Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis

Vol. 126, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

February 15, 1985

Pages

1154-1160

CLONING AND EXPRESSION OF THE Escherichia coli D-XYLOSE ISOMERASE GENE IN --Bacillus subti=J.J. Laboratory

Received

January

4,

Huang,

and N.W.Y.

Ho*

of Renewable Resources Engineering Purdue University West Lafayette, IN 47907

1985

SUMMARY: A DNA fragment containing the Escheria coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomefase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. s bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene structural gene downin B. subtilis by fusion of the E. coli xylose isomerase stream to the promoter of the pezicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able 0 1985 Academic Press, Inc. to be expressed in both B. subtills and --E. coli.

An 5. --coli (x&A)

DNA fragment

and the

D-xylulokinase

laboratory

(1).

separated

from

Expression vector

the

has also D-xylose

xylose is

fructose ally

syrup

used for

expression

*

for

of the -E. --coli

To whom correspondence

0006-291X/85 Copyright All rights

and processes

in a yeast-E.

coli

(2).

shuttle

enzyme product responsible

of the xylA -

for

the

D-glucose

to D-fructose

process

currently

used for

the production

is a safe

microorganism

which

subtilis

of industrial

gene in -B. -___ subtilis

and reprint

requests

$1.50 Inc. reserved.

enzymes.

1154

should

Hence, are

gene,

conversion

isomerizes

xylA

0 1985 by Academic Press, of reproduction m any form

in this

also

the production

gene

(3).

is

-.B

recently

of subcloning

promoter

enzyme which It

isomerase

gene has been characterized

the

industrial

the D-xylose

has been cloned

(EC5.3.1.5),

(4).

(5,6).

(&B)

by a yeast

been documented

to D-xylulose

both

gene by a series

gene

industrial

contains

isomerase

xylulokinase

isomerase

the basis

gene

The xylose

of the -xylA

an important

which

cloning

desirable.

be addressed.

is

is

of Dand this of high traditionand

Vol.

126,

Recently,

we have

shuttle

plasmid.

tilis. I_-

However,

was not

expressed

enzymatic

original

promoter

The cloning

been reported tion

(7-9),

in both

describe formis expression

of the

xylA

xsA

xylA

for

of the

E. coli -__

gene

a Bacillus-E. -~

-B. --sub-

coli -E. --_

most likely

is

coli -.--

-

xylA

gene

due to the

in the new host.

gene

promoter

the

COMMUNICATIONS

to transform

the intact This

gene

in _. B. ----9 subtilis with

(penP)

has also

(10-11).

of the -~__ Bacillus

suitable

gene into

gene has to be replaced

penicillinase

to make it

RESEARCH

was used

that

promoter

and B. subtilis

the modification

plasmid

transformants.

and the &

g. --coli

-xylA

indicated

the -E. --_ coli

for

BIOPHYSICAL

the intact

assay

in ___Bacillus

to express

AND

hybrid

of the E. coli

In order

moter.

subcloned

The resulting

ineffectiveness

the

BIOCHEMICAL

No. 3. 1985

a ---Bacillus

that pro-

from g. -----licheniformis been shown able

In this

of --penP-xylA __

has

to func-

communication,

penP gene isolated

construction

we believe

we

from B-. lichenifusions

for

the

in -B. -Isubtilis.

MATERIALS

AND METHODS

Strains J --) media and ----plasmids Bacterial strains, media, and the growth _--__ -_ conditions for E. -coli have been described previously (1,2). B. --_____ licheniformis 749/C was obtained from -_I_ Bacillus genetic stock center, Ohio State University, and B. -__subtilis PSLl was from A. Aronson, Purdue University. The growth of Bacillus was carried out as described by Ehrlich (12). The vector plasmids ~used for E. --coli were pBR322 (13) and pHSG415 (14), and those used for Bacillus were pC194 (12) and pUBll0 (15). wePreparation amplified and pUB110, pC194 Helinski was as described

of plasmid and chromosomal DNA. Plasmid DNA from E. --coli was --__I_--prepared as described previously-(16). For the preparation of and their derivatives, the clear lysate procedure of Clewell and followed (17). Chromosomal DNA of g. --_____ licheniformis was prepared by Marmur (18).

Enzymes __ and -----chemicals.Restriction endonucleases, T4-DNA ligase and Ba131 --exonuclease were purchased from Bethesda Research Laboratory. Media and antibiotics were from Sigma. All other chemicals were obtained from commercial sources. out

Transformation. Transformation of E. coli by previouslydescribed procedures (19).

and -B. ____ subtilis

were

Detection of penicillinase-positive colonies. Penicillinase-positive -___-___-_ colonies were selected on LB agar, containing ampicillin, and were onto LB plus polyvinyl alcohol (plate method) for quick detection triction analysis of plasmid DNA isolated from individual colonies quick lysate procedure was used to verify the insertional fragment

carried

replicated (20). Resby a miro(21).

Complementation xylose ---__ isomerase mutants The analysis of plasmid--___ -of ~---mediate complementation of D-xylose isomerase mutation was carried out according to the procedures reported previously (1,2). 1155

Vol.

126,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Enzymatic assays. Preparation of cell extracts and the E. coli xylose isomerase assay were carried out as previously described (1,2). Xylose isomerase activity of --. Bacillus was measured according to the procedure described by Danno (6).

RESULTS AND DISCUSSION It 4.2

has been reported

kb EcoRI

restriction

EcoRI-digested agarose

plasmid

with

PstI

ligase

pJH415 which

fragment

designated

was selected

tle

pJH831

to introduce

order

plasmid

kb which

pJH302

carries

resistant

gene from

pJH302Aa.

resistant

pUBi

fragment

was deleted

The EP

gene

plasmid

site

of pJH302Aa.

The resulting

ried

the En-P

was designated

shown

gene

in Fig.

and confers

2.

Plasmid

upon both

The construction pJH1107

was modified gene without

StKUCtUKe

pLX25

(2)

could

hosts

its

be inserted

pBR322

coli __

ampicillin

at either

in both

fusion

(the

is

and Kanamycin

1156

of the

plasmid

gene

which

EcoRI car-

8.0 kb plasmid

is

and B. subtilis,

resistance.

depicted

promoterless OK

4.2 kb

to the unique

E. --coli

fragment,

the PstI

of 6

was designated

shuttle

and Kanamycin

the XhoI-BglII

the

shut-

has a size

A map of this

replicates

--coli

-

resistant

plasmid

as pJH1107.

promoter

(3)

pJH831 was subcloned

of the --penP-xylA so that

pJH302

the ampicillin

Bacillus-E. ~-_-

pJH1107

contained One plasmid

the subcloning

and the resulting

from

plasmid

all

a I__Bacillus-E.

Plasmid

1).

pJH831,

they

(14)

studies.

gene from

from

-E.

and T4 DNA

orientations.

To facilitate

(15).

HO

pHSG415

of the

that

to -B. ___. subtilis, (Fig.

of

EcoRl-treated

Gel analysis

subsequent

penP gene

ampicillin

on pJH302

for

on 0.7%

Sl nuclease

showed

on a

the penP gene, -

plasmid

with

two different

was constructed

EcoRI -_ penP containing cloned

clones

but with

with

by digesting

treatment

positive

located

by the method

ligated

the AmpK gene of pHSG415.

penicillinase

is

to isolate

recovered

than

was constructed

same 4.2 kb EcoRI

In

were

gene

DNA was fractionated

of 4-5 kb were

by the

=P

In order

chromosomal

followed

to abolish from

(8-11).

DNA fragments

and BstEll

prepared

fragment

and DNA fragments

The recovered

coli

the g. licheniformis

l_icheniformis

-B.

gel

(22).

that

AvaI

in Fig.

containing

2.

Plasmid

the __ xylA

-xylA),

isolated

from

site

of -penP.

This

was

Vol.

126.

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MboI

pJH302Aa

pJHIIO7

Figure

1.

The

strategies

for

by first

converting in

the

either

ments

were from

containing led

the

PstI

digested

JH1107-HX the

-E. coli -

of

site

site

of EP

and JH1107-HA

promoterless

-xylA

were structural

of plasmids fused

the -__ E. coli transformants

genes xylA

cloned

mutation. harboring

pJH217

isolated

by

resulting These

XhoI-BamHl

by the XhoI-BglIl

fragfragment,

from

pLX25.

were

found

This

and pJH110. and pJH217

The D-xylose

1157

site,

and pJH1107-HA.

gene,

on pJH110

pJHll0

to BamHl followed

to a XhoI

and the smaller

replaced

The detail

2.

of EP

pJH1107-HP

BamHl and XhoI

pJH1107 .

plasmid

Hind111

or the AvaI

with

to the construction

complement

the

of two new plasmids

The =P-xylA__

the

converting

construction

plasmids

construction

map of pJH1107 is shown in Figure

restriction

accomplished

the

and pJH217

able

isomerase

activities

have

been compared

to of with

Vol. 126, No. 3. 1985

8lOCHEMlCAL

Hmdlll - BomHI PstI-XhoI JHIIOI-HP

Figure

that

2.

of the

studies dent

untransformed

demonstrated

the -penP-aA

the

Plasmids

pJHll0

The resulting

found

to have higher

cells

(Table pJHll0

gene

cannot.

the

in -.E fact

of other

PSLl.

both

Hmd Ul - BO~HI AvovoI-XhoI JHllO?-HA

as shown

for

coli that

induction.

the expression genetic

and pJH217 were transformants

This

and pJH217

previous

in --E. coli

We found

that

of xylose

is

the

expression

induction.

of EP-xylA-

depenof

This

in --E. coli

is

under

signals.

PSLl(pJH110) activities that

can be expressed

Furthermore,

x&A

used to transform

isomerase

demonstrates

1.

of intact

Is independent

heterologus

xylose

in Table

expression

of xylose

the

2).

cells

that

fusions

confirms

control

RESEARCH COMMUNICATIONS

The protocol for the construction of hybrid plasmids containing penP-*A gene fusions. The strategies and the detail manipulation for the construction of pJHll0 and pJH217 have been described in the text.

on the presence

further

AND BIOPHYSICAL

and PSLl(pJH217) than

the =P-xylAin -.B

1158

-B. subtilis

subtilis

the

strain were

also

untransformed

fused

genes

while

the

cloned intact

PSLl on -xylA

Vol.

126,

No. 3, 1985

Table

1.

BIOCHEMICAL

Comparison

of xylose

AND

isomerase

SRIb

0.0

SRI(pJH1lO)C

6

SRI(pJH217)d

17

still

One is

natural

ribosomal

the

ribosomal

gene was inserted

binding

either

the structural

that

the x&A

site

is

natural

ribosomal for

has been demonstrated in a fused

may still

be substantially

Table

2.

Comparison

the

leader

the AvaI

site)

binding

site

of xylose

sequence

rather

(at

and the fact

the

we believe

functional

binding

ribosomal of the fusion transformants

of one of the

ribosomal

isomerase

activities

in various

B. subtilis -___

Enzyme Activity nmol/min/mg protein

PSI,1

4

PSL(pUBllO)a

6

PSL(pJHllO)a

23

PSL(pJH217)a

41 harboring

or

fusions.

Strains

aPSLl transformants

site)

in -B. subtilis.

of the Bacillus removal

is

the -xylA

the -penP ribosomal

the expression

activity

other

that Pstl

translation

of a second

by the

-xylA

binding

of the -penP gene,

hinder

improved

for

ribosomal

Due to the

of *A

isomerase

the EP-xylA-

site

-penP.

the presence

the xylose

from

for

gene may severly

Hence,

two functional

binding

the initiation that

(22,24).

site

(at

pJHll0 pJH217

contain

site

within gene

responsible

binding

strains

14

fusions

site

g. g

GMBa

the natural

ing

in various

COMMUNICATIONS

Enzyme Activity nmol/min/mg protein

The --penP-xylA

within

activities

RESEARCH

Strains

aE. -- coli wide type bA xylose isomerase mutant of GM8* 'SRI transformants harboring plasmid dSRI transformants harboring plasmid

sites.

BIOPHYSICAL

various

plasmids. 1159

strains

It bind-

Vol.

126,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENT This 0797,

work

was supported

and SERI Subcontract

by NSF Grant

PCM-8305043,

USDA Grant

81-CRST-2-

xX-3-03116.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Ho, N.W.Y., Rosenfeld, S.A., Stevis, P., and Tsao, G.T. (1983) Enzyme Microb. Technol., 5, 417-420. Rosenfeld, S.A., Stevis, P.E., and Ho., N.W.Y. (1984) Mol. Gen. Genet., 194, 410-415. Ho, N.W.Y., Stevis, P., Rosenfeld, S.A., Huang, J.J., and Tsao, G.T. (1983) Biotechnol. Bioeng. Symp., 13, 245-250. Yamanaka, K. (1966) Methods Enzymol., 2, 528-593. Bucke, C. (1977) Topics in Enzyme and Fermentation Technology, Part B, pp. 147-171, Halstead Press, England. Danno, G. (1970) Agri. Biol. Chem., 2, 1795-1804. Imahaka, T., Tanaka, T., Tsunekawa, H., and Aiba, S. (1981) J. Bacteriol., 147, 776-786. Brammar, W.J., Muir, S., and McMorris, A. (1980) Molec. Gen. Genet., 178, 217-224. Kroyer, J., and Chang, S. (1981) Gene, Is, 343-347. Acid Neugebauer, K., Sprengel, R., and Schaller, H. (1981) Nucleic Res., 2, 2577-2588. 145, 422-428. Gray, D., and Chang, S. (1981) J. Bacterial., S.D. (1977) Proc. Nat-l. Acad. Sci. UK 74, 1680-1682. Ehrlich, Bolivar, F., Rodriquez, R.L., Betlach, M.C., and Boyer, H.W. (1977) Gene, 2, 95-113. Hashimoto-Gotch, T., Franklin, F.C.H., Nordheim, A., and Timmis, K.N. (1981) Gene, 2, 227-235. Keggins, K.M., Lovett, P.S., and Duvall, E.J. (1978) Proc. Nat'l. Acad. Sci. USA, 2, 1423-1427. Clewell, D.B. (1972) J. Bacterial., 110, 667-676. Clewell, D.B., and Helinski, D.R. (lm) Proc. Nat-l. Acad. Sci. USA, 62, 1159-1166. Marmur, J. (1961) J. Mol. Biol., 3, 208-218. Yang, M., Galissi, A., and Henner, D. (1983) Nucleic Acid Res., 11, 237-249.

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J. (1973) J. Gen. Microbial., 76, 217-230. Sherratt, D., Collins, Cumer. J.C.. and Nester, P.W. (1976) Anal. Biochem., --70. 441-443. Ho, N;W.Y. (1983) Electrophoresis, I, 168-170. Zitomer, A.!?., Rymand, B.C., Schumperli, D., and Rosenberg, M.J. (1983) Gene Expression, pp. 523-541, Alan R. Liss, Inc., New York. Emerick, A.W., Bertolani, B.L., Ben-Bassat, A., White, T.J., and Konrad. M.W. (1984) Biotechnolonv. 165-168.

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