Molecular cloning of a Cellulomonas fimi cellulase gene in Escherichia coli

Gene, 17 (1982) 139-145 Elsevier Biomedical Press

139

Molecular cloning of a Cellulomonas fimi cellulase gene in Escherichia coli (Recombinant

DNA: plasmid pBR322; immunoassay)

D.J. Whittle, D.G. Kilbum,

R.A.J. Warren and R.C. Miller Jr.

Department of Microbiology, Universityof British Columbia, Vancouver, BC, V6T I W5 (Canada)

(Received November 14th, 1981) (Accepted December 2nd, 1981)

SUMMARY A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi. The enzyme present in extracts of E. colt’ carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars. -

INTRODUCTION Microorganisms

capable

have a great economic cipal and industrial

of

potential

degrading

cellulose

for converting muni-

wastes into substrates suitable for

fermentation. However, the insoluble crystalline nature of cellulose makes it difficult to degrade by direct microbial action. Furthermore, the association of cellulose with other complex compounds such as lignin makes it refractory to microbial invasion. Bacteria like C. jimi secrete cellulases that penetrate the wood matrix and attack the cellulose extracellu-

Abbreviations: Ap, ampiciIIin; BSA, bovine serum albumin; CMC, carboxymethylcellulose; CNBr, cyanogen bromide; kb, kilobase pairs; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; Tc, tetracycline; [I, indicates plasmid carrier state.

0378-1119/82/0000-0000/$02.75

0 Elsevier Biomedical Press

larly. Usually, the amount of cellulase secreted and the subsequent rate of cellulose digestion are low. One approach to increasing cellulase production by cellulolytic organisms would be to isolate the gene(s) coding for cellulase(s) on a recombinant DNA plasmid and then to modify the expression of these genes by current molecular genetic techniques. These could include coupling the genes to strong promoters, eliminating operators sensitive to repression, and increasing the efficiency

of translation.

This paper reports the successful molecular cloning of a cellulase gene from C, fimi on plasmid pBR322 in E. coli. Active cellulase was detected in extracts of one E. coli transformant. To obtain these clones, we developed a sensitive screening procedure for E. coli colonies carrying cellulase genes. The screening procedure is based on binding cellulase to antibodies followed by detection of the antigen-antibody complex with 1251-labeled protein A from Staphylococcus

140

aureus.

We report

here the details of the screening,

molecular cloning, and cellulase assay procedures.

ultrafiltration Residual

(Amicon

cells

concentrate

were

through

PM10 membrane) removed

by

a 0.45 pm

pore). l-ml samples, containing MATERIALS

at 4°C.

passage

of the

membrane

(Milli-

approx. 1500 units of

total cellulase activity, were mixed with 1 ml complete Freund’s adjuvant and injected into mature

AND METHODS

white

New Zealand

rabbits.

Booster

injections

of

1500 units of cellulase were given in the same manner

(a) Bacterial strains and media

4 and 6 weeks later. The rabbits were killed, and the The bacterial

strains used were: C. fimi ATCC484;

E. coli SF8[pBR322]

(Dunn

coli C600.

(d) Isolation of DNA

C. fimi was grown in basal medium

(1 g NaNOa,

1 g KzHPO‘,, 0.5 g KCl, 0.5 g MgS04 . 7 H?O, 0.5 g yeast extract, per liter, pH 7.0) and either 1 g glucose or 1 g CMC (Sigma) per liter as an energy source (Hitchner and Leatherwood, 1980). E. coli strains were grown in LB (10 g tryptone, 5 g yeast extract, 5 g NaCI, 1 g glucose, per liter pH 7.4) or M9S medium (Champe and Benzer, 1962) supplemented with uridine (200 /.&ml), thiamine (20 &ml) and thymidine (10 /.&ml). Ampicillin (50 pg/ml) or tetracycline (20pg/ml) was used when growing bacteria containing plasmids. Solid media contained 11 g agar per liter. Top agar contained 10 g tryptone, 5 g NaCl, and 7 g agar per liter, pH 7.4. (b) Calorimetric cellulase assay Cellulase activity was analyzed by measuring the increase in reducing groups released by the hydrolysis of a carboxymethylcellulose substrate (Stewart and Leatherwood, 1976). Reaction mixtures contained 0.5 ml of appropriately

sera were collected a week after the last injection.

et al., 1979); and E.

diluted enzyme

solution

and

1 .O ml of 4% CMC in 100 mM phosphate buffer, pH 7.0. After 30 min at 37”C, 1.5 ml dinitrosalicylic acid reagent (Miller, 1959) were added, and the tubes were placed in boiling water for 15 min. The absorbance was read at 550 nm against blanks containing equivalent amounts of boiled enzyme. One unit of enzyme released 1 pg of glucose equivalents per min by reference to a standard curve. Protein concentrations were determined by the Bio-Rad protein assay. (c) Preparation of antisera

The

isolation

previously

of plasmid

DNA

was described

(Dunn et al., 1979). C. fimi DNA was ob-

tained by a lysozyme-SDS-pronase

procedure (Lovett

and Keggins, 1979). It was purified by phenol extraction and banding

in CsCl density gradients (see Dunn

et al., 1979). (e) Cloning of BumHI fragments of C fimi DNA into E. coli Purified pBR322 DNA was linearized with BarnHI under the buffering conditions recommended by the supplier. C. fimi chromosomal DNA was partially digested with BarnHI under similar buffering conditions. Digestions were terminated by phenol extraction. The extent of digestion was monitored by horizontal agarose gel electrophoresis. A 1 pg sample of linearized pBR322 DNA was mixed with 5 pg of a BamHI partial digest of C. fimi DNA; and the DNA was precipitated with ethanol. After adding 50 ~1 ligase buffer (66 mM Tris * HCl, pH 7.8, 10 mM MgCl?, 20 mM dithiothreitol, 1 n&l ATP, and 5Opg gelatin/ml), the mixture was kept at 0°C for 8 h. T4 DNA ligase was added, and the mixture was held at 4°C for 14 h, at 14°C for 8 h, and then used to transform E. coli C600 to ampicillin resistance (see Cohen and Chang, 1973). The transformed cells were stored at -20°C in LB containing 40% glycerol. The proportion of cells carrying recombinant plasmids was determined by plating appropriately diluted samples medium + tetracycline.

on

ampicillincontaining

(f) Enzymes and reagents C. fimi was grown to late log phase in CMC basal medium. The cells were removed by centrifugation, and the supernatant was concentrated lOO-fold by

T4 DNA ligase and restriction endonuclease BamHI were from New England Biolabs. ‘*‘I-labeled

protein A was prepared using the enzymobead iodination

reagent

(Bio-Rad)

radio-

and protein A (Phar-

macia). All dilutions of antiserum and 12SI-labeled protein A were made in PBS containing 1% BSA. The specific activity of the 12SI-labeled protein A was 5 X lo6 cpm/pg. PBS contained

8 g NaCl, 0.2 g KCl, 0.2 g

KH2P04, 2.17 g Na2HP04 . 7 HsO, per litre, pH 7.4.

RESULTS

(a) Development of a sensitive immunological ing method to detect cellulase enzymes

screen-

The goal of this work was to clone the C. fimi cellulase genes into the E. coZi plasmid pBR322. The initial step was the development of a sensitive and simple screening method for cellulase gene products. The approach adopted was immunological. Proteins from lysed colonies were transferred to CNBr-activated futer papers, which then were treated with anticellulase antibody. Antibody bound to the filters was detected by autoradiography using [1251]protein A, which binds specifically to the F, portion of IgG

Fig. 1. Autoradiogram of C. fimi colonies. This autoradiogram represents the results of an experiment designed to demonstrate the specificity and sensitivity of the immunological screening method described in RESULTS. Uninduced C. jimi and E. coli colonies were grown on LB plates and screened using (A) CNBr-activated paper and (B) normal paper. The location of E. coli colonies is indicated by circles. Induced C. fimi colonies were grown on CMC-plates and screened using (C) CNBractivated paper and (D) normal paper.

molecules. Log-phase culture supernatants ing on CMC medium activity

contained

per ml. Concentrates

from C, fimi grow20 units of cellulase

of such supernatants

were injected into rabbits to elicit antibody production (see MATERIALS AND METHODS). Log phase rather than stationary phase cultures were chosen to limit contamination with intracellular proteins

lysed appeared clear against a darker background.

The

darker background was assumed to be caused by the presence of contaminating anti-yeast antibodies bind-

released by cell death and lysis. These contaminating

ing to components

proteins would greatly increase the number of falsepositive clones detected during screening. The presence of anti-cellulase antibodies in the rabbit

medium. The background was eliminated by diluting the antiserum l/1000 or by excluding yeast extract from the medium. Using the transfer system it was

serum was demonstrated by using it to prepare an immunoadsorbent column that was subsequently shown to remove cellulase activity from C. fimi culture supernatant. A control column prepared using normal rabbit serum did not adsorb the enzyme activ-

then shown that E. coli C600 colonies gave a negative C. fimi a weak positive, and response, uninduced

ity. Experiments with phage T7 and anti-T7 serum showed that antigen could be bound directly to CNBr-activated paper and that less than 1 ng of bound antigen could be detected with antibody and [‘2SI]protein A. With an anti-cellulase serum dilution of l/250, sites where E. coli C600 colonies had been

of the yeast-extract

present in the

induced C. fimi colonies a strong positive response to the anti-cellulase serum when screened in the same manner (Fig. 1). Some cellulases have a high affinity for cellulose. Induced colonies of C. fimi still gave a strong positive response by this screening method when the filter paper was used without CNBr-activation. However, since it was not known if all C. fimi cellulases would bind strongly to native paper, the CNBr-activated paper was used routinely in the present work.

142

(b) Constructrion

and identification

of cellulase-pro-

(from CMC plates) were spotted at defined positions as markers

ducing recombinant clones

and as positive controls.

covered, incubated On the assumption tain internal

that cellulase genes might con-

BarnHI restriction

sites, a partial BarnHI

digest of C. fimi DNA was prepared construction

of recombinant

(see MATERIALS

AND

plasmids from pBR322

Colonies

Some 30% of

METHODS).

the E. coZi ApR transformants an indication

and used for the

obtained

were TcS.

of the presence of inserts. containing

recombinant

plasmids (ApR, with

ampicillin and grown overnight at 30°C. Colonies were lysed in situ by overlaying the agar with 2 ml top agar containing

SDS (0.25 mg/ml) and lysozyme (0.5

mgjml)

et al., 1978).

Induced

C. fimi

cells

cases, the disks were used without

cellulase.

incubated

at 37°C for 14 h. Colonies

dilution

carried

of l/250.

a recombinant

Two C. firm’ colonies plasmid

(pDW1)

E. coli C600

were used as references

encoding

at 30°C for

and placed

in

1M

overnight at 4°C. The disks were washed with PBS. then incubated with rabbit anti-cellulase serum (diluted l/250-1/1000) for 3 h at room temperature. The disks then were washed, suction filtered with

producing

which

removed

glycine-phosphate buffer, pH 7.4 at 30°C for 5 h to inactivate residual protein binding sites, then stored

with

at a serum

prior activation.

The plates were covered, and incubated

of an E. coli colony and

pH

7.4, then placed on top of the lysed colonies. In some

L-plates

ampicillin

paper disks (Clarke

et al,, 1979) were soaked in 25 mM phosphate,

Fig. 2. Autoradiogram supplemented

paper disks for 15 min at

CNBr-activated

3 h. The disks were

TcS) were picked onto LB plates supplemented

(Erlich

ed over chloroform-saturated room temperature.

The plates were

at 30°C for 30 min and then invert-

C. firm’ cellulase.

containing

recombinant

then were screened

and positive

controls.

1plasmids were spotted as outlined

One E. coli colony

on

in RESUI .TS was detec ted

143

P,BS, and incubated

with [‘2SI]protein

A (diluted

to 10

5 X 10’ cpm/ml) for 3 h at room temperature. Finally, the disks were washed, suction filtered, dried at 41°C for 1 h and placed over Kddak XRP-1 X-ray film for lo-20 the colonies

h. Orientation

was accomplished

tions of the induced producing

colonies

a

of the paper disks to by noting

the posi-

C. fimi cells. Potential

celIulase-

were picked either from replica

plates or from the original lysed plate and re-streaked on LB plates (Erlich

et al., 1978). These colonies

were re-screened as outlined One positive screening

clone

above.

was obtained

in the initial

2

of 1000 clones (Fig. 2). Upon re-streaking

and repeated

screening,

munoreactive, bacteriophage

recombinant clone. Phage typing with T7 confirmed that the bacterium was

it was confirmed

E. coli. The plasmid it contained

as an im-

was given the desig-

nation pDW1.

(c) Characterization of the recombinant plasmid

10

20 Cell

30

40

extractcpl)

Fig. 3. Cellulase activity of E. coli[pDWl]. E. coli C600 carrying recombinant plasmid pDW1 DNA was grown to 5 X lO*/ml in L-broth. The cells were collected by centrifugation, lysed in a French press and assayed for cellulase activity by the calorimetric assay outlined in MATERIALS AND METHODS (circles). The experiment was repeated with E. coli C600[pBR322] (squares).

The doubly-screened, positive recombinant clone was grown overnight in LB supplemented with ampicillin. Cells were concentrated loo-fold in 100 mM phosphate buffer pH 7.0. DNase (25 &ml) was added, and the cells were ruptured by passing them twice through a French Press (12 000 lb/in?). The extract was centrifuged at 25 000 rev./min in a Beckman type 40 rotor for 60 min. Supernatants were collected and assayed for cellulase activity as outlined previously. Cell-free tained

extracts

5 units

of E. coli C600[pDWl]

of cellulase/ml

of original

conculture

volume. The activity was approximately linear with respect to amounts of extract added over the range of 1.5 mg to 6.0 mg protein (lO/..d-40~1 (Fig. 3). Na activity

was detectable

40

0

10

of extract)

in an extract of

E. coli C600[pBR322] over an identical range of added protein. As was the case with C. _timiculture supernatants, the cellulase activity in the E. coli C600[pDWl] could be adsorbed onto immobilized anti< fimi cellulase antibodies (Fig. 4). The plasmid from E. coli C600[pDWl] was purified and digested with BarnHI. The digest was analyzed by agarose-gel electrophoresis. It contained four components: one, of 4.4 kb, corresponded to linear pBR322; the other three, of 1.6,6.6 and 12 kb, presumably were C’.fimi DNA.

2

4 Fraction

6

8

10

no.

Fig. 4. Immunoadsorption of E. coli[pDWl] cellulase enzymes by anticellulase antibody bound to a protein A column. Normal serum was added to a protein ASepharose CL4B column. After washing with PBS, 350 units of E. coli[pDWl] cellulase were added to the column. Fractions of 1.5 ml were collected and assayed calorimetrically for cellulase activity (circles). The column was eluted with glycine . HCl buffer (pH 3.0) and re-equilibrated with PBS. The experiment was repeated using anti-ceUulase serum instead of normal serum (squares).

144

viscometric

DISCUSSION

indicate The work reported here is part of a comprehensive program

using

recombinant

develop an economical to fermentable

DNA

technology

process to hydrolyse

to

cellulose

sugars. The first steps in this approach

have been accomplished. A sensitive and simple screening method has been developed for detecting recombinant DNA plasmids carrying cellulase genes from C. fimi, and one such plasmid has been isolated. The screening

method

described

here has several

assay (data

the presence

not

shown).

These results

of an endoglucanase

activity,

but do not preclude the presence of an exoglucanase. Further

studies

are in progress

to characterize

the

nature of this enzyme(s). A number

of factors could influence

of cellulase production apparent bers;

factors

regulation

stability

in E. coli[pDWl].

include:

cellulase

or inducibility

the amount The most

gene copy num-

of cellulase

genes;

of cellulase in E. coli; and, transcriptional

translational

differences

between

Gram-positive

or and

advantages. First, the use of CNBractivated paper to bind proteins released from lysed colonies renders

Gram-negative

the technique

present in E. coZi[pDWl]. It is interesting that E. coZi[pDWl] carried pBR322 with a 20.2.kb insert without any apparent

generally

of any recombinant which an antibody

applicable

for the screening

clones that express a peptide for is available, The use of [12’1]-

protein A to detect bound antibody makes possible the use of one labeled probe for many different screenings. A similar technique has recently been developed independently and described by Kemp and Cowman (1981). In the present studies, CNBr-activated paper was used routinely,

although

the affinity of cellulases for their substrate, activated

paper

can be omitted.

because of the use of

This simplifies

the

systems. Any one or a combination

the above factors could influence

of

the level of cellulase

abnormalities in the growth of the bacteria or the plasmid. Since we used a partial BamHI cleavage of Cellulomonas DNA in the original recombinant DNA formation, it is not possible to say whether or not the insert represents a contiguous portion of the Cellulomonas DNA. At present, other E, coli clones carrying C. fimi DNA are being screened for additional cellulolytic activities. Ultimately, we plan to combine

procedure when applied in genetic engineering experiments designed to increase yield or to develop export

appropriate cellulase genes with a suitable vector in a host that will efficiently produce and export large

of the cellulase encoded by pDW 1. The sensitivity of the technique was estimated to be in the nanogram range, certainly adequate to detect the production of cellulase in uninduced Cellulomonas. In addition, the technique is responsive to

quantities

the amount of cellulase in induced Cellulomonas, which represents an increase of at least two orders of magnitude compared to the uninduced state. This will facilitate experiments designed to increase levels of cellulase production. Another advantage of the procedure described here is the ability to recover live cells from the bottom of colonies on the original lysed plate. This eliminates the necessity of replica plating (Erlich et al., 1978). Hydrolysis of cellulose to glucose usually requires at least three types of enzymes: endo,4glucanase, exoQl,4glucanase and @lucosidase. We have determined that at least one enzyme possessing cellulolytic activity is present in E. coli extracts carrying pDW1. This was indicated by the production of reducing sugars in the calorimetric cellulase assay. Cellulase activity was also observed in these extracts using a

of cellulase.

ACKNOWLEDGEMENTS

This

study

was supported

by

Strategic

Grant

NSERC GO.475 from the Natural Science and Engineering Research Council of Canada.

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