Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-β-1,4-glucanase gene

Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-/%l,4-glucanase gene *Prince Sharma, J. K. Gupta, D. V. Vadehra and D. K. Dube Dept o f Mtcrobtology, PunJab Umverstty, Chandlgarh-160014, Indta

(Recewed 18 February 1987, revised 26 May 1987)

The gene encodmg endoglucanase m thermophthc Bacillus sp PDV was cloned m Eschenchla coil stram TB1 usmg p UC 8 as vector The cloned 3 1 kb Pstl DNA fragment was found to express the endoglucanase acttvtty m either ortentatton The deletton analysts ofpSD 81 suggested that the Bacillus endoglucanase gene expressed m E coil under the control of tts own natural promoter, contamed putattvely m the 0 2 kb HlndllIfragment at the 5' end of the msert The relative level of endoglucanase expresston tn E coh was about three ttmes htgher than that m parent Bacdlus sp PD V The cloned organtsm secreted about 84% of the total synthestzed CMCase mto the culture medtum The CMCase was stable up to 60°C and m the p H range of 4-10

Keywords:Cloning, endoglucanase expressmn, Bacdlu~ sp PDV, CMCase gene Introduction The members of the family Bacdlaceae are well known for their ablhtles to produce extracellular fl-glucanases and flxylanases, enzymes I which are important from an industrial pomt of wew because of their potentml use m the brewing industry 2'3 and m bmconverslon of agricultural waste materml to more useful products such as smgle cell protein, fuels and chemical feed stocks 4 6 Several Bacdlus strains have been identified that produce fl-l,4glucanase and the genomes of some similar alkalophlhc Bacdlus s p - - s t r a m N-4, B subtdts and Bacdlus sp D L G - - h a v e been used as sources for clonmg of the respective genes 7-11 We have isolated from soft a thermophfllC organism, Bacdlus sp P D V that produced extracellular, constltutwe endoglucanase and xylanase maximally within 8-12 h of the onset of growth The filter paper and avlcelase activities were also found m the extracellular culture supernatant and fl-glucos~dase m the cell extract The presence of three bands of CMCase activity in the zymogram of Bacdlus sp P D V culture supernatant showed that the orgamsm produced CMCase m multiple forms, a common feature of endoglucanases of m a n y organisms 12 14 The extracellular, purified endo-l,4-flglucanase (E C 3 2 1 4) had a strong actwlty toward noncrystalhne cellulosic substrates such as carboxymethylcellulose and methylcellulose and a weak activity toward more crystalhne forms such as avlcel, filter paper and cotton Th~s enzyme worked optimally at 60°C temperature and in the p H range of 4-8 This study deals with the molecular cloning and expressmn m E colt of this thermostable endoglucanase and a high level of secretmn of the synthesized enzyme m the clone The efficient tTo whom correspondence should be addressed

602

Enzyme Microb Technol, 1987, vol 9, October

expression and secretmn of thermostable cellulase may find use m industry because of~ts abdlty to functmn under a w~de variety of conditions

Materials and methods Bactertal stratus and p l a s m M Bacdlus sp P D V was a soil ~solate E cob strata TBI

(F', r - , m +, ara, Alac-pro, str A, tha, 0 80d lac Z AM15) is a derivative of E colt K-12 strata JM83 constructed by Dr Thomas O Baldwin (Texas A&M University, College Station, TX) and kindly supphed to us by Dr Dennis T Trent (Immunochemlstry Branch Center, Ft Colhns, CO) The plasmld p U C 8 was used as vector ~5 Growth medla

The media 16 used were YT broth supplemented with 0 1% glucose (YTG) and M9 medium containing 0 5 % glucose, 10#g vRamln BI and 4 0 p g prohne m1-1 (M9 medium modified by Baldwin for E cob strain TB1, personal communlcaUon) F o r the detection of cellulolytlc colonies, the agar m e d m m consisted of YT broth, 1 5 % agar and 0 5 % carboxymethylcellulose, sodium salt (CMC, low viscosity, Sigma Chemical C o ) Amplcllhn, streptomycin and chloramphemcol were used at a concentratlon of 50, 25 and 170 pg m l - ~ respectively Preparatton of DNA

For large-scale preparations, the plasmld D N A was amphfied with chloramphenlcol, then extracted from the l y s o z y m ~ S D S cell lysates and purified by lsopycnlc centnfugatlon m a cesium chlorIdemthldlum bromide density gradient 17 For small-scale Isolation, plasmld D N A was prepared from unamphfied overmght culture 0141-0229/87/100602 05 $03 O0 © 1987 Butterworth Pubhshers

Clonmg and expresston tn E Col/ Prmce Sharma et al

using alkallne-lysIs method is C h r o m o s o m a l D N A was prepared by total lysis with lysozyme and SDS 17

Construction o f genomzc hbrary C h r o m o s o m a l D N A of Bacillus sp P D V was partially digested with PstI and the fragments between 2-10 kb in size were collected by passing the digested D N A through Sephacryl-S-1000, equilibrated with Tris-Cl E D T A buffer 19 The plasmid p U C 8 was hnearlzed with PstI and dephosphorylated with bacterial alkahne phosphatase by incubating at 68°C for 1 h in the buffer recommended by suppher (50 mM Trls-Cl, pH 8 0, 1 mM MgC12, 0 1 mM ZnCI 2 and 1 mM spermldlne) The digestions and dephosphorylatlon were terminated by phenol/chloroform extraction and the extent of digestion was monitored by horizontal agarose gel electrophoresls 16 The digested plasmld and chromosomal D N A s were mixed in the ratio of 1 4 and hgated with T4 D N A hgase at 4°C for 16 h in the hgase buffer (50 mM Trls CI, pH 7.6, 10 mM MgC12, 10 mM dlthlothreltol, 1 mM spermldlne and 1 mM ATP) The hgatlon mixture was used to transform E colt TB1 by the method of Trent

Transformation An overnight culture of E colt TB1 (0.5ml) was inoculated to 250 ml of Y T G broth in a 1 liter flask and mcubated at 37°C m a rotary shaker (200 rev m l n - 1) till the O D55 o of the culture reached 0 2. The culture was chilled in ice water for 20 mln and centrifuged at 60009 for 6 min The pelleted cells were suspended in 50 ml of the solution containing 50 mM MnClz, and 20mM sodium acetate, pH 5 6 and incubated on ice for 25 min The cells were centrifuged at 60009 for 6 m l n and the pellet resuspended in 2 m l of a solution containing 75mM CaCI2, 100 mM MnC12 and 20 mM sodium acetate, p H 5 6 These competent cells (0 2 ml) were mixed with 40 ng of the hgated D N A m a 1 5 ml slhconlzed and autoclaved Eppendorf tube and incubated for 40 min in ice with regular mixing The cells were subsequently heat shocked for 2 5 mln at 37°C and transferred to 10 ml ofprewarmed (37°C) Y T G broth and then incubated for 60 mIn at 37°C with shaking This transformed culture (0 2 ml) was mixed with 30/d of 5-bromo-4-chloro-3-1ndolyl-fl-D-galactopyranoside (2 % in N,N'-dimethylformamide) and plated on Y T - C M C agar containing amplcllhn

DetectLon o f endoglucanase positive transformants Transformants were selected for their ability to grow on amplcllhn containing Y T G plates Clones carrying plasmld p U C 8 with inserts were identified as white colonies, whereas clones without inserts gave blue colonies White colonies were toothpicked, in duphcate, onto YT plates containing C M C and amplcilhn, incubated overmght at 37°C and subsequently stained with Congo Red (1 mg m1-1) for 15mln 2o The plates were washed thoroughly with 1 M NaCI The CMCase producing transformant was identified as the colony wth a yellow halo around it

Cell fractlonatlon and endoglucanase assay 0 5 ml of the overnight cultures in Y T G broth of

Bacdlus sp P D V and E colt TB1 (harboring the recombinant plasmids) were seeded to 50 ml of M9 medium in 250 ml Erlenmeyer flasks and incubated for 18 h at 37°C

on a rotary shaker (200 rev m l n - 1) The M9 medium for the growth of E coh-TB1 cells was supplemented with streptomycin and amplcllhn The cells were harvested (90009, 15 mln, 4°C), washed twice and suspended in 5 ml of 0 1 M citrate-phosphate buffer, pH 5 0 The cell extract was obtained by somcatlng the cells (150 mA, 15 mln) and centrifuging the homogenate at 10000 9 for 30 min at 4°C The cell debris was similarly washed twice and suspended in 5 ml of the same buffer 100 #1 of each of the extracellular supernatant, cell extract and cell debris suspension were used for the assay of CMCase activity by the method of Berghem and Pettersson 13 The reducing sugars released were quantltated by the dlnltrosahcyhc acid method 21 One unit of activity was defined as ktmoles of reducing sugar estimated as glucose liberated per ml of enzyme solution per mln

Gel electrophorests 16 Electrophoresls of D N A was performed on agarose slab gels (08 1 5%) prepared in Trls-borate E D T A buffer (0 089 M Tris-borate, p H 8 0, 0 089 M boric acid and 0 002 M EDTA) Gels were stained with ethldlum bromide (0 5/~g m1-1) and D N A bands were observed with a short wavelength u v -transillummator (Fotodyne) D N A fragments of 2-phage digested with HmdIII were used as molecular size references

Nick translation The recombinant plasmld pSD 81 was digested exhaustively with PstI and subjected to analytical electrophoresls on 1% agarose gel The 3 l k b Insert was recovered from the gel by electroelutlon and purified 22 For making the gene probe, 20 ng of the cloned insert was radlolabeled using 50 p moles of [~-32p]-dCTP (3000 CI per mmol from Amersham) and nick translation kit (Bethesda Research L a b , MD) by the method of Rlgby et a123 The level of incorporation of the radtolabel was of the order of 108 cpm # g - 1 of D N A

Results Clonmg o f Bacillus sp. PD V C M C a s e gene m E. c o b A gene bank ofBactllus sp P D V D N A was constructed by llgatlng the PstI partially digested chromosomal D N A with PstI exhaustively digested p U C 8 and using this hgatlon mixture to transform E colt TB1 Out of 6 × 10 3 transformants screened, one formed a large yellow zone around the colony on the C M C - C o n g o Red assay plate The recombinant plasmid pSD 81 was obtained from this Ap r CMCase ÷ transformant and was found to contain an additional 3 1 kb PstI fragment

Restrwtton map o f p S D 81 The hybrid plasmld pSD was digested with several restriction endonucleases either individually or in combinations of two or three enzymes acting together or one after another.i 6 The digests were analyzed by agarose gel electrophoresls The restriction m a p is shown in Figure 1 Out of a multitude of enzymes tested, Bali, EcoRI, AccI and HmeII had unique sites in the insert while HmdIII and TaqI had two and five sites, respectively. The enzymes PstI, BamHI, Sail, Bell, PvuI, XhoI, Sinai and KpnI had no site in the insert

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603

Papers Homology among the cloned fragment and chromosomal D N A To confirm ]ts origin, the 3 1 kb insert was labeled with

32p by nick translation (as described m Materials and methods) and hybridized 24 w]th the Bacillus sp PDV and E. colt TB1 chromosomal DNAs Both Bactllus and E colt genomes were digested exhaustively w]th PstI and electrophoresed on 1% agarose gel The recombinant plasmld

T L 0

T

TT

I 1

i 2

l i 3

F i g u r e 1 RestnctMon enzyme map of the endoglucanase gene of Bacdlus sp PDV from recombinant plasm~d pSD 81 Symbols Pstl, © Hmdlll, • Taql, /k Ball, [ ] Hmcll, A Accl, • EcoRI, [ ] the putative promoter The scale at the bottom us kb

pSD 81 was digested partially with PstI and electrophoresed s~mllarly thus giving three bands--undigested pSD 81 (5 8 kb), cloned fragment (3 1 kb) and pUC 8 (2 7 kb) The electrophoresed DNAs were transferred and lmmoblhzed on the nitrocellulose paper (Ftgure 2) Rad]olabeled insert hybridized to pSD 81 and unlabeled 3 1 kb fragment (lane 1) but not to pUC 8 It hybridized to the fragment m the digested chromosomal DNA of Bactllus sp PDV (lane 2) There was no sequence homology among the insert and digested E cob DNA (lane not shown)

Expression and locahzatton o f CMCase m E coh The level of expression in E coh TB1 ofendoglucanase activity was approximately three times higher than that m Bacillus sp PDV (Table 1) Almost 84% of the total actw~ty was exported into the extraeellular medium, 10 % was found in the mtracellular mdleu and the remaining 6 % assocmted with cell debris

2 Subclonmg o f the 3 1 kb Pstl fragment ~L

/

5.84. ~2

~,

3.1 --

F)gure2 Hybndlzationof32p-dCTPlabeled31 k b m s e r t w l t h p S D 81 (5 8 kb) and unlabeled cloned fragment (3 1 kb) (lane 1 ), and with Pstl exhaustively digested Bacdlus DNA (lane 2)

Various subclones were constructed to determine the minimum size of inserted DNA that was necessary for endoglucanase activity (Figure 3) The 3 1 kb fragment containing the CMCase gene was cloned in pUC 8 in opposite ( - ) orientation The plasmld pSD 83 was obtained by the partial digestion of pSD 81 with HmdIII and hgatlon w]th PstI and HmdIII cut pUC 8 The 2 9 kb insert m pSD 83 was subeloned in correct ( + ) orientation and was devoid of 0 2 kb HmdIII region at the 3' end of the cloned fragment The expression of CMCase activity in pSD 83 was comparable with that m pSD 81 The plasmld pSD 9 was constructed by the deletion of 0 2 kb HmdIII regions from both ends of the 3 1 kb fragment and contained the 2 7 kb insert in correct ( + ) orientation down stream the lac Z promoter of pUC 8 Plasmld pSD 9 expressed shghtly more endoglucanase actwlty The subcloned plasmld pSD 1 was ]dentlcal to pSD 9 but had the insert in opposite ( - ) orientation and did not express any activity at all These results suggest that the cloned CMCase gene was expressed under the control of its own natural promoter m pSD 81 and pSD 83 while in pSD 9 it was under the control of lac Z promoter o f p U C 8 The 0 2 kb HmdIII region at the 5' end of insert

1 Expression and localization of endo-/~-l,4-glucanase m Bacdlus sp PDV and Eschenchta Coh TB1 (harboring recombinant plasmlds)

Table

Organism

Plasmid

Bacdlus sp PDV

-

E coh TB1

pUC 8 pSD 81

Specific endoglucanase activity a Extracellular

Intracellular

Cell debr)s

Total

36 66 (100) b 0 97 12

0

0

36 66

0 721 (6) 314 (2) 2 73 (2) 0

0 11608

(84) pSD 83 pSD 9

89 72 (87) 11836

(87) pSD 1

0

0 11 75 (10) 1212 (11) 14 96 (11) 0

a/%glucanase activity was determined at pH 5 0 and temperature 55°C bF~gures m parentheses indicate percent of the total specific activity in the respective cell fraction

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Enzyme Microb Technol, 1987, vol 9, October

10298 136 05 0

C l o n m g a n d expresston tn E Cob Prmce Sharma et al CI=nCKI Subdon~J mm't

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Rimmct~n enzyme map

, ?

T

~zl~e(kb) ~ f i c l l c t ~ t y a

Orientation

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31

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?

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2?

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27

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0,po,,t, (-) 15s

w pSD 9

?

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pso~

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~

? ~' 'r

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=%

T T ~'

~

1

~ ~

co..t

{.)

~zs

CorrKt

(*)

185

-

Figure 3 The subcloned fragments used to construct the plasmlds pSD81,pSD 83, pSD 9, pSD 1,pSD 81S, pSD81L, pSD84and pSD 85 Symbols ® Pstl, 0 Hmdlll, • Taql, /k Ball, [] Hmcll, A Accl, • EcoRI, [] the putat=ve promoter T h e scale at the bottom ~s kb aTotal specff)c endoglucanase act~wty m extracellular med=um, cell extract and cell debns

in pSD 81 contained the putative promoter and regulated the C M C a s e expression Because this 0 2 kb fragment was absent and the insert was in opposite ( - ) orientation in pSD l, the endoglucanase gene did not express at all None of the subclones pSD 84, pSD 85, pSD 81L and pSD 81S were found to express any endoglucanase activity E n z y m a t z c p r o p e r t t e s o f the c l o n e d C M C a s e

The synthesis of CMCase in E coh TB1 carrying the recombinant plasmld pSD 81 was constitutive as in Bacdlus sp P D V and no effect of C M C supplementation to the medium was observed The CMCase was active over a broad range of temperature and p H It worked well in the temperature range of 40-70°C with an optimum at 60°C and p H range of 4 0-8 0 with p H 5 0 as optimum Metal ions such as Cd + +, Ag + +, Zn + +, Ba ++, Cu +, Mn ++, Co ++ , Mg ++, Ca ++ , Fe +++ and other compounds such as phenylmethyl sulfonylfluorlde (PMSF), sodium azlde, a m m o n i u m molybdate, sodium arsenate, various oxidizing and reducing agents and chelators like E D T A N a 2 had no effect on enzyme activity at 1-10 mM concentration, while Hg ++ inhibited the activity completely at 1 mM concentration

Discussion

Cloning of the thermostable cellulases is the first step required to engineer a thermophllhc organism capable of producing high levels of enzymes The gene encoding CMCase in Bactllus sp P D V was cloned in E colt TB1 The cloned fragment hybridized with the exhaustively digested Bacdlus chromosomal D N A and ht up only one band corresponding to the 3 1 kb position in gel, thus suggesting the presence of either one gene coding for CMCase or more besides this 3 1 kb fragment, but having no homology with the latter However, Sashlhara et al 7

suggested the presence of two genes in the chromosomal D N A of alkalophdhc Bacdlus sp strain N-4 coding for two different endoglucanases The CMCase gene contained in the 3 1 kb insert was believed to be expressed under the control of its own natural promoter since the cloned fragment expressed the enzyme activity in equal amounts in both the orientations The putative promoter regulated the CMCase synthesis in pSD 81 and pSD 83, while the CMCase g e n e m pSD 9 was believed to express under the control of lac Z promoter of p U C 8 The almost equal levels of expression of CMCase in pSD 81 and pSD 9, in terms of specific activity of enzyme, indicated that the RNA polymerase of E coh recognizes the Bactllus CMCase promoter and lac Z promoter of p U C 8 with equal efficiency Robson and Chambhss 9 showed that the 3 0 kb E c o R I fragment of Bactllus strain D L G DNA, cloned in B subtths PSL 1, contained the fl-glucanase promoter E colt TB1 harboring the recombinant plasmid pSD 81 secreted about 84 % of the total CMCase synthesized into the extracellular medium, thus indicating that in E colt the secretion signal sequence of Bactllus sp P D V can function efficiently and the enzyme is correctly processed for secretion Hinchcllffe 3 found that the cloned B subtdlS endo- 1, 3-1, 4-fl-D-glucanase (an enzyme hydrolyzlng fl-1, 4-linkage in hchenan) was located primarily (57%) in periplasmlc space of E colt host cells with 26 % of the enzyme extracellular and only 17 % intracellular Sashlhara et al 7 found that the two CMCase genes of alkalophllic Bacillus strained cloned in E colt resulted in 74 and 34 % respectively of the enzyme extracellular. Robson and Chambllss 9 determined that 63 % of CMCase, cloned in E colt C600 SF8, was retained m cytoplasm, 37% was loosely associated with cell and that there was no activity in culture supernatant, all of which suggested that E colt may not correctly process the enzyme for secretion An investigation of secretion mechanism of cloned enzymes through the cell envelope of E colt will be useful for developing a secretion vector for Gramnegat]ve bacteria It must be taken into account that the culture supernatant of Bacdlus sp P D V contains Avicel activity besides multiple forms of endoglucanase and that the intracellular milieu contains fl-glucosldase activity, whereas E coh produces only one component of the system Cloning of all the components wdl allow reconstltutlon of the system and elucidation of the synergism between the various products of the cloned genes References

1 Priest, F Bactertol Rev 1977, 41, 711-753 2 Enan, T M and Markkanen, P H Proc Am Soc Brew Chem 1975, 33, 13 17 3 Hmchchffe,E J Gen Mwrobtol 1984, 130, 1285-1291 4 Fogarty, W M, Grlffith, P J and Joyce, A M Process Btochem 1974, 9, 11 18 5 Ryu,D D Y and Mandels, M Enzyme Mwrob Technol 1980, 2, 91-102 6 Sandhu, J S and Kennedy, J F Enzyme Mwrob Technol 1984,6, 271-274 7 Sashlhara, N, Kudo, T and Honkoshl, K J Baetenol 1984, 158, 503-506 8 Kolde,Yet al Agrtc Blol Chem 1986,50, 233-237 9 Robson, L M and Chambhss, G H J Bactertol 1986, 165, 612-619 10 Borns, R, Baumleln, H and Hofemelster, J J Appl Mzcroblol Btotechnol 1985, 22, 63-71 11 Mackay,R M et al Nucl Aczds Res 1986, 14, 9159-9170

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Papers 12 Robson, L M and Chambhss, G H Appl Envzron Mtcrobtol 1984, 47, 1039 1046 13 Berghem, L E R, Pettersson, L G and Axlo-Frednksson, U B Eur J Btochem 1976, 61 621-630 14 Ramasamy, K and Verachtert, H J Gen Mtcrobtol 1980, 117, 181-191 15 Messing, J m Methodv m Enzymology (Wu, R, Grossman, L and Moldave, K eds), Academic Press, New York, 1983, vol 101, pp 2(Y78 16 Mamatls, T, Frltsch, E F and Sambrook, J Molecular Clomng, A Laboratory Manual, Cold Spring Harbor, New York, 1982 17 Godson, G N and Vapnek, D Btochlm Blophyv Acta 1973, 299, 516-520

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18 Blrnbolm, H C and Doly, J Nucl Actds Res 1979, 7, 1513-1523 19 Bywater, M, Bywater, R and Hellman, L Anal Btochem 1983, 132, 219-224 20 Teather, R M and Wood, P J Appl Environ Mtcrobtol 1982,43, 777-780 21 Mdler, G L, Blum, R Glennon, W E and Burton, A L Anal Btochem 1960, 1, 127 132 22 McDonell, M W, Simon, M N and Studler, F W J Mol Btol 1977, 110, 119-146 23 Rlgby, P W J et al J Mol Bzol 1977, 113, 237 251 24 Southern, E M J Mol Btol 1975, 98, 503-517