Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

175

Gene, 45 (1986) 175-181 Elsevier GENE

1698

Sequencing sphaericus

and heterologous

expression of the gene encoding penicillin V amidase from Bacillus

DNA; nucleotide sequence; ribosome-binding site; promoter; Escherichia coli host)

(Recombinant

Anders Olsson*

and Mathias UhlCn

Department of Biochemistry, Royal Institute of Technology, S-100 44 Stockholm (Sweden) Tel. + 468 787 7513

1lth, 1986)

(Received

April

(Revision

received

(Accepted

May 28th, 1986)

May 30th, 1986)

SUMMARY

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5’- and 3’-flanking regions, was determined using an improved sequencing strategy. The deduced ammo acid sequence suggests a protein consisting of 338 residues with an M, of 37 500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.

INTRODUCTION

Penicillin amidases, often called acylases, are enzymes that catalyze the hydrolysis of penicillins. Although a wide range of bacteria and fungi produce these enzymes, their physiological role is still unclear (Vandamme and Voets, 1974). Little is known about the structure and function of these enzymes and their corresponding genes, despite their use in very large * To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

aa, amino

acid(s);

propyl-j?-D-thiogalactopyranoside; otide(s);

ORF,

open reading

ribosome-binding sulfate;

u, units;

0378-l I19/86/$03.50

bp, base pair(s); kb,

frame;

iso-

nt,

nucle-

PA, polyacrylamide;

RBS,

site; ss, single stranded;

[ 1, designates

IPTG,

1000 bp;

SDS, sodium dodecyl

plasmid-carrier

state.

0 1986 El sevier Science Publishers

B.V. (Biomedical

amounts for the production of semisynthetic penicillins. Best characterized is the penicillin G amidase from E. coli which has been cloned and partially sequenced (Bruns et al., 1985). This revealed arather complex posttranslational processing of a 90-kD primary translation product reminiscent of the processing of some eukaryotic hormones. Recently, the penicillin G amidase of Proteus rettgeri has been shown tb have a subunit structure similar to that of E. coli (Daumy et al., 1985) and the subunits have been shown to play different functional roles in catalysis. We have earlier described a penicillin amidase from B. sphaericus (Olsson et al., 1985). Biochemical characterization revealed that the enzyme differs both structurally and functionally from the other two amidases. The preferred substrate is penicillin V Division)

176

instead

of penicillin

G and the active enzyme

sists of four identical 35 000. Here

we report

subunits

con-

the complete

sequence

of this

penicillin

amidase

complete

DNA sequence from this class of enzymes.

(a) Characterization V amidase

of the gene encoding penicillin

gene which is the first report of a

It is also the first gene B. sphaericus, a bacillus esting biological

RESULTS

with a M, of approx.

products,

sequence described synthesizing many

from inter-

such as toxins with high

biocidal activity against several species of pathogenic mosquitoes

(Louis et al., 1984).

MATERIALS

AND METHODS

(a) Strains and plasmids The bacterial strains used were E. coli JM 103 and E. cofi RR1 (Messing, 1983). The vectors employed were pEMBL 8 + , 8 - and 9 (Dente et al., 1983) M13mp8 and M13mp19 (Messing, 1983). (b) General techniques Restriction endonucleases, T4 DNA ligase and BAL 3 1 exonuclease were purchased from Boehringer Mannheim and New England Biolabs and used according to the suppliers recommendations. [a-35S]dATP was obtained from Amersham International. The M 13 RIT universal sequencing primer (5’-AGGGTTTTCCCAGTCACGAC-3’) was obtained from KabiGen AB, Stockholm (Sweden). Deoxy, dideoxy and oligonucleotides were from PL-Biochemicals, Uppsala, (Sweden). Agarose and PA gel electrophoresis, ligation and transformation of E. coli were all standard procedures according to Maniatis et al. (1982). SDS-PA electrophoresis was performed as described by Laemmli (1970). Penicillin amidase according to Kornfeld to Bradford (1976). detect activity whole chromogenic substrate benzoic acid (Olsson

We have

earlier

reported

that

amidase gene from B. sphaericus located on a 2.2-kb HindHI-PstI

the penicillin

V

ATCC14577 is fragment which

was cloned into an E. cofi plasmid 1985). The plasmid was designated

(Olsson pOH35

et al., and a

more detailed restriction map of the 2.2-kb DNA fragment is shown in Fig. la. To facilitate further characterization of the gene, deletion mapping by BAL 3 1 exonuclease treatment was performed starting from the PstI site. A set of plasmids containing the insert with differently sized deletions were obtained. The result is shown in Fig. lb. Construction of this set of deletions had two purposes. First, one border of the gene required to get a functional enzyme could be defined, thus simplifying the characterization of the gene. Second, the deletions yielded overlapping inserts allowing a simple and quick sequencing strategy. Taking advantage of the pEMBL-vectors no subcloning was needed as the ss templates could be obtained simply by superinfection with fl phage (Dente et al., 1983). In addition, several restriction fragments of the inserts were subP&I

0

+

b

1

2

t

I

t

-

I I

i

+I -

activity was determined (1978) and protein according As an alternative method to cells were resuspended in the 6-nitro-3-phenoxyacetamido et al., 1984).

-

C

Fig.

1. Restriction

map, deletions

and sequencing

penicillin V amidase gene. (a) partial restriction HindIII-PstI

fragment

harboring

(shown as bar); (b) schematic and restriction indicating

fragment

enzymatic

1.6 kb P.~tl-AhaIII

the penicillin

representation

subclone

activity.

fragment.

(lower)

(c) sequencing

strategy

of the

map ofthe 2.2-kb V amidase

gene

ofBAL31

products

with (+

) and (- )

strategy

for the

177

cloned

and one of those,

the

1.3-kb AhaIII-PstI

fragment, is shown in Fig. lb. The plasmids by the BAL 3 1 treatment

obtained

sequence complementary

to the 3’ end of 16s rRNA

of B. subtilis. For Bacillus and Staphylococcus genes,

were tested for enzymatic

activity. The results (Fig. 1b) suggest that the borders of the gene are situated between position 0.0 kb and the AhaIII

the PstI site at site at 1.3 kb

(Fig. la). (b) Improved sequencing strategy We have earlier described

a new method to sepa-

300

rate sequencing reactions using a field strength gradient denaturing PA gel (Olsson et al., 1984). However, to obtain an even more versatile sequencing system we decided to introduce a few improvements. First, a new sequencing primer was synthesized, complementary to the ss DNA and hybridizing further away from the multilinker region of the Ml3 and pEMBL vectors. Second, 4% PA gels were used thus giving better separation of large fragments. Using this system an improved separation of DNA fragments is obtained between 60 and 300 nt in length (Fig. 2). The new RIT primer ensures that no sequence information inserted in the multilinker region is lost. Thus a single separation on 55 cm PA gel can be performed to obtain at least 300 nt of inserted DNA. (c) The nt and deduced aa sequence Using the described sequencing strategy the entire nt sequence of the 1.6-kb Pstl-AhaIII fragment was determined (Fig. 3). A sequence analysis using the University of Wisconsin Genetics Computer Group sequence analysis soft-ware package (Devereux et al., 1984) revealed only one large ORF starting with an ATG initiator codon at nt 326 and terminating in a TAA stop codon at nt 1339. This corresponds well with the borders determined by the deletion analysis. The deduced protein consists of 338 aa with a predicted M, of 37 459. This is in close agreement with the experimental value of approx. 35000 found by SDS-PA gel electrophoresis (Olsson et al., 1985). As expected, the analysis of codon preference according to Gribskov and Devereux (1984) revealed an increased number of rare codons in the two other reading frames as well as immediately outside the proposed structural gene. The putative ATG initiator codon is preceded by a

Fig. 2. Example strength

gradient

of an autoradiogram sequencing

the size of the elongated 20’-mer

universal

C. Nucleotide Sanger

oligodeoxynucleotide

RIT primer.

sequencing

of a wedge-shaped

gel (4% PA). The numbers

sequencing

primer.

by Olsson

followed the procedures

The extended

on a wedge-shaped

system.

Single-stranded or M 13 vectors.

DNA

the

estabished

by

and the 20-nt long RIT

oligodeoxynucleotide

chains

PA gel as described

et al. (1984) using the thermostatic

pEMBL

including

Lanes are in order G, A, T and

et al. (1977) using [a-%]dATP

were separated

tieldrefer to

was prepared

earlier

LKB Macrophor using

either

the

178

Ah.111 TTTAAAAATACTACTTCATAGTATAGAAATAATAGTAACGCCAAAAAATGACGGTGT

57

TTA Leu

AGA ArS

GCT Ala

TAC Tyr

ATT Ile

GGT Gly

GTC Val

ACA Thr

CCA Pro

AAT Asn

CCG Pro

CCA Pro

CAA Gin

GAT Aep

ATA II.

934

ATGTGGCGCGATCTGGCGTTATTGCATGGGATTGGAAATTTCAGTCTTAAAAAAAGGTGT

117

ATG Met

ATG Met

GGA Gly

GAC Asp

TTG Leu

GAT Aep

TTG Leu

ACA Thr

CCG Pro

TTT Phe

GGG Gly

CAA Gin

GGG Gly

GCA Ala

GGG Gly

979

ATGACCGCCAAAAAACGGCGTAAATTTATCCTTT

177

GGC Gly

TTA Leu

GGA Gly

TTA Leu

CCA Pro

GGT GLy

CAT Aep

TTT Phe

ACG Thr

CCG Pro

TCA Ser

GCA AIo

CGT ArS

TTT Phe

CTT Leu

1024

TGGTTTTGGAAGCGGAATAAATCTATTTTTAGTTATAGTGAAG

237

CGG hrg

GTA Vol

GCA Al.

TAC Tyr

TGG Trp

AAA Lye

hhh Lye

TAT Tyr

ACT Thr

GAA Glu

AAA Lye

GCC Ala

AAA Lye

AAT Asn

Ghh Glu

,069

297

ACA Thr

Ghh Glu

GGC Gly

GTA Vol

ACA Thr

AAC hen

TTG Leu

TTC Pho

CAT H>s

ATT IIe

CTA Leu

TCT Ser

TCT SW

GTA V-1

AAT Aon

1114

AhoIII TAAATTCTTTATTATGAAGATACGTTAGTTGATTTAAAAATAATTCCGTTACATTTTTTT

l *

AhcrIII

l ***

AAAATACTTTTTCAAGGGAGTGTTTTTT

ATG Met

TTA Leu

GGT Gly

TGC cys

AGT Ser

AGC Ser

TTA Leu

TCA Ser

349

ATC Ile

CCA Pm

AAA Lye

GGT Gly

GTT Val

GTT Vol

TTC Leu

ACA Thr

AAT Asn

GAG Glu

GGG Gly

AAA Lye

ACG Thr

GAT hap

TAT Tyr

1159

ACC Thr

ATC Ile

TAT Tyr

ACC Thr

TCA Ser

GCT Alo

ATG Met

TGT Cys

GCA Ala

CAA Gin

AGT Ser

AAA Lys

AAC hen

TAT Tyr

TAC Tyr

1204

Ah.111 TTT AAA Phe Lye

CTG LPU

TAT Tyr

GAC Aep

AAT Asn

AGT Ser

CGA ArS

ATT Ile

TCA Ser

GCC Al.

GTT Vol

TCC Ser

TTA Lsu

ATG Met

1249

ATT

11~ Arg Thr

CGT

ACA

ACA Thr

GAT Aep

GAT A.p

AAA Lye

ACT Ser

TTA Leu

TTC Phe

GCT Al.

CGC Arg

ACA ATG Thr: Met

GAT Asp

394

TTT phe

ACA Thr

ATG Met

GAA GIu

CCA Pro

GAT A-p

AGT Ser

AAA Lya

GTG V-1

ATT IIe

ATT II.

GTC V-1

CCA Pro

CGT Arg

AAT Aon

439

TAC Tyr

GGC Gly

ATT Ile

CGA ArQ

TTG Leu

TTA Leu

GAA Glu

AAA Lye

GAA Glu

AAT Aen

GTA Val

GTC V-1

ATT Il.

AAC Awn

AAT Aen

484

GCT hi.

GAA Glu

Ah.111 AAT TTA AAT Am Leu Aen

AGT Ser

CAA Gin

GAT Asp

TTA Leu

ATT Ile

ACA Thr

TTT Phe

GAG Glu

TGG Trp

GAT Asp

1294

TCA Sar

TAT Tyv

GCT Al.

TIT Phe

GTT V.1

GGA GIy

ATG Met

GGA Gly

AGC Ser

ACT Thr

GAC Asp

ATT 11.

ACA Thr

TCA Ser

CCA Pro

529

CGT ArQ

AAA Lye

CAA Gin

AAG Lye

Chh Gin

TTA Leu

AAT Asn

CAA Gin

GTA VoI

AAT Aen

GTA Vol

ATG Met

AGC Ser

1339

GTT VaI

CTC Leu

TAT Tyr

GAT Asp

GGG Gly

GTA "al

AAC Asn

GAA Glu

AAG Lye

GGA Gly

TTA Leu

ATG Met

GGC Gly

GCA hlo

ATG Met

574

TAhAAATTGCCTATTATATAGTACAAGGTATTAAAAAATGCCCCCGATTGTTAGATATAT

CTT Leu

TAC Tyr

TAT Tyr

GCT

ACA Thr

TTT Phe

GCG Al.

ACT Thr

TAT Tyr

GCT Alo

GAC Asp

GAA Glv

CCT Pro

AAA Lyb

AAA Lye

619

GAACAATCGGGGGCTCTTTTTCGATAGTAAAATACACAAAGTCATTAGAATTAAAAAGAT

1459

Ala

GGC Gly

ACA Thr

ACA Thr

GGC Gly

ATC Ile

AAT hen

CCC Pro

GTG Vol

TAT Tyr

GTA Val

ATT Ile

TCT Ser

CAA Gin

GTT "01

TTA' Leu

664

TTGTGGAATGTTAATATATTGTTAGAAATTATTTCACTGTAAAGATAGGAAAGTATCCGA

1519

GGA Gly

AAT Asn

TGT Cye

GTA Val

ACT Thr

GTC GAT r/al A.p

GAT Asp

GTT Vnl

ATT Iie

GAA Glu

AAA Lye

TTA Leu

AC? Thr

TCT Ser

709

AAAAGCTCATTGTGGTTGTGAGGATTGCCAACTTTTCGCTAAGCAAATTCTATATGCAAG

1579

TAT Tyr

ACA Thr

TTA Leu

TTG Leu

AAT Aan

GAG Glu

GCC Alo

AAT hen

ATA Il.

ATA Ile

CTT Leu

GGC Gly

TTT Phe

GCA Ala

CCC Pro

754

TCCACAAGTTTTGGATTTCTTTAGCAGAGGTCTGCAG

CCA pro

CTT Leu

CAC Hts

TAT Tyr

ACA Thr

TTT Phe

ACA Thr

GAT Aep

GCT Al.

TCT Ser

GGT Gly

Cl.1 GAA TCG GIu Ser

ATT Ile

GTT "al

799

ATT Ile

GAA Glu

CCG Pro

GAT Amp

Ahh Lye

ACA Thr

CGC Gly

ATT Ile

ACC Thr

ATT Ile

CAT His

CGA ArS

AAA Lys

ACG Thr

ATT Ile

844

GCC GIy

GTC Val

ATG Met

ACG Thr

AAT Aon

AGC Ser

CCT Pro

GGC Gly

TAT Tyr

GAA Glu

TGG Trp

CAT His

CAG Gin

ACA Thr

AAT Asn

889

GAT Asp

ATT Ilc

________-----,399

__________-___

Fig. 3. Nucleotide

and deduced

aa sequence

(*) and a possible

transcription

terminator

of the penicillin V amidase sequence

gene. Nucleotides

P&I

complementary

1616

to the 16 S rRNA ofB. subtilis

(----) are indicated.

the distance between the last G in the Shine-Dalgamo sequence and the start codon is usually 8-9 nt (McLaughlin et al., 1981) and the region often consists of a high content of A + T residues (Band and Henner, 1984). Both these criteria are fulfilled for the ATG codon at nt 326. In conclusion, although we have not shown that the codon at this position is used to initiate translation, these structural con-

siderations as well as the codon preference analysis and the size of the deduced protein all strongly suggest that this is the start of translation. A number of sequences similar to promoters known to be functional in B. subtilis(Johnson et al., 1983) can be found in the 5’-flanking region of the structural gene. Some resemble sigma-43 promoters with sequences similar to the consensus sequence

179

TTGACA (-35) and TATAAT (-lo), but also sequences resembling sigma-37 promoters with the consensus sequence AGG-TTT (-35) and GGAATTATTT (-lo), can be observed. Nothing is known about promoter sequences in B. sphaericus but it is reasonable to expect similarities to the promoters found in B. subtilis. As the penicillin V amidase gene is the first gene to be sequenced from this species, more work is required to define the promoter of this gene. The structural gene is followed by a palindromic sequence suggesting a transcription termination signal (Fig. 3). This sequence is followed by a stretch of T residues and the free energy of the hairpin structure is -33.1 kcal/mol, similar to other termination signals (Rosenberg and Court, 1979). Hydrophobicity predictions according to Kyte and Dolittle (1982) of the deduced aa sequence did not reveal any marked hydrophobic regions that could suggest the presence of a signal sequence or TABLE Codon aa Phe Leu

Be

I usage for the penicillin

V amidase

codon uuu

10

uuc

2

UUA

gene

aa

15

UUG

6

CUG

4

cut

1

CUA

1

CUG

1

AUU

18

AUC

3

AUA

3

Met

AUG

12

Val

GUU

8

GUC

5

GUA

9

GUG

2

ucu

5

ucc

1

UCA

6

UCG

1

Ser

membrane spanning region (Silhavy et al., 1983). This is consistent with an intracellular location. To clarify any structural homologies with the penicillin G amidase of E. coli, a homology plot analysis was performed (Uhlen et al., 1984). No region with significant homology was observed (not shown). This and the fact that the E. coli enzyme is secreted and consists of processed subunits of different sizes supports the conclusion that these enzymes have evolved along different phylogenetic paths. The codon usage frequency for the different aa is presented in Table I. The utilisation of codons in the penicillin V amidase gene is unusual and probably influenced by the low G + C content, (36.5 %), of the B. sphaericus genome (Fasman, 1975). In fact the preference for A + T in the wobble positions of the codons of the gene is as high as 75 y,. One exception from this is glycine codons, where a majority of triplets have a C in the third position.

Tyr Term

codon

aa

UAU

2

Term

UGA

UAC

1

Trp

UGG

3

Arg

CGU

4

UAA

-

UAG

-

His

CAU CAC

1

Gln

CAA

8

CAG

1

Asn

AAU

18

AAC

4

LYS Asp Glu CYS Thr

codon

3 Ser Arg Gly

-

CGC

1

CGA

3

CGG

1

AGU

6

AGC

4

AGA

1

AGG

0

AAA

17

GGU

5

AAG

2

GGC

10

GAU

16

GGA

6

GAC

4

GGA

5

GAA

13

ecu

2

Pro

GAG

3

ccc

2

UGU

2

CCA

8

UGC

1

CCG

4

ACC

5

ACC

3

ACA ACG

Ala

GCU

8

GCC

3

19

GCA

6

4

GCG

1

Sum 338 % G + C” 34.7 a % G + C in the third degenerate

base omitting

the codons

AUG (Met), UGG

(Trp) and AUA (Be).

180

(c) Expression

of the gene in E. coli

b

a

cde

We have previously shown that a functional penicillin V amidase can be expressed from the cloned gene in E. coli minicells.

However,

in whole cells of E. coli was found to be low,

despite

the fact that a high-copy-number

was used. In fact, E. colicells containing gene produce

45

plasmid the plasmid-

less enzyme than the original

B. sphaericus strain containing (Olsson

one copy of the gene

et al., 1985). This reduction

in expression

can be at the level of transcription, translation or posttranslation. As the minicell analysis suggested that the gene product is relatively stable in E. co& we decided to try to enhance the expression by inserting the gene after a suitable E. coli promoter. To facilitate such expression studies the AhaIII site at nt 300 in Fig. 3 was converted into a BamHI site using BamHI oligodeoxynucleotide linkers. The resulting 1.3-kb BamHI-PstI fragment was inserted into the BamHI/PstI sites of pEMBL8 giving the plasmid pOH50 in which the penicillin V amidase gene with the putative RBS has been fused directly downstream from the luc promoter. The plasmid was introduced into E. coli RR1 and cells were grown in the presence or absence of 1 mM IPTG. The results (Table II) show that the cells produce high amounts of enzymatically active protein and that induction of the lac promoter gives approximately a 200-fold increase in yield. Analysis of the total protein content of the E. coli cells, with or without inducer, is shown in Fig. 4. A major band comigrating with purified penicillin V of B. sphaericus can be seen in the induced

amidase

TABLE

V amidase

activity

of induced

and uninduced

cells E. coli RRl[pOHSO]

were grown

in the presence

1 mM IPTG. The cells were disrupted collected.

Enzymatic

activity

Kornfeld

(1978)

the

described

by Bradford

Cells

and

total

was

or absence

and the supernatant determined

protein

was

Specific

Noninduced

0.016

Induced

3.20

of was

according

to

determined

as

(1976). activity

(u/mg)

21 14

Fig. 4. An SDS-13% [pOH50].

PA gel of total

Cells were grown to A,,,

IPTG. Cells were harvested extract corresponding Lanes:

b, total

extract

from induced

B. sphaericus

of E. coli by 1 mM

after 5 h and homogenised.

25 ~1 of

to 250 ~1 culture was applied to each lane.

cell extract

(Olsson

cell extracts

= 0.5 and induced

from uninduced

cells; d, purified

cells; c, total

penicillin V amidase

cell from

et al., 1985). Lanes a and e are marker

proteins

(phosphorylase

B, bovine serum

soybean

trypsin inhibitor

and lysozyme).

albumin,

ovalbumin,

Sizes are given in kDa.

cells. From the SDS-PA gel, the amount of enzyme produced is estimated to be approx. j-10% of the total protein content (Fig. 4). (d) Conclusions

II

Specific penicillin

66

the level of pro-

duction

encoded

92

In this paper, we have characterized the gene encoding penicillin V amidase from B. sphaericus. We have also shown that this enzyme can be produced efficiently in E. coli using a host promoter allowing inducible expression. It is now possible to perform refined genetic approaches to study the effect of specific changes in the primary sequence of the enzyme. Such studies might create a new enzyme engineered to perform specific reactions. In addition, gene fusions to genes encoding ‘affinity tails’ can be performed, thus facilitating the immobilization and purification of the enzyme (Brewer and Sassenfeld, 1985).

181

Kornfeld,

ACKNOWLEDGEMENTS

J.M.: A new calorimetric

of 6-aminopenicillanic

This investigation was supported by grants from the Swedish Board for Technical Development and Fermenta

AB, Sweden.

Gatenbeck,

Bj6rn

fruitful discussions Benson

We wish to thank Drs Sten

Nilsson

and

Andras

Gaal

and critical comments

for patient

secretarial

method for the determination

acid.

Anal.

Biochem.

86 (1978)

118-126. Kyte, J. and Doolittle, hydropathic

R.T.: A simple method

character

of a protein.

for displaying

the

J. Mol. Biol. 157 (1982)

105-132.

for

Laemmli, U.K.: Cleavage bly of the head

and Gerd

of structural

proteins

of bacteriophage

during the assem-

T4. Nature

227 (1970)

680-685.

help.

Louis, J., Jayaraman,

K. and Szulmajser,

J.: Biocide gene(s) and

biocidal activity in different strains ofBacillus sphaericus. Mol. Gen. Genet. Maniatis,

125 (1984) 23-28.

T., Fritsch,

A Laboratory

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