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.
20. 21. 22. 23. 24.
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.
1160