (Aldrich Chem. Co., Milwaukee, WI). Colonies producing xylanase hydrolyze this substrate creating clearing zones. Th (obtained as gift from Squibb Canada Inc., Montreal, Que., Canada) dissolved in DMSO, was added where required to a final concentration of 50 pg/ml. (d) DNA preparation and analysis Chromosomal DNA was extracted from S. lividans 1326 according to the method of Hopwood et al. (1985). Large-scale purification of plasmid DNA was performed by the alkaline method of Kendall and Cullum (1984) and hybrid plasmids were analyzed by the micro-extraction technique of Thompson et al. (1982). Restriction enzymes, T4

325

DNA ligase and nick-translation reagents were purchased from Bethesda Research Laboratories (BRL, Burlington, Ont., Canada) and used according to the manufacturer’s instructions. The DNA probe was nick-translated with [a-32P]dATP from New England Nuclear. Calf intestine alkaline phosphatase (molecular biology grade) was from Boehringer Mannheim Co. (Dorval, Que., Canada). Agarose gel electrophoresis of restriction fragments was performed using Tris-borate-EDTA buffer (Maniatis et al., 1982) and Southern blotting and hybridization conditions were as described by Hopwood et al. (1985). (e) Construction of a gene bank of Streptomyces lividans 1326 Chromosomal DNA (600 pg) was partially digested with Sau3A and the reaction was followed by agarose gel electrophoresis. The resulting fragments were fractionated on a linear 10 to 40% sucrose gradient (Hopwood et al., 1985). The enriched fractions containing 4 to 10 kb fragments were pooled, diluted with Tris 10 mM-EDTA 1 mM buffer at pH 8 and ethanol-precipitated. Plasmid pIJ702 was digested to completion by BglII and the enzyme was removed by phenol-chloroform extraction. The plasmid was ethanol-precipitated and dephosphorylated as described by Kendall and Cullum (1984). For ligation, a mixture of partially digested chromosomal DNA fragments and dephosphorylated pIJ702 at a ratio of 5 : 1, was treated with 0.1 unit of T4 DNA ligase overnight at room temperature at a concentration of 37.5 pg/ml. Protoplasting and transformation of the mutant lo-164 was performed as described by Chater et al. (1982). Transformed protoplasts were plated on R5 medium supplemented for melanin expression as described above and overlayed with 3 ml of R5 medium containing 0.6% agar at 42°C. Transformants were selected for Th resistance after 16 h at 30” C according to the procedure of Kendall and Cullum (1984). (f) SDS-PAGE and Western blotting Extracellular proteins from culture supematants were concentrated on a microconcentrator (Amicon Co., Danvers, MA) and were run on a 9%

SDS-PAGE slab gel (Laemmli, 1970). The gels were further transblotted onto nitrocellulose:sheets and processed according to Towbin et al. (1979) using rabbit antibodies raised against the purified xylanase (Morosoli et al., 1986) combined with [ 1251]protein A (New England Nuclear Co., Boston, MA). The blots were exposed to Kodak X-Omat AR film with intensifying screens for three days at -70°C. (g) Enzyme studies The transformed strains of S. lividans lo- 164 were grown on slants containing Bennett or YEME agar to which 50 pg Th/ml had been added. Spore or mycelial suspensions or homogenates of the mycelium were prepared from such slants and used as inoculum for submerged cultures in TSB or xylan medium (Kluepfel et al., 1986) containing 5 c(g Th/ml. The incubation was carried out on a rotary shaker at 240 rev./min at 34°C. Intracellular and extracellular xylanase activities were analyzed after 24 to 72 h as described by Kluepfel et al. (1986). The enzyme activities were expressed in IU (1 PM of reducing sugars released in 1 min, using D-xylose as standard).

RESULTS

AND DISCUSSION

(a) Cloning of the xln gene of S. lividans 1326 A gene bank of S. lividans 1326 was constructed by shotgun cloning in the xylanase- and endocellulase-negative lo-164 using the BglII site of the vector ~13702. Approximately 24000 ThR transformants were obtained of which 22000 (90%) were melanin-negative indicating the presence of inserts. All the resulting recombinants were spot-plated on Bennett agar containing 50 pg Th/ml and grown at 30°C for 3 days before further analysis. Replica plating was performed on minimal salt medium containing RBB-xylan and the plates were incubated for 2 to 5 days at 30°C. The enzymatic activity was revealed by the appearance of a clearing zone surrounding the presumptive positive clones (Fig. 1). Three clones were isolated from the screening,

326

xln

1 kb -

Fig. 1. Xylanase production by wild-type strain, presumptive clones and xylanase-negative mutant on RBB-xylan medium aRer incubation for 2 days at 30°C. Lanes : (1) 5’. lividam3 13 1; (2) clone X31; (3) clone X30; (4) clone X18; (5) retransformant harbouring pIAFI8; (6) 5’. ~i~~du~ IO-164 (xylanase-negative mutant).

purified on the same medium and further analyzed for plasmid DNA content. This gene bank was also screened for cellulase activity and four positive clones were isolated (F.S., FM., R.M. and D-K., manuscript in preparation). The three xylanasepositive clones obtained did not show any cellulase activity. (b) Characterization and pIAF31

of plasmids

pIAF18, pIAF30

Preliminary analysis by agarose gel electrophoresis showed that all plasmid preparations were larger than pIJ702, i.e. clones X18, X30 and X3 1 had inserts of 5.7, 6.7 and 7.0 kb, respectively. These hybrid plasmids were used to retransform the mutant 10-164. In all cases, 100% ofthe transformants were positive for the enzymatic activity previously scored indicating that the presumptive xln gene was plasmid-linked. Restriction mapping of the plasmids pIAF18, pIAF30 and pIAF31 revealed a discrepancy between the three inserts as to the restriction sites, probably because of the simultaneous cloning of two fragments in pIAF18 and pIAF3 1. However,

Fig. 2. Restriction maps of the xfn clones. The single line represents the plasmid pIJ702. The double line represents the chromosomal DNA inserts. The hatched box near the top represents the BumHI-SphI restriction fragment of piasmid pIAF18 used as a probe in hybridization experiments. The dotted line indicates the region of homology between the three plasmids and the approximate location ofthe xln gene. The arrow indicates the direction of the transcription of the tyrosinase gene in plasmid pIJ702. Ba, BamHI; Bg, BgIII; Ps, PstI; Sa, Suu3A; Sp, SphI.

Southern blotting, using as a probe the BarnHI-SphI restriction fragment internal to the insert pIAF18, showed that about 2 kb of DNA was common to the three inserts (Fig. 2). (c) Expression fividuns

of the xln gene in Stre@myces

lo-164

The expression of the xln gene was studied by submerged cultures of the difYerent clones in TSB and in xylan medium and compared to the wild-type strains S. iivid~ 1326 and 3131. An incubation temperature of 34°C as chosen since it afforded a good ratio of growth to xylanase activity (Kluepfel et al., 1986). The results are shown in Table I. The wild-type strain, whether it carried the plasmid pIJ702 or not, produced no enzyme activity when grown in TSB and the levels produced in xylan medium varied between 3 and 6 W/ml of culture filtrate. The clones X18, X30 and X31 all showed significant activity in TSB (0.9-4.5 W/ml) even in the absence of any inducer. Clones Xl8 and X30 grown in xylan medium produced very high levels of xylanase, e.g., Xl8 reached 380 IUjml. The xylanase production of clone X31 was unexpectedly low in

327

TABLE I Comparison of xylanase production by Streptomyces lividans1326 and 3131 and by clones X18 and X30 in submerged cultures in the absence and presence of xylan, after 48 and 72 h of incubation at 34°C. The activity is expressed in III/ml of culture filtrate. Strain

Xylanase activity in III” TSB

1326 3131 X18 x30 x31

Xylan medium

48 h

72 h

48 h

72 h

0 0 4.4 1.4 0.9

0 0 4.5 1.4 1.1

3.1 3.7 293.0 219.0 1.8

5.8 6.3 380.0 305.0 n.d. b

* The assay for xylanase was carried out by incubating 1 ml of enzyme solution appropriately diluted in 0.1 M McIlvain buffer pH 6.0 with 1 ml of an aqueous suspension of 1% xyian at 60°C for 10 min. The reaction was terminated by the addition of 2 ml of dinitro-salicylic acid reagent and by heating for 15 min in boiling water. The amount of reducing sugars released was determined with D-xylose as standard. The blanks consisted of 1 ml xylan suspension incubated in the same manner to which 2 ml of the dinitro-salicylic acid solution and 1 ml of enzyme were added (see MATERIALS AND METHODS, section g). b nd., not done.

xylan medium. Apparently, this strain rapidly lost its activity during the passages required for the inoculum build-up. This is probably caused by an instability of the insert, since freshly retr~sfo~ed colonies exhibited as much activity as clone X30 (Fig. 1). In the intracellular fraction of all cultures, only trace amounts of xylanase activity were detected, indicating an efficient secretion of the enzyme. (d) Analysis of the xylanase produced by clones X18 and X30

The enzymes secreted by the clones Xl8 and X30 into the culture medium were analyzed by SDS-PAGE as shown in Fig. 3A. The xylanase-like products of these clones had an estimated M, of 43 000. This value corresponded exactly to the M, of the native purified xylanase (Morosoli et al., 1986)

A Fig. 3. Analysis of culture supernatant concentrates of strain S. lfviduns3 131, clones X 18 and X30 aAer induction with xylan. (A) SDS-g% PAGE after staining with Coomassie blue. Lanes: (1) Prestained M, standard proteins (in kDa); (2) clone X18; (3) clone X30; (4)s. lividans 3131. (B)Autoradiogram of a Western blot probed with antixylanase antibodies combined with [ “‘I]protein A. Lanes: (5) native purified xylanase isolated from S. iividuns 1326; (6) clone X18; (7) clone X30.

indicating that all the clones had inserts coding at least for the complete structural gene. Furthermore, the identity of these proteins was confirmed immunologically in Western blotting experiments using antixylanase antibodies, as shown in Fig. 3B. (e) Conclusions

The xln gene of S. lividuns has been cloned by functionally complementing the xylanase- and endocellulase-negative mutant 10-164. The xylanase protein expressed by these clones appeared as a M, 43 000 band in SDS-PAGE which corresponded to the M, of the mature protein purified from S. lividans and it reacted immunologically with antixylanase antibodies. A structural gene for such a protein would be approximately 1200 bp in length. Such a coding sequence is presumably within the 2 kb common to the three cloned inserts. Because the

328

orientation of the inserts in pIJ702 can not be correlated to the level of expression of the xylanase activity in the transformants, the cloned DNA fragment probably contains the natural promoter signal of the xln gene. Although the exact mechanism of re~lation of xylanase synthesis is not presently known in S. ~~vidans,the expression of the xfn gene cloned in the multicopy plasmid pIJ702 did not appear to be constitutive as has been reported for the agarase gene also cloned in pIJ702 (Kendall and Cullum, 1984). Instead, the xln gene was inducible in strains containing any of the three plasmids which suggests that the regulatory gene was cloned together with the structural gene. Although, it is possible that a gene present in the genome regulates the expression of the cloned xln gene. The clones produced signilicant levels of xylanase without induction, which might be caused by a gene dosage effect, since plasmid pIJ702 is probably present in 40 to 300 copies per chromosome (Kieser et al., 1982). It is of particular interest to compare the present results with those reported previously on the xylanase genes from Bacillus species cloned into E. coli, a heterologous host (Bernier et al., 1983; Panbangred et al., 1983). In both cases, only low xylanase expression of about 1 IU was obtained and no secretion of the enzyme was observed. The cloning of the xln gene of S. lividans in a homologous system not only allowed excellent secretion but yielded enzyme levels that were more than 60 times higher than those of the wild type, and this even before a proper physiological strain development had been carried out. Studies in this direction will be undertaken shortly and should lead to even higher xylanase levels. The present enzyme values are higher than any reported in the literature. The high expression of the xln gene and the secretion of the xylanase in streptomycetes will open the possibility to investigate further the mechanism of secretion of proteins by comparison with other genes coding for extracellular proteins, e.g., tyrosinase (Bernan et al., 1985). It also will be possible to develop ~tre~to~yces vectors and strains suitable for the introduction, controlled expression and secretion of genetically engineered proteins (Gray et al., 1984). The expression and secretion of the xyhmase are currently under investigation to elucidate the mechanism of regulation of the enzyme.

ACKNOWLEDGEMENTS

This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada and by Esso Imperial Oil Canada Ltd. We also wish to thank Liette Biron, Denise Bienvenue and Jean-Louis Bertrand for their excellent technical assistance and collaboration.

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